American Pharmaceutical Review | Volume 22 Issue 3 | April 2019

American Pharmaceutical

The Review of American Pharmaceutical Business & Technology

Volume 22 Issue 3 | April 2019

SHOW ISSUE:
CPhI North America
BIO International Convention

The Application
of Risk Assessments
for the Design and
Development of Devices
for Biological Products

Low-Frequency Raman
Mapping and Multivariate
Image Analysis for
Complex Drug Products
www.americanpharmaceuticalreview.com

Our Complicated
Relationship with Fungi
www.americanpharmaceuticalreview.com
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 Corporate
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2 | | April 2019
 April 2019 | Volume 22, Issue 3

COVER FEATURES
28 MICROBIOLOGY
Our Complicated Relationship with Fungi
Jeanne Moldenhauer
Excellent Pharma Consulting, Inc.

40 BIOPHARM DEVELOPMENT
The Application of Risk Assessments for the Design and Development of Devices for Biological Products
Manfred Maeder, PhD
Global Head Device Development & Commercialization
Global Drug Development/Technical Research & Development
Novartis

48 SPECTROSCOPY
Low-Frequency Raman Mapping and Multivariate Image Analysis for Complex Drug Products
Daniel R. Willett, Huzeyfe Yilmaz, Anna M. Wokovich, Jason D. Rodriguez
Food and Drug Administration (FDA)/Center for Drug Evaluation and Research (CDER)/Office of Pharmaceutical Quality
(OPQ)/Office of Testing and Research (OTR)/Division of Pharmaceutical Analysis (DPA), St. Louis, MO

www.americanpharmaceuticalreview.com | | 3
IN THIS ISSUE »
12 MICROBIOLOGY 44 ROUNDTABLE

Focusing on the Operator: Reducing Facility Environmental Contamination Contamination Control

Tim Sandle, PhD
Head of Microbiology and Sterility Assurance, 52 ROUNDTABLE
Bio Products Laboratory Limited Raw Materials and Functional Excipients
Elstree, Hertfordshire, United Kingdom

18 FORMULATION & DEVELOPMENT SPECIAL FEATURES

Bioavailability Enhancement By Attenuating Presystemic Metabolism
Phillip M. Gerk, Pharm.D., PhD
10 Social Media Connections
Associate Professor, Virginia Commonwealth University School of Pharmacy,
Department of Pharmaceutics
11 CNPerspectives
24 BIOPHARM PROCESSING
Single-Use Systems Continue to Gain Traction Among CMOs
Ilene Roizman
Communications Manager REGULAR FEATURES
BioPlan Associates

32 FORMULATION AND DEVELOPMENT 6 A Message from the Editor

Polymeric Particles as Cancer Vaccine Vectors
Suhaila O. Suliman, Graduate Student 8 Editorial Advisory Board
Emad I. Wafa, Graduate Student
Sean M. Geary, Assistant Research Scientist
54 P.I.N. Points
Aliasger K. Salem, Professor
Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy
University of Iowa 56 Advertiser's Index

4 | | April 2019
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 » A Message from the Editor »
Collecting Data for Data’s Sake

Way back when, pre-Internet, the publishing company I worked for produced about 21 different magazines. All were new product tabloids
covering every imaginable industry. As a result, we had a large editorial department, since this was pre-internet, and pre-email, everything
was sent to us via regular mail. Editors had to sort through piles of mail, choose products that were appropriate to their markets and write
up new product reviews, oftentimes more than a hundred for each issue.
After an issue was complete, one of the tasks each editor had to do every month was to take a printed issue, and assign a code to every
product and every advertisement. This was a tedious task that involved looking through a massive printout of product categories, finding
the appropriate code, and writing that code on every piece of content in the issue. Depending on the size of the issue – this task could take
hours to complete.
Once finished, the marked-up issue was sent to Data Entry for processing. As Editors we all assumed this data was being used for some
purpose. When, finally, the inevitable happened, and someone asked what the data was being used for, the answer came back, “We aren’t
using it for anything, but keep doing it in case we need it, because we might.”
It wasn’t long after this, that we were allowed to end this task. To this day, I have no idea if that data collected was ever used for anything
worthwhile. Who knows, all this data could be sitting in a cloud somewhere.
I imagine the pharmaceutical industry has operated in this way for some time. Collect as much data as possible, store it, because who knows
someday it might be needed.
This is a wasteful, time-consuming and inefficient practice. As a traditionally conservative industry, you can sort of understand why it has
been this way for so long, but finally there are some initiatives taking hold that will hopefully transform the collection of data into truly
actionable information.
First is the promise of Big Data. With all of the data companies have been collecting, the time has finally come where this data can be used
for advancing programs and products. Without going into details leveraging Big Data is finding applications in advancing clinical trials,
increasing industry collaboration, streamlining sales and marketing, and developing apps for consumers.
On the heels of the Big Data push is the Pharm 4.0 initiative – which will essentially tie all resources - human, data, and physical machines –
in one virtual network.
Pharma 4.0 combines diverse technologies, including big data analytics and cloud computing. The ability to analyze the enormous amount
of data collected and then share insights will allow faster innovation and a quicker response to changing market dynamics.
It’s encouraging to see data used for a purpose, other than just for “in case”. Technology has advanced to the point where billions of pieces
of data can be collected and stored for every operation. It’s time that data was leveraged for good.

Mike Auerbach
Editor-In-Chief
mauerbach@comparenetworks.com

6 | | April 2019
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 » Editorial Advisory Board »
Shaukat Ali, PhD John Finkbohner, PhD Daniel L. Norwood, MSPH, PhD
Technical Support Manager Director, Regulatory Affairs Distinguished Research Fellow
BASF Corporation MedImmune Boehringer Ingelheim Pharmaceuticals, Inc.

Ghulam Shabir Arain, PhD, CSci, CChem, Adam S. Goldstein Mehul Patel
FRSC, FCQI Senior Manager, Clinical Purification, Operations/Development Global Marketing Director
Managing Director Genentech Endotoxin and Microbial Detection
DGS Pharma Consulting Ltd., UK Charles River
Davy Guillarme, PhD
Katherine Bakeev, PhD Senior Lecturer, School of Pharmaceutical Sciences David Radspinner
Director of Analytical Services and Support University of Geneva, University of Lausanne Director of Marketing and Applications Support for BioProcess
B&W Tek, Inc. Production
Chris Halling
Douglas J. Ball, MS Thermo Fisher Scientific
Senior Manager, Global Communications
Diplomate, American Board of Toxicology; Research Fellow, and European Marketing
Drug Safety Research & Development Aniruddha M. Railkar, PhD
Catalent Pharma Solutions
Pfizer Global Research & Development Director of CMC
Tarsa Therapeutics
Brian Lingfeng He
Suraj Baloda, PhD
Research Investigator Gary E. Ritchie
Founder and President
Bristol-Myers Squibb President
SARMICON, LLC.
Council For Near Infrared Spectroscopy
Ronald Iacocca, PhD
Rory Budihandojo
Senior Research Advisor, Product and Process Performance Rodolfo J. Romañach, PhD
Director, Quality Systems Audit
Eli Lilly & Co. Professor of Chemistry
Boehringer Ingelheim Shanghai Pharmaceuticals
Co., Ltd. University of Puerto Rico, Mayagüez Campus
Maik W. Jornitz
Vice President of Business Development Shouvik Roy, PhD
Harsh Chauhan, PhD
G-Con Manufacturing, LLC Principal Scientist, Organizational Unit Leader in
Assistant Professor
School of Pharmacy & Health Professions Hemant N. Joshi, PhD, MBA Drug Product Engineering
Creighton University Principal Amgen
Tara Innovations LLC Jim Rydzak
Robert V. Chimenti
Sr. Strategic Applications Engineer Ian Lewis, PhD Investigator, Strategic Technology Division
Innovative Photonic Solutions Director of Marketing GlaxoSmithKline
Adjunct Professor Kaiser Optical Systems, Inc. Ronak Savla
Rowan University
Ralph Lipp, PhD Fellow
Emil W. Ciurczak, PhD President and CEO Catalent Applied Drug Delivery Institute
Doramaxx Consulting Lipp Life Sciences LLC
Ken Seufert
Rick E. Cooley Jack Lysfjord Managing Director, North America
Retired Principal Consultant MEGGLE USA Inc.
Eli Lilly & Co., Inc. Lysfjord Consulting LLC
Jaleel Shujath
Weiguo Dai, PhD Steven R. Maple, PhD Industry Strategist, Life Sciences
Scientific Director, Janssen Fellow, Drug Product Development Head of Pharmaceutical Technology Development Dept. OpenText
Johnson and Johnson Lilly Research Laboratories, Eli Lilly & Co., Inc.
Donald C. Singer
Nila Das, PhD Jerold M. Martin GSK Senior Fellow, R&D
Senior Research Investigator Senior Vice President, Scientific Affairs GlaxoSmithKline
Bristol-Myers Squibb Pall Life Sciences
Onkar N. Singh, PhD, MBA
Vivek Dave, PhD John P. Mayer Director, Pharmaceutical Development at CONRAD
Assistant Professor, Pharmaceutical Sciences Senior Research Scientist Eastern Virginia Medical School
St. John Fisher College, Wegmans School of Pharmacy Indiana University
Allen Templeton, PhD
Michael Dong, PhD Michael J. Miller, PhD Associate Vice President
Consultant President Formulation Sciences Merck Research Laboratories
MWD Consulting Microbiology Consultants, LLC
Zhenyu Wang, PhD
Dr. Thomas Dürig Ronald W. Miller, PhD, MBA Associate Principle Scientist and Group Leader
Sr. R&D Director, Pharmaceutical and Food Ingredients President , Technology Consultant Respiratory Product Development
Ashland Inc. Miller Pharmaceutical Merck & Co.

Walter Dziki, PhD Ganapathy Mohan, PhD Wayne K. Way, PhD

Associate Research Fellow Head of Global CMC Regulatory Affairs Analytical Business Marketing Manager
Abbott Laboratories Merck, Sharp and Dohme Corp. MilliporeSigma

Stuart Farquharson, PhD Shane R. Needham, PhD Larry Wigman, PhD

President & CEO Laboratory Director Principal Scientific Manager
Real-Time Analyzers, Inc. Alturas Analytics, Inc. Genentech

8 | | April 2019
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 AMERICAN PHARMACEUTICAL REVIEW

Social Media Connections

Weill Cornell Medicine CDC

@WeillCornell @DanaFarber
The Starr Foundation is continuing its commitment to stem
We can end the #HIV epidemic in the US. CDC’s new
cell research in #NYC with a $50 million gift in support of
#VitalSigns report shows why immediate, effective treatment
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collaboration between @sloan_kettering,
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@RockefellerUniv & #WCM. https://bit.ly/2IdG36r

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"We need to shorten the time it takes from idea to
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“By the year 2030, colon cancer is estimated to rise 90% and
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Celgene Corporation
Memorial Sloan Kettering @Celgene
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@sloan_kettering #NEWS: #Celgene and @AcceleronPharma announce
submission of a Biologics License Application to the U.S. FDA
Researchers have redesigned CAR T cells in hopes of in both #MDS & Beta-Thalassemia for an investigational agent.
improving immunotherapy treatment for multiple myeloma. #thalassemia http://bit.ly/2uNaQ27
https://t.co/pQsM9Y2yFx

Connect with American Pharmaceutical Review at:

facebook.com/AmericanPharmaceuticalReview
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linkedin.com/groups/American-Pharmaceutical-
Review-3889659

10 | | April 2019
CNPerspectives
American Pharmaceutical Review is one of several outstanding publications available from CompareNetworks, Inc.
Here is a look at the insightful content our readers may enjoy from four of our sister publications: Pharmaceutical Outsourcing,
Labcompare, American Laboratory and Biocompare.

Assessing Cold Chain Control for Clinical Trial Success

Preclinical research provides a great deal of insight into a new treatment’s safety, but clinical studies are vital to determining how a drug will affect
the human body, enabling researchers to gauge toxicity and overall efficacy of the treatment to achieve the desired patient outcome. While there
is always risk associated with a clinical trial, there are elements that give the study its best possible chance of success. Paramount among these
factors is the storage and handling of clinical compounds at each clinical trial site. Segregation and control of clinical compounds from approved
drugs can be a heavy burden at the trial sites, especially considering the increasing volume of approved temperature-sensitive drugs.
https://bit.ly/2I6w57Y

What Are Emerging Contaminants and Why Are

We Concerned?
The term “compounds of emerging concern” (CEC) is used to identify chemical contaminants that have no regulatory standard, have been found
in environmental samples, and have potentially adverse effects on aquatic life. Of particular concern are compounds that may have always been
present, but at concentrations previously below limits of detection, or compounds that pass through water treatment processes. One may ask
why so many of these CECs remain CECs even though we knew about them years ago. Contrast current CEC with the original priority pollutants,
or, in particular, the routinely monitored conventional pollutants, trace metals, radionuclides, organic solvents, and pesticides. At one time, each
of the mentioned classes of compounds were CEC.
https://bit.ly/2I88hQS

Expansion of Human Bone Marrow-Derived

Mesenchymal Stem Cells in a Single-Use Bioreactor

One of the challenges in providing human mesenchymal stem cells (hMSCs) for curative use is the production of large quantities of cells in a
robust manner. Whatever the tissue source, the number of hMSCs extracted will not be sufficient for clinical use; hMSCs have to be expanded
following isolation. Besides providing the needed cell quantities, hMSC production must also comply with the manufacturing process regulations
required of a fully controlled production system. hMSC expansion in stirred-tank bioreactors can be monitored and is scalable, and hence can
fulfill these requirements, from experimental quantities to production.
https://bit.ly/2thktph

Alternative Biomanufacturing Platforms

The manufacture of therapeutic proteins, particularly monoclonal antibodies, has evolved into reliable, robust protocols characterized by
platform processes and standardized unit operations. These production methods, and their attendant efficiencies, have been disruptive in the
traditional sense but took decades before they were universally accepted. For example, single-use processing took years to reach its current
status, and has perhaps reached an adoption steady state. Similarly, the roughly tenfold improvement in titers for fed-batch CHO (Chinese
hamster ovary) cell cultures—a significant driver for disposable manufacturing—has been transformative, but took decades to achieve.
https://bit.ly/2uVoZKG

www.americanpharmaceuticalreview.com | | 11
» MICROBIOLOGY »

Focusing on the Introduction

The majority of contamination in a pharmaceutical facility, presuming

Operator: Reducing that the air handling system is functioning as designed, that water
systems are low in bioburden and are not leaking, and there is good
control of in-coming materials, will derive from people. It follows

Facility Environmental that an effective contamination control strategy will focus on the
control of operators. Control extends to behaviors, gowning and
having the correct equipment, and this will be supported by periodic

Contamination qualifications and underpinned by audits.

Tim Sandle, PhD
Head of Microbiology and Sterility Assurance,
Bio Products Laboratory Limited
Elstree, Hertfordshire, United Kingdom
These requirements are spelt out in EU GMP Annex1, which states1:
“The manufacture of sterile products is subject to special requirements
in order to minimize risks of microbiological contamination, and
of particulate and pyrogen contamination. Much depends on the
skill, training and attitudes of the personnel involved. Quality
Assurance is particularly important, and this type of manufacture
must strictly follow carefully established and validated methods
of preparation and procedure.” And the FDA guidance for sterile
product manufacture2: “As operator activities increase in an aseptic
processing operation, the risk to finished product sterility also
increases. To ensure maintenance of product sterility, it is critical for
operators involved in aseptic activities to use aseptic technique at
all times” (my emphasis).
The traditional model of environmental control and environmental
monitoring is a reactive one. Incidents happen, trends are assessed,
microorganisms are identified and (ideally) effective corrective
and preventive actions are set. While this model will always need
to exist, it is far better to focus on preventative strategies to reduce
microbial contamination. Given the association between microbial
contamination in the cleanroom and people, the most useful
preventive strategy involves improving controls around personnel.
This article addresses some of the key concerns relating to operators
within cleanrooms, in the context of the risk operators pose in terms of
microbial contamination. The article is divided into three sections. The
first part looks at means to improve operator behaviors, the second
part looks at how regular reviews by plant microbiologists or quality
assurance staff can help to reinforce these behaviors, and the third
part considers the idea of having ‘environmental control champions’
within the production facility.

12 | | April 2019
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» MICROBIOLOGY »
Improving Operator Behaviors
The proactive detection of contamination issues in a facility will need
to include cleanroom operators, and their behaviors and practices.
Improving knowledge of operators with respect to microbial
contamination risks and the role that they play in increasing or
decreasing these risks is also of importance.
The focus of people is necessary because within a typical
cleanroom suite:
• Majority of contamination comes from human operators.
This is supported in terms of our increased understanding of
the human skin microbiome3, in relation to what is recovered
from cleanroom environments4.
• This is further shown through benchmarking of cleanroom
microbiota trends. This author’s ten-year study, for example,
showed that 80 percent of cleanroom microorganisms were
transient or residential to human skin5.
• Furthermore, studies indicate that a human sheds ~1000
million skin cells/ day and around 10% of skin shed is
expected to harbor microorganisms (which men shedding
slightly more skin cells than women, thereby potentially
contributing slightly more microorganisms)6.
• Cleanroom clothing can reduce ≥0.5µm particles by 50%,
and by a corresponding 9-fold reduction in ≥0.5µm particles
– which leads to microbial reductions given the proportion
of these particles that will be microbial carrying7. However,
to achieve this gowns must be processed, stored and
donned/worn correctly. In addition, the use of cleanroom
clothing alone is not sufficient and human operators
will also affect microbial levels if they do not behave
appropriately.
To maintain a high standard of personal hygiene and cleanliness it is
important that operators do not enter cleanrooms wear wristwatches,
make-up or jewelry and that they report any condition which may
cause abnormal levels of shedding. Furthermore, staff that have
handled animal tissue materials or live cultures should not enter
cleanrooms unless rigorous and clearly defined entry procedures have
been followed.
Therefore, a preventive contamination control strategy should focus
on increased awareness of contamination issues and seek to drive
improvements to operator behaviors and practices. There are two
ways to address this, through more focused and relevant training and
education (for which ‘environmental control champions’ can be used)
and through a specific approach to self-inspection.
Hygiene Walkthroughs
In terms of walkthroughs of the facility, examples of the types of
things that plant microbiologists or quality assurance staff should be
looking for in relation to personnel are presented below. As well as the
14 | | April 2019
personnel related items a number of other aspects of environmental
control need to be considered, such as cleanroom fabric, cleanability
of surfaces, use of transfer hatches and so on. These fall outside the
scope of this article.
Operator personal protective equipment
With gowning, the clothing and its quality should be appropriate for
the process and the grade of the working area. When conducting
audits of operator clothing, areas to focus on include:
• Are operators washing their hands prior to entry to the
facility?
• Are operators gowned appropriately for the grade
of cleanroom?
• Are all buttons fastened correctly?
• Avoidance of unraveled stitching extending from hoods,
zippers, and pants.
• Are operators wearing the correct suit, with masks, gloves,
facemasks and goggles as required?
• Are appropriate cleanroom suit sizes being worn?
There should be good written procedure for changing/washing,
designed to minimize contamination of clean area clothing/transfer of
contaminants to the clean areas.
Changing room controls
All staff entering cleanrooms should be trained in changing
procedures and cleanroom behaviors. Training should include GMP,
principles of clean manufacture, aseptic technique, hygiene and basic
microbiology, as well as task-related training. Areas to consider with
changing areas include:
• Assess the number of personnel present, do the numbers
exceed the maximum assessed during qualification and
which will impact upon airborne particulates?
• Is there sufficient demarcation between ‘clean’ and ‘dirty’
sides and are personnel adhering to this?
• Has outdoor clothing been removed prior to entry to
the cleanroom?
• Are gowns correctly presented? The presentation (folding) of
garments can help or hinder good gowning technique.
• How long are personnel taking to get changed?
• Is the correct gowning procedure being followed?
Does the gown touch the floor?
• How are used gown disposed of? Could used gowns
contaminate clean gowns?
• Are bins full?
• Are all shoes captive to the change facility?
• Is shoe disinfection regularly undertaken?
• Are interlocks in place between the cleanroom and the
process area?
 When finding nothing means everything.
When it comes to patient safety, should close
enough ever be good enough? In an industry
that relies on cutting edge technology to stay
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» MICROBIOLOGY »
Operator behaviors
Operators must not engage in any activity that poses an unreasonable
contamination risk to the gown. Areas to focus on in an audit include:
• Are operators moving slowly and carefully within
cleanrooms?
• Are operators standing still when no activities are taking
place?
• Is glove sanitization being performed regularly? (especially
prior to any open processing or before taking QC samples).
• Are sufficient glove sanitizers in place (and are these within
expiry date)?
• Are gloves regularly changed?
• Are samples being taken correctly, minimizing the risk of
cross-contamination?
If an element of a gown is found to be torn or defective, it should
be changed immediately. It is additionally important that operators
observe the principle of slow, careful movement throughout the
cleanroom. Rapid movements can create turbulence and disrupt
the airflow, and generate particles, presenting a challenge beyond
intended cleanroom design and control parameters8.
Cleaning and disinfection
Cleaning and disinfection are key steps for maintaining contamination
control. Areas to consider here are:
• Are operators cleaning prior to disinfection?
• Is there evidence of residues remaining on surfaces?
• Is the correct disinfectant rotation frequency being
followed?
• Are contact times being observed?
• Is a double or triple bucket method being used?
• Are mop heads fitted correctly?
• Are mop heads changed between rooms?
• Are cleanrooms being cleaned and disinfected from the
back to the front?
• Is the wiping technique appropriate and conducted
according to disinfectant qualification studies?
(e.g. four-fold wipe method).
• Are room log books completed?
Use of equipment
When carrying out activities, personnel should not directly contact
products, containers, closures, or critical surfaces with any part of their
gown or gloves. Areas to focus on in relation to the use of equipment
and tools within the cleanroom include:
• Is equipment stored correctly?
16 | | April 2019
• Is the appropriate equipment being used? (e.g. sterile
where required)
• Is equipment of a suitable standard (that is, not damaged or
with evidence of contamination)?
• Are operators checking status labels of clean and
dirty equipment?
• Is clean and dirty equipment appropriately segregated?
• When unidirectional airflow devices are used, is the air
path impeded?
• Are cart wheels disinfected prior to movement between
cleanrooms?
In terms of frequency of these types of inspections, this will depend on
the size of the facility. However, weekly or monthly are they levels of
frequency that will begin to yield positive results. It is important that
regular communication and feedback is passed directly to operators,
highlighting not only negative aspects but positive aspects as well as
so to reinforce good behaviors.
The performance of areas can also be tracked over time, through the
use of bar charts or pie charts, and also through the use of heat maps
outlining cleanrooms. Color coding can also be useful, such as ‘red’
for a poorly performing area; ‘green’ for an area with few incidences;
and ‘yellow’ for a moderately performing area. This enables plant
microbiologists and operators to carry out additional visits to areas
that require additional attention. Such approaches can also be used to
compare different shift teams of operators.
Environmental Control Champions
Select or ask for volunteers on the production floor to help to drive
improvements. These will be enthusiastic individuals who will
work with the quality unit and plant microbiologists to drive better
practices in terms of operator behaviors and aspects like cleaning
and disinfection. It is useful if there are sufficient champions to be
present in each manufacturing area and across different shifts, to act
as ambassadors for contamination control.
Champions work best when they also come together as a team and
have shared objectives. The aims of the team could be to:
• Increase knowledge and understanding of
contamination control
• Be proactive as to the detection of issues that could cause
a contamination event, and to report these to maintenance
(for repair) and to microbiology (to assist with root cause
analysis).
• To help to teach best practices for environmental
monitoring.
• To increase awareness of aseptic behaviors and how good
practices help to reduce contamination events.
These aims can be supported through the use of posters highlighting
good practices and display environmental monitoring data trends

(and status indicators for departments); through training courses; and
through short and targeted coaching sessions (such as at the time of a
shift handover). Comparing the performance of different departments
through the use of metrics is also a possibility, if competition is deemed
to be something that helps to drive improvement.
As an example, a poster campaign could center on improving glove
management and hand sanitization. This could be supported by visual
boards; creating training videos; adding photographs to procedures;
and a renewed training program. A training program should be rolled
out to all personnel with access to the manufacturing area to educate
personnel on the importance of hand-washing and hand hygiene
Such key messages should also be incorporated into site induction
programs and as part of the training package delivered to new starters,
prior to going through gowning qualification. The training can also be
re-delivered should any operator be excluded from a cleanroom and
be required to requalify.
The effectiveness of updates to procedures, alterations to induction
processes, and training can be measured through the incident rate
of glove plate microbial excursions, with the expectation that the
incidence rate will decrease.
Summary
While the GMP expectations concerning control of microbial
contamination have generally remained constant over the past
decade, the number of warning letters and audit observations
pertaining to operator related microbial contamination in cleanrooms
appear to have increased over the last number of years. This trend
suggests that need for greater vigilance on operator behavior so to
« MICROBIOLOGY »
reduce the impact upon the cleanroom environment.
The approach outlined in this article, centered on education, audit
and changing the culture can prove to be effective, not only in driving
changes to behavior but also in providing examples so that training
systems can be updated and procedures relating to gowning and
practices can be improved.
References
1. EudraLex. The Rules Governing Medicinal Products in the European Union, Volume 4 - EU
Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary
Use, Annex 1, Manufacture of Sterile Medicinal Products, Brussels, Belgium, 2009: https://
ec.europa.eu/health/sites/health/files/files/eudralex/vol-4/2008_11_25_gmp-an1_
en.pdf
2. FDA. Guidance for Industry Sterile Drug Products Produced by Aseptic Processing —
Current Good Manufacturing Practice, U.S. Department of Health and Human Services,
2004: https://www.fda.gov/downloads/Drugs/Guidances/ucm070342.pdf
3. Hannigan, G.D. and Grice, E.A. Microbial ecology of the skin in the era of metagenomics
and molecular microbiology, Cold Spring Harb Perspect Med. 2013 Dec 1;3(12):a015362
4. Kooken, J.M., Fox, K.F., and Fox, A. Characterization of Micrococcus strains isolated from
indoor air, Mol Cell Probes. 2012 ;26(1):1-5
5. Sandle, T. A Review of Cleanroom Microflora: Types, Trends, and Patterns, PDA Journal of
Pharmaceutical Science and Technology, 2011, 65 (4): 392-403
6. Mackintosh, C.A. , Lidwell, O.M., Towers, A.G., and Marples, R.R. The dimensions of skin
fragments dispersed into the air during activity, The Journal of Hygiene. 1978, 81(3)471-
479
7. Whyte, W. and Hejab, M. European Journal of Parenteral and Pharmaceutical Sciences,
2007, 12 (2). pp. 39-46
8. Eudy J. Human Contamination. A2C2 Magazine. April 2003
www.americanpharmaceuticalreview.com | | 17
» FORMULATION & DEVELOPMENT »
Bioavailability Enhancement By Attenuating
Presystemic Metabolism
Phillip M. Gerk, Pharm.D., PhD
Associate Professor, Virginia Commonwealth University School of Pharmacy,
Department of Pharmaceutics
In regards to oral bioavailability, much research and discussion have
been levied against two issues: solubility and permeability. Depending
on the drug molecule, its dose, and its application, release from the oral
dosage form, dissolution into gastrointestinal fluids, and avoidance
of precipitation may each require significant formulation efforts and
strategies. Permeability is also a considerable problem for highly
hydrophilic or highly hydrophobic molecules (i.e. logP values <1.5 or
>5). To handle these issues better, several systems have been devised
to guide drug developers. The first common one was Lipinski’s “Rule
of 5”,1 followed by Amidon’s “Biopharmaceutical Classification System”
(widely known as BCS),2 Benet’s “Biopharmaceutical Biopharmaceutics
Drug Disposition Classification System” (BDDCS),3 and most recently a
“refined Developability Classification System” (rDCS).4 Although these
frameworks vary in purposes and methods, their common theme is to
understand the limiting factors determining oral drug bioavailability.
Overall, absolute oral bioavailability (Fpo) may be estimated by
multiplying the fraction absorbed (Fa), the intestinal availability (Fg),
and the hepatic availability (Fh), as shown in Equation 1 below. For
many pharmaceuticals, considerations of solubility and permeability
(determinants of Fa) may suffice for bioavailability estimations and
comparisons, if Fg and Fh are close to 1. However, on their way to
systemic blood circulation, some drugs are extensively metabolized
in the intestine and the liver. This presystemic metabolism can
deliver a “one, two punch” resulting in low and variable oral absolute
bioavailability. Thus, oral bioavailability could be maximized by
improving Fa, Fg, and Fh.
Equation 1: Fpo = Fa*Fg*Fh
One common approach to overcoming rapid presystemic metabolism
is through structural modification of the active compound. Functional
18 | | April 2019
groups susceptible to metabolism (such as phenols) could be
concealed through ester formation, as in some prodrug approaches.
The structure of the molecule could also be redesigned to avoid the
susceptible group followed by additional activity testing. However,
both of these approaches require the generation of new molecules
and would necessitate expensive toxicology testing in addition to
clinical studies. The advantage of our patented approach5 is that with
a relatively small amount of preclinical and formulation work, the
approach can be tested clinically.
This article will discuss the approach as applied to three different types
of compounds.
Application 1: Buprenorphine
Several buprenorphine products are marketed in the US to treat
the epidemic of opioid use disorder, including sublingual or buccal
products, and a monthly depot injectable formulation. However,
there has been no orally swallowed formulation of buprenorphine
itself. Recently, an ester prodrug of buprenorphine (buprenorphine
hemiadipate) was under development for oral dosing, but failed
in Phase 1 clinical trials.6 Buprenorphine itself has very low
oral bioavailability7,8 despite good solubility and permeability.8
Buprenorphine has a high intrinsic hepatic clearance suggesting a
high hepatic extraction ratio, thus a low hepatic availability (Fh), which
we estimated to be 0.29.8,9 Additionally, the intestinal metabolism
of buprenorphine is also extensive, resulting in a low intestinal
availability (Fg), which we estimated as 0.04.8,9 Even granting a fraction
absorbed (Fa) of 1, the absolute oral bioavailability of buprenorphine
in humans is estimated between 1 and 3%.8-10 Therefore, the very low
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» FORMULATION & DEVELOPMENT »
Fg is the major factor causing low Fpo of buprenorphine. Meanwhile,
high oral doses of buprenorphine would not feasibly overcome its
low oral bioavailability due to the high cost of pharmaceutical grade
buprenorphine (~$800/g).
Thus, both intestinal and hepatic presystemic metabolism are
problems to be overcome in attempts to formulate an orally swallowed
buprenorphine product. However, buprenorphine metabolism is
complex, involving both oxidation by CYP isoforms 3A4, 2C8, and
2D6, and glucuronidation by UGT isoforms 1A1, 2B7, and 1A3.8,9,11,12
So overcoming these multiple metabolic barriers presents quite a
challenge. To address this challenge, we screened many substances
which have GRAS or dietary supplement status for their abilities
to inhibit the oxidation and/or glucuronidation of buprenorphine
in pooled human intestinal or hepatic microsomes.8 We also
considered their dosing ranges and likely BCS class assignments, and
further investigated their efficacies and potencies for both routes
of metabolism in both organs. The results led us to a short list of
compounds that would have the greatest in vivo potential, including
pterostilbene, α-mangostin, chrysin, silybin, and ginger extract.
From further studies, we calculated that doses of pterostilbene in
the range of 20-25mg would boost buprenorphine Fpo to about 80%,
although we also estimated an increase in variability in AUC. We then
performed simulations and extrapolations, predicting that a dose of
~2mg of pterostilbene is expected to boost the oral bioavailability
of buprenorphine to approximately 35%, on par with a sublingual
product, meanwhile minimizing variability.10 We look forward to the
opportunity to perform clinical testing on prototypes containing
combinations of pterostilbene and other GRAS or dietary compounds.
Application 2: Phenylephrine
In the United States, the Combat Methamphetamine Epidemic Act of
2005 has made access to pseudoephedrine-containing decongestants
more difficult, and many products replacing pseudoephedrine with
phenylephrine have been marketed. Meanwhile, the efficacy of
phenylephrine has been doubted.13,14 The absolute oral bioavailability
of phenylephrine is commonly listed as 38±14%, based upon a study
with only three subjects in the oral dosing phase.15 Although widely
quoted,16-18 the lack of scientific rigor and reproducibility in this study
is obvious. We hypothesize that the absolute oral bioavailability of
phenylephrine may be much lower, and that this low and variable
bioavailability may be one significant reason for its questionable
clinical efficacy.
With phenylephrine being an OTC drug, many drug products are mar-
keted, but an opportunity may exist for a more reliable phenylephrine
product. Our data have shown that combinations of certain GRAS and
dietary compounds can inhibit the main routes of phenylephrine pre-
systemic metabolism, namely sulfation and oxidation.5,19 Specifically,
these data show that certain combinations of GRAS and dietary com-
pounds (such as resveratrol, zingerone, quercetin, and vanillin) can
strongly inhibit phenylephrine presystemic metabolism.
20 | | April 2019
Opportunities and Challenges:
Besides the applications for the drugs discussed above, the same
approach could be applied to other compounds with extensive phase
1 or phase 2 presystemic metabolism. These could include other
opioids and other adrenergics, as well as estrogen-related compounds
such as 2-methoxyestradiol and raloxifene. Additionally, the approach
could potentially be adapted to compounds with high efflux transport
by P-glycoprotein and the Breast Cancer Resistance Pump. Variability
is another issue to be overcome; for example, morphine sulfate oral
bioavailability is well-known to be variable, and new formulations
solving this problem could be produced.
Another opportunity is in the area of nutraceuticals. Many natural
products are also quickly metabolized presystemically resulting
low oral bioavailability, as is the case with buprenorphine and
phenylephrine. The dietary supplement market in the US is estimated
to grow from $31.7B in 2016 to $56.7B in 2024.20 The abundance of
natural products and interest in tapping this market has resulted in
numerous preclinical studies for potential treatments of common
diseases such as diabetes, obesity, dyslipidemia, hypertension,
cancers, and even longevity. Unfortunately, many clinical studies
have been performed on various pure natural compounds or herbal
extracts have failed to show encouraging results. However, many
of these studies have failed to consider the biopharmaceutical and
pharmacokinetic issues associated with the natural products being
tested, thus contributing to clinical failures. Those planning clinical
studies would do well to reverse that trend.
Meanwhile being lured by the hopes of promising in vitro and
preclinical activities but recognizing potential bioavailability issues,
several complex and costly strategies have been attempted such
as prodrug approaches and nanoparticle formulations, as with
curcumin.21,22 However, unless prescription drug status can be obtained
for these products, their high costs may limit their marketability.
We have applied our approach using in vitro studies, demonstrating
its feasibility with several phenolic natural molecules (unpublished
data). Although further studies are needed to refine the approach for
individual applications, we anticipate this approach would result in
cost-effective clinically reproducible dietary supplements.
In the case of the KALETRA® (lopinavir/ritonavir) and STRIBILD® (el-
vitegravir, cobicistat, emtricitabine, tenofovir disoproxil fumarate)
products, CYP3A inhibitors (ritonavir and cobicistat, respectively) are
included in the formulation and are known to have systemic effects
on drug metabolism, as indicated in their prescribing information. As
a result, patients and healthcare providers using these medications
need to be especially vigilant in avoiding drug-drug interactions. By
inhibiting presystemic metabolism using strategically minimal doses
of GRAS or dietary compounds which are themselves quickly elimi-
nated presystemically, unwanted systemic drug-drug interactions
could be avoided clinically by separating dosing times. In the case of
some dietary compounds, it may be challenging to obtain excipient-
grade material for inclusion in the finished products.
 « FORMULATION & DEVELOPMENT »

Meanwhile, clinical studies have shown
mixed results in the use of dietary com-
pounds for bioavailability enhancement. For
example, piperine (from black pepper) in-
hibits human P-glycoprotein and CYP3A4,23
and enhances the oral bioavailability of the-
ophylline and propranolol as demonstrated
in humans.24 In animal models, piperine in-
creases the oral bioavailability of curcumin,
resveratrol,epigallocatechin gallate, and
amoxicillin.25-28 Additionally, curcumin has
also been shown to inhibit numerous en-
zymes involved in presystemic elimination.29
However, in a clinical study with healthy
volunteers, piperine and curcumin at fairly
high doses failed to change the AUC values
for acetaminophen, midazolam, or flurbi-
profen, but modestly increased Cmax for
acetaminophen.30 As noted in the study,
circulating plasma concentrations of uncon-
jugated piperine or curcumin were unde-
tectable (<0.6μM or < 0.05μM, respectively)
despite the high doses, but conjugated me-
tabolites were abundant. Therefore, to make
an oral bioavailability enhancement strategy
clinically successful, one would need to pay
careful attention to the biopharmaceutics
and disposition of the enhancers as well as
the active compounds. Particularly, it would
be best to maximize the exposure of the in-
testinal and hepatic enzymes to the enzyme
inhibitors during the absorption phase of
the active ingredient.
References:
1. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ.
Experimental and computational approaches to
estimate solubility and permeability in drug discovery
and development settings. Adv Drug Del Rev
2001;46(1–3):3-26.
2. Amidon GL, Lennernas H, Shah VP, Crison JR.
A theoretical basis for a biopharmaceutic drug
classification: The correlation of in vitro drug product
dissolution and in vivo bioavailability. Pharm Res.
1995;12(3):413-420.
3. Wu CY, Benet LZ. Predicting drug disposition via
application of BCS: transport/absorption/ elimination
interplay and development of a biopharmaceutics
drug disposition classification system. Pharm Res.
2005;22(1):11-23.
4. Rosenberger J, Butler J, Dressman J. A refined
developability classification system. J Pharm Sci.
2018;107(8):2020-2032.
5. Gerk PM, Barr WH, Ritter JK, Inventors; Virginia
Commonwealth University, assignee. Selective
metabolic approach to increasing oral bioavailability
of phenylephrine and other phenolic bioactivities. US
patent 9,616,0332017.
6. Indivior. Result of Phase 1 trial of RBP-6300
Buprenorphine Hemiadipate. Investor Information
2016; http://www.indivior.com/investor-news/
result-phase-1-trial-rbp-6300-buprenorphine-
hemiadipate/. Accessed 2019/03/28, 2019.
7. Jeffcoat AR, Cook CE, Perez-Reyes M, et al. Human
disposition of intravenous, oral and sublingual [3H]
buprenorphine. In: Harris L, ed. Problems of Drug
Dependence, 1992: Proceeding of the 54th Annual
Scientific Meeting The College on Problems of Drug
Dependence, Inc. Vol 132. Keystone, CO: US Department
of Health and Human Services; 1992.
8. Maharao NV, Joshi AA, Gerk PM. Inhibition of
glucuronidation and oxidative metabolism of
buprenorphine using GRAS compounds or dietary
constituents/supplements: in vitro proof of concept.
Biopharm Drug Dispos. 2017;38(2):139-154.
9. Cubitt HE, Houston JB, Galetin A. Relative importance
of intestinal and hepatic glucuronidation-impact
on the prediction of drug clearance. Pharm Res.
2009;26(5):1073-1083.
10. Maharao N, Venitz J, Gerk PM. Use of generally
recognized as safe or dietary compounds to inhibit
buprenorphine metabolism: potential to improve
buprenorphine oral bioavailability. Biopharm Drug
Dispos. 2019;40(1):18-31.

Conclusion
Despite the wealth of available literature
regarding solubility and permeability en-
hancement approaches, relatively little is
established regarding bioavailability en-
hancement by inhibition of presystemic me-
tabolism. Our patented approach works to
address the issues discussed above by using
RELIABLE
combinations of enzyme inhibitors directed
toward multiple metabolic pathways.5 This
DOMESTIC Your Integrated Drug
Substance Partner
approach would be well-suited for com-
pounds with rapid intestinal and/or hepatic
SUPPLY Meet us at
metabolism resulting in low and variable More than 40 Years of Experience CPhI North America,
oral bioavailability, especially for older drug April 30 - May 2, 2019
molecules like buprenorphine and phen- Chicago, IL
ylephrine and nutraceutical compounds. Booth 207
Hopefully, more partnerships between in-
www.ALBcustom.com
dustry and academia will help to bring many
new products to market.

www.americanpharmaceuticalreview.com | | 21
» FORMULATION & DEVELOPMENT »
11. Picard N, Cresteil T, Djebli N, Marquet P. In vitro metabolism study of buprenorphine:
evidence for new metabolic pathways. Drug Metab Dispos. 2005;33(5):689-695.
12. Rouguieg K, Picard N, Sauvage FL, Gaulier JM, Marquet P. Contribution of the different
UDP-glucuronosyltransferase (UGT) isoforms to buprenorphine and norbuprenorphine
metabolism and relationship with the main UGT polymorphisms in a bank of human liver
microsomes. Drug Metab Dispos. 2010;38(1):40-45.
13. Hendeles L, Hatton RC. Oral phenylephrine: an ineffective replacement for
pseudoephedrine? J Allergy Clin Immunol. 2006;118(1):279-280.
14. Eccles R. Substitution of phenylephrine for pseudoephedrine as a nasal decongeststant. An
illogical way to control methamphetamine abuse. British journal of clinical pharmacology.
2007;63(1):10-14.
15. Hengstmann JH, Goronzy J. Pharmacokinetics of 3H-phenylephrine in man. Eur J Clin
Pharmacol. 1982;21(4):335-341.
16. McEnvoy GK, ed AHFS Drug Information. 1999 ed. Bethesda: American Society of Health-
System Pharmacists, Inc.; 1999.
17. Kanfer I, Dowse R, Vuma V. Pharmacokinetics of oral decongestants. Pharmacotherapy.
1993;13(6 Pt 2):116S-128S; discussion 143S-146S.
18. Lexi-Comp. Phenylephrine. Lexi-Comp Online; 2015: https://online.lexi.com/lco/action/
doc/retrieve/docid/patch_f/1799564. Accessed 4/3/2015.
19. Zhang Z, Gerk PM. Effects of generally recognized as safe (GRAS) and dietary compounds
on phenylephrine metabolism in LS180 human intestinal cells. Biopharm Drug Dispos.
2018;39(9):443-447.
20. Statista. Total U.S. dietary supplements market size from 2016 to 2024 (in billion U.S.
dollars). The Statistics Portal 2019; https://www.statista.com/statistics/828481/total-
dietary-supplements-market-size-in-the-us/. Accessed 2019/03/28, 2019.
21. Jaisamut P, Wiwattanawongsa K, Graidist P, Sangsen Y, Wiwattanapatapee R. Enhanced
oral bioavailability of curcumin using a supersaturatable self-microemulsifying
system incorporating a hydrophilic polymer; in vitro and in vivo investigations. AAPS
PharmSciTech. 2018;19(2):730-740.
22. Lu P, Tong Q, Jiang F, et al. Preparation of curcumin prodrugs and their in vitro anti-tumor
activities. Journal of Huazhong University of Science and Technology Medical Sciences.
2005;25(6):668-670,678.
23. Bhardwaj RK, Glaeser H, Becquemont L, Klotz U, Gupta SK, Fromm MF. Piperine, a major
constituent of black pepper, inhibits human P-glycoprotein and CYP3A4. J Pharmacol Exp
Ther. 2002;302(2):645-650.
22 | | April 2019
24. Bano G, Raina RK, Zutshi U, Bedi KL, Johri RK, Sharma SC. Effect of piperine on bioavailability
and pharmacokinetics of propranolol and theophylline in healthy volunteers. Eur J Clin
Pharmacol. 1991;41(6):615-617.
25. Barve K, Ruparel, K. Effect of bioenhancers on amoxicillin bioavailability. ADMET & DMPK.
2015;3(1):45-50.
26. Johnson JJ, Nihal M, Siddiqui IA, et al. Enhancing the bioavailability of resveratrol by
combining it with piperine. Mol Nutr Food Res. 2011;55(8):1169-1176.
27. Lambert JD, Hong J, Kim DH, Mishin VM, Yang CS. Piperine enhances the bioavailability of
the tea polyphenol (-)-epigallocatechin-3-gallate in mice. J Nutr. 2004;134(8):1948-1952.
28. Zeng X, Cai D, Zeng Q, et al. Selective reduction in the expression of UGTs and SULTs, a
novel mechanism by which piperine enhances the bioavailability of curcumin in rat.
biopharmaceutics & drug disposition. 2017;38(1):3-19.
29. Volak LP, Ghirmai S, Cashman JR, Court MH. Curcuminoids inhibit multiple human
cytochromes P450, UDP-glucuronosyltransferase, and sulfotransferase enzymes, whereas
piperine is a relatively selective CYP3A4 inhibitor. Drug Metab Dispos. 2008;36(8):1594-
1605.
30. Volak LP, Hanley MJ, Masse G, et al. Effect of a herbal extract containing curcumin and
piperine on midazolam, flurbiprofen and paracetamol (acetaminophen) pharmacokinetics
in healthy volunteers. Br J Clin Pharmacol. 2013;75(2):450-462.
About the Author:
Phillip M. Gerk received his Doctor of Pharmacy
(Pharm.D.) degree at the University of Illinois at
Chicago, then did a clinical research fellowship at
Auburn University. He then attended the University
of Kentucky where he received his Ph.D. and
stayed to perform postdoctoral research. He has been a faculty
member at Virginia Commonwealth University in the Department of
Pharmaceutics since 2004, where he performs his research on drug
metabolism, transport, and oral bioavailability.
» BIOPHARM PROCESSING »
Single-Use Systems Continue to Gain Traction
Among CMOs
Ilene Roizman
Communications Manager
BioPlan Associates
Single-use bioprocessing equipment has made considerable progress
in the past 10 years. An estimated 10% of current total worldwide
bioprocessing capacity, or about 1.7 million liters, involves primarily
single-use system (SUS) process lines. The market has grown from a
few legacy products, such as plastic serum and media storage bags,
tubing, and filter membranes, to the current situation where single-use
technologies represent the majority of non-commercial applications
in bioprocessing. This includes a wide variety of products and novel
technologies currently available and in development.
BioPlan’s 15th Annual Report and Survey of Biopharmaceutical
Manufacturing, 2018,1 which included responses from 222 biopharma-
ceutical manufacturers and CMOs in 22 countries and 130 bioprocess-
ing suppliers or vendors, shows that single-use equipment is domi-
nating small- and mid-scale bioprocessing and is being adopted for
larger-scale commercial manufacturing.
With advancing technology, knowledge, and experience, future
progress and market expansion are expected. In addition, the
expansions and adaptations now being implemented in mainstream
biologics production are also being seen in adjacent sectors, including
cell and gene therapy, and even in the production of cosmetics and
food derived from plant cell and tissue cultures, which are now being
produced at manufacturing scales.
Single-use equipment is generally made of plastic parts that are
sterilized by gamma irradiation, then used once and discarded.
More than a decade of combined industry experience has shown
the benefits of single-use versus fixed stainless steel, including lower
capital investment and operational costs, as well as flexibility. SUS
enable rapid setup of bioprocessing and progressive manufacture
of multiple products at multiple scales in the same areas. Single-use
product lines have expanded from basic storage bags to complex
bioreactors. Now, essentially all bioprocessing, particularly upstream,
can be done with single-use systems.
Less than 20 years ago, just a few single-use systems were available,
generally limited in size to 100 liters. Now single-use bioreactors and
mixers are available at ≤2,000 L scale. Due to engineering limitations,
systems above 2,000 L are generally not practical or cost-effective.
Currently, 1,000 L bioreactors are on track to be become the industry
standard for new product large-scale and much commercial-scale
manufacturing, with a growing number of companies offering an
increasing range of product options.
In terms of revenue, stainless steel facilities currently account for
~85% of the market and single-use ~15%. This is projected to change
24 | | April 2019
in 5 to 10 years to about 70-75% stainless and 25-30% single-use. This
includes growth in the SUS market of up to 300% and market share
growing to ~25-30%. These data reflect downstream applications
remaining limited over the next 5 years, with some increased
adoption of SUS continuous chromatography and increased use
for commercial manufacturing. These projections also reflect likely
market growth from many new players entering bioprocessing,
including in developing countries and biosimilar developers.
Market for Single-Use Equipment
The most dramatic growth will be for commercial-scale GMP manufac-
turing. The single-use market for commercial applications is projected
to grow to over $1 billion/year in the next five years. This will be driven
by products currently in development using single-use systems receiv-
ing approval and graduating to commercial-scale manufacturing. New
products will continue to mostly be monoclonal antibodies, which will
generally continue to require manufacture of 100 kilograms or more.
This will increasingly involve parallel tracks, continuous bioprocessing,
and/or multiple facilities worldwide anchored by 500-2,000 L SUS bio-
reactors. But even with this growth, fixed stainless steel systems will
still dominate GMP/commercial and thus the overall market.
The BioPlan annual survey and other sources confirm that among the
facilities surveyed, mostly in the U.S. and Europe, average purchases of
single-use equipment are over $1 million/year. Bioprocessing profes-
sionals realize that SUS has improved bioprocessing. Over two-thirds
(68.8%) of survey respondents cited SUS as providing “some” or “sig-
nificant” improvement in their bioprocessing within the past year.
But end users recognize that they are often using first-generation
or other less than optimal equipment. Our survey indicates that
over the past five years, end users have continued to express a de-
sire for improved single-use systems. As with most emerging tech-
nology, once new and better products and systems are introduced
and become the norm, demand for further improvement continues
until a general level of satisfaction is reached. In the bioprocess-
ing industry, end users are apparently concerned that innovation in
single-use bioprocessing supplies has been slow in coming. This is
fully expected and normal in such a highly regulated environment,
and demand for better products continues.
Single-use products with the highest rate of use at any stage/scale
are shown in Figure 1. The most commonly used products are simpler
devices, such as bags and connectors. But it is significant that more
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than 70% of facilities use major bioprocessing
systems, including bioreactors, mixers, and
tangential flow filtration, and are more likely
to be fully dedicated to using SUS.
Among the reasons for adopting single-
use described as “very important,” 46.2%
of respondents said it decreases the risk of
cross-product contamination, 41.2% said it
eliminates cleaning requirements, 44.1% said
it reduces the time to get a facility up and
running, and 40.4% said it reduces capital
investments in facilities and equipment. With
regard to concerns or potential problems,
five were mentioned by more than 50% of
respondents: “Breakage of bags and loss of
production material,” 75.0%; “Leachables and
extractables,” 73.3%; “High cost of dispos-
ables,” 68.8%; and “Material incompatibility
with process fluids” and “We do not want to
become vendor-dependent (single-source is-
sues),” 56.7% (tie).
Figure 1. Usage of Disposables in Biopharmaceutical Manufacturing at
Any Stage of Manufacture
26 | | April 2019
Concerns Over SUS
As use of SUS increases, concerns about cost
can be expected to increase, particularly
as more bioprocessing professionals only
familiar with stainless steel equipment
purchase SUS. However, from other survey
research, we found that price is not a
primary concern among purchasers of any
bioprocessing equipment. End users prefer
to purchase the best equipment they can get
and are willing to pay more, as long as the
premium is not excessive.
Single-use systems manufacturing will
advance and continue to proliferate, with
more diverse technologies and products
becoming available. However, in terms of
capacity, most of the largest facilities are
likely to remain in use. New capacity will
be added at smaller scales, with most new
commercial manufacturing facilities still
using stainless steel. As such, stainless steel
will continue to dominate bioprocessing in
terms of capacity and number of large-scale
and commercial facilities.
A number of individualized biologics and
personalized medicines are in active develop-
ment, including patient-specific cellular and
gene therapies, cultured tissues and organs,
and cancer and other therapeutic vaccines.
With their one-off nature and need for sterility,
these products can be expected to be manu-
factured using single-use equipment. BioPlan
has projected significant growth in cellular and
gene therapy capacity and facilities, despite a
current shortage that will become an actual
capacity crunch in coming years.
We are already seeing another trend: modular
facilities, with processes housed in connectable,
portable clean rooms or isolator units, and
whole portable manufacturing facilities that
can be constructed and operational in a
matter of months or even weeks. With modular
systems using SUS equipment and providing
much the same advantages as single-use,
the market is expected to grow dramatically.
This includes “plug-and-play” factories, with
whole production lines and facilities fully
clonable. With an increasing demand for
domestic biopharmaceutical manufacture,
modular facilities will become more common
in developing countries, particularly China and
India, in the next decade.
Besides inherent conservatism in this highly
regulated industry, an issue restricting
progress in the expansion of single-use
systems is vendors’ hesitancy and end users’
distress over incremental improvements and
other changes in established product lines.
This is because any changes in bioprocessing
products already in use can require extensive
validation studies, more regulatory filings,
costly testing, new SOPs, etc. Innovations
in single-use equipment will more likely
originate from small companies or new major
suppliers and developers less attached to
aging technologies. New technologies in this
industry are generally implemented by totally
new facilities and process lines, not retrofitting.
Fastest Growing Areas of
SUS Development
The fastest growing segment of the single-
use equipment market will be upstream
 « BIOPHARM PROCESSING »

bioprocessing at large and commercial scales as products now in

development using SUS move up to commercial manufacturing. As
demonstrated by the annual BioPlan survey and confirmed by many
other sources, adoption of single-use systems, generally in place of
fixed stainless steel systems, will continue. Modular systems may be
the next technology to experience such increased and rapid adoption
over the next decade. And as cellular and gene therapies emerge, we
will likely see SUS technologies created and adapted explicitly for
these personalized applications.
Most of the growth in SUS will be in developed countries, but
developing countries will see much faster growth rates. For example,
many of the new bioprocessing facilities expected to be constructed
in China to meet both domestic demand and its desire to become an
exporter of biopharmaceuticals will be primarily SUS facilities.2 The
future for SUS technologies remains defined by the expanding global
need for better, cheaper, and faster biologics production.
About the Author
Ilene Roizman is communications manager at BioPlan Associates, Inc. She
is a skilled writer and medical communicator, able to distill highly technical
information in medicine and science. Her medical expertise includes editing
of textbooks, as well as research papers for international science and
medical journals. + 1 301-921-5979, www.bioplanassociates.com
Survey Methodology: The 2018 Fifteenth Annual Report and Survey of
Biopharmaceutical Manufacturing Capacity and Production yields a
composite view and trend analysis from 222 responsible individuals
at biopharmaceutical manufacturers and contract manufacturing
organizations (CMOs) in 22 countries. The methodology also included
over 130 direct suppliers of materials, services, and equipment to this
industry. This year’s study covers such issues as: new product needs,
facility budget changes, current capacity, future capacity constraints,

References expansions, use of disposables, trends and budgets in disposables, trends

1. Langer, E.S., et al, 15th Annual Report and Survey of Biopharmaceutical Manufacturing
Capacity and Production, BioPlan Associates, Rockville, MD, USA April 2018.
2. Directory of Top 60 Biopharmaceutical Manufacturers in China, 2nd edition, BioPlan
Associates, Feb. 2017, 357 pages (also online database).
in downstream purification, quality management and control, hiring
issues, and employment. The quantitative trend analysis provides details
and comparisons of production by biotherapeutic developers and CMOs.
It also evaluates trends over time and assesses differences in the major
markets in the U.S. and Europe.

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» MICROBIOLOGY »

Our Complicated Relationship with Fungi

Jeanne Moldenhauer
Excellent Pharma Consulting, Inc.

wheat both grow with more carbohydrate and phosphorous content

Abstract than is present with non-infected plants. (Dhande, 2019)
Other fungi are effective as root nibblers. These fungi result in root
If you have been in the pharmaceutical industry for any length of
fibers that increase the uptake of nutrients and water resulting in
time, you have probably been exposed to some concern in your
facility regarding fungi, i.e., yeasts and molds. The New England higher yields. (Dhande, 2019)
Compounding Center (NECC), of Framingham, MA had a fungal
contamination that led to a significant outbreak of meningitis in
2012. Sixty-four deaths and numerous non-fatal injuries occurred Bioherbicides
as a result of this contamination. This outbreak changed regulatory
controls for compounding pharmacies and a heightened regulatory A bioherbicide is an agent for controlling weeds. It is biologically
concern about fungal contamination in manufacturing facilities. based.
(Anonymous, 2019)
Fungi are well-known for their specific and effective actions against
Concerns about molds specifically have resulted in companies weeds. Additionally, they have low levels of residues compared to
establishing zero-tolerance policies for mold in their facilities although synthetic pesticides. (Dhande, 2019)
mold is present in the air and may be carried on humans. It is generally
recognized that mold should not be routinely present in aseptic areas.
However, fungi play an important role in many areas of our lives. They
impact the foods we eat and drink, provide important pharmaceutical
Biofertilizers
agents, are used as a source of enzymes, as well as negatively affecting Some fungi have symbiotic relationships with plants, via vesicular
the structural integrity of buildings, can poison us, cause diseases, and
arbuscular mycorrhizae. The roots of higher plants are benefited by
are a significant risk for plant diseases that can threaten the security of
the fungi. This results in increased phosphate nutrition of the plant
food worldwide. (Idnurm and Meyer, 2014)
and protection of the roots by developing a mantle. (Dhande, 2019)
This article talks about some of the “positive” uses and potential uses
of fungi. Dhande (2019) refers to the “mind blowing potential” of
fungal biotechnology! Fungal investigation and research are at an all
time high! Biodegradation and Remediation
Fungi have significant roles in various types of biodegradation. White
rot fungi degrades toxic pesticides, e.g., DDT, PCB, and Lindane. Other
Food and Beverages toxic chemicals can also be degraded, e.g., dioxin, cyanides, and
azides. Petroleum can be degraded using several types of fungi in
There are a variety of ways in which fungi are important in the
contaminated soils. (Dhande, 2019)
food and beverage industry. Most microbiologists are aware of the
common uses of fungi in the production of beer, wine, yogurts and Cellulose can be degraded by fungi. This is useful in resolving
cheese production. (Godani, 2019) There are many other fermentation agricultural residues. This degradation process can result in the
products also produced. production of bioenergy. (Dhande, 2019)

Some fungi are used directly as food, e.g., mushrooms, as they are high Even hazardous wastes can be remediated using fungi in contaminated
in protein and low in calories. (Dhande, 2019) soils. (Dhande, 2019)

Enhancing Produce Quality Biomineralization

We commonly think that fungal infections are always damaging to Myceliar beads of Penicillium have been shown to play a key role in
crops. However, some fungal diseases can have a beneficial effect on the removal and recovery of heavy metals from wastewater. Some of
product. Corn that is smutted (black powdery mold) and rust infected the minerals removed include: Hg, Cu, Ni, Pb, and Cd. (Dhande, 2019)

28 | | April 2019
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numerous types of fungi available and their capabilities will provide

Single-Cell Proteins (SCPs) and research opportunities for many years.

Fungal Metabolites
A single-cell protein (SCP) is a protein which has been derived from
a culture of single-celled organisms, frequently used as a food
supplement. Several species of fungi (both yeasts and molds) have
been used as single-cell proteins. SCPs can be used to increase the
nutritional value of foods, e.g., adding vitamins, amino acids, and
lipids. (Godani, 2019 and Dhande, 2019)
Conclusion
This partial listing of some of the real and potential uses of fungi barely
touch the surface of the possibilities. The research in fungi applications
has the potential to totally change how we live in the world. The
Literature Cited
Anonymous (2019) New England Compounding Center Meningitis Outbreak. Wikipedia. Downloaded
from: https://en.wikipedia.org/wiki/New_England_Compounding_Center_meningitis_outbreak
on March 27, 2019.
Dhande, R. (2019) Potential Applications of Fungi; A Biotechnological Approach. Street Directory.
Downloaded from: https://www.streetdirectory.com/travel_guide/119227/science/potential_
applications_of_fungi_a_biotechnological_aproach.html on March 27, 2019.
Godani, K. (2019) Top 6 Fungal Products Obtained from Fungal Biotechnology. Biology Discussion.
Downloaded from: http://www.biologydiscussion.com/biotechnology/fungal-biotechnology/top-6-
fungal-products-obtained-from-fungal-biotechnology/8530 on March 27, 2019.
Idnurm, A. and Meyer, V. (2014) Welcome to Fungal Biology and Biotechnology. Fungal Biology
and Biotechnology 1:8-9. Downloaded from: https://fungalbiolbiotech.biomedcentral.com/track/
pdf/10.1186/s40694-014-0008-5 on March 27, 2019.

Table 1. Some Common Uses of Fungi

Type of Application Product Fungal Source Uses
Agriculture

Various Names Saprophytic fungi Aid in the decay of dead animals and plants

Septagloeum gills, Wallrothiella arecuthobii, Colletotrichum

Biopesticides glocosporiordes, phyllosticta, Leptosphaerulina trifolia, Weed killers
Cercospor ageratinae

Symbiotic relationship with fungi and plants result in

Biofertilizers Various
phosphate nutrition and root protection.

Amino Acids

L-glutamate Corynebacterium glutamicum

Antibiotics

Cephalosporin Cephalsporium Antibacterial, for Gram positives

Griseofulvin Penicillium griseofluvum Fungistatic treatment of dermatophytes

Used against Mycobacterium tuberculosis, Listeria sp. And

Lentinan Lentinus sp.
Herpes simplex virus-1 (HSV-1)

Penicillin Penicillium chrysogenum Antibacterial, for Gram positives

Schizophullan Schizophyllum Antibacterial against Staphylococcus aureus

Bakeries

Baked Goods Saccharomyces cerevisiae Food

Breweries

Ethyl alcohol Saccharomyces cerevisiae Alcohols, wines

Cheese Production

Various Types Penicillium, Aspergillus Food

Enzymes

Aspergillus oryzae, Aspergillus niger, Aspergillus flavus,

Acid, alkaline and neutral proteases Meat tenderizer, Bakeries
Aspergillus sojae

Cellulase Trichoderma koningi Paper industry, detergent, coffer

Diastase Aspergillus oryzae Acid reflux, food supplement

Glucoamylase Aspergillus niger, Aspergillus oryzae Sweeteners

Invertase Saccharomyces cerevisiae Sugar, candles

Lactase Saccharomyces lactis, Rhizopus oryzae Lactose intolerant patients

Ligninase Phanerochaete chrysoporium Pulp and paper industry

Lipase Rhizopus sp. Baking, pancreatic disorders

Pectinase Aspergillus niger, Sclerotinia libertine Fruit juices

30 | | April 2019
 « MICROBIOLOGY »

Table 1. Some Common Uses of Fungi (continued)

Type of Application Product Fungal Source Uses
Foods

Agaricus compestris, Volvariella, Morchella, Pleurotus

Varieties of Mushrooms High protein, low calorie foods
sp.,Agaricus bisporus

Medicines (Recombinant Proteins)

Angiostatin Pichia pastoris EntreMed’s Angiostatin, anti-angiogenic

Elastase Pichia pastoris Dyax’s drug for cystic fibrosis

Endostatin Pichia pastoris EntreMed’s anti-angiogenic

Epidermal Growth Factor Pichia pastoris Transition Therapeutic’s treatment for diabetes

Glucagon Sacchromyces cerevisiase Novo Nordisk’s Glucagen

Hepatitis-B Sacchromyces cerevisiase Glaxo Smith Kline’s Ambrix and Pharma’s HBVAXPRO

Hepatitis-B Surface Antigen Pichia pastoris Glaxo Smith Kline’s treatment for serum hepatitis

Mitusbishi Pharma’s product for stabilitizing blood in burn/shock

Human serum albumin Pichia pastoris
patients

Insulin Sacchromyces cerevisiase NovoNordisk’s Actrpid

Insulin-like growth factor 1 Pichia pastoris Cdphalon’s product for insulin-like growth factor 1 deficiencies

Macrophage CSF rh Platelet-derived Sacchromyces cerevisiase Ortho’s Regranex

Surface antigen Sacchromyces cerevisiase Merck’s Comvax

Urate oxidase Sacchromyces cerevisiase Syntiabo’s Elitex

Organic Acids

Citric Acid Aspergillus niger Food preservation, powerful cleaning agent, and in cosmetic products

Fumaric Acid Rhizopus nigricans Used in food and beverage products, oral pharmaceutical formulations

Gluconic Acid Aspergillus niger Acidity regulator for food additives, cleaning products

Prepare acrylic fibers and rubbers, reinforced glass fiber, water treatment
Itaconic Acid Aspergillus terreus
systems, artificial diamonds and lenses

Kojic Acid Aspergillus oryzae Food and cosmetics

Perfumes

Perfumes Lichens Cosmetics

Recycling

Single Cell Proteins (SCPs)

Sacharomyces cerevisae, Aspergillus niger, Penicillium

Various Products Used in production of food
chrysogenum, Fusarium avenacum

Vaccines (anti-fungal vaccines)

NDV-3 Agglutin-like sequences for

Candida sp. Anti-fungal vaccines
Candida sp.

Heat Killed Saccharomyces Protection against fungal infections of Coccidiodes posandasii, C.

Saccharomyces cerevisiae
cerevisiae (HSK) vaccine albicans, Aspergilllus fumigatus

Whole recombinant S. cerevisiae- Treatment of cancer and viral diseases together with cytotoxic drugs to
Saccharomyces cerevisiae
based method increase clinical responses.

Vitamins

Glycolipid mannosylerythritotol
Ustilago scitaminea NBRC 32730 Biosurfactant
lipids

Treating high blood pressure, high cholesterol, depression, and

Nicotinamide adenine dinucleotide
Candida boldinii Parkinson’s disease, reducing the signs of aging, and protecting against
(NADH)
the side effects of an AIDS drug (zidovudine, aka AZT)

Used orally for osteoarthritis, rheumatoid arthritis, Parkinson’s disease,

Panthothenic acid (Vitamin B5) Fusarium oxysporum
nerve pain, premenstrual syndrome (PMS), enlarged prostate

Increasing energy levels, boosting immune system function,

Riboflavin (Vitamin B2) Ashbya gossypii, Candida famata maintaining healthy hair, skin, mucous membranes, and nails, slowing
aging

Used for depression, osteoarthritic, chronic lower back pain, Alzheimer’s

S-adenosylmethionine (S AMe) Sacchromyces sake disease,, slowing the aging process, attention deficit-hyperactivity
disorder (ADHD), migraines, headaches, and lead poisoning

www.americanpharmaceuticalreview.com | | 31
» FORMULATION AND DEVELOPMENT »

Polymeric Particles as
Vaccines have been successful at preventing a range of diseases
including diphtheria, polio, whooping cough, measles and tetanus;
whereby incidences of such diseases are now rare in developed

Cancer Vaccine Vectors

countries. In fact, thanks to vaccines, smallpox has been successfully
eradicated worldwide whilst polio is on the verge of global eradication.
However, the incidence rate of other diseases remains high owing to a
lack of efficacious vaccines or therapies to prevent or treat them. This is
often because the pathogen or diseased cell, as in cancer, has evolved
ways of evading immune detection or suppressing immune-based
attack. Despite the substantial progress in cancer care (including early
diagnosis), cancer continues to be a challenging and devastating
health problem and is still the second leading cause of death in the
Suhaila O. Suliman, Graduate Student, United States, exceeded only by heart disease. According to the 2018
Emad I. Wafa, Graduate Student, National Vital Statistics Report issued by Centers for Disease Control
Sean M. Geary, Assistant Research Scientist, and Prevention, nearly 3 million deaths due to cancer were reported in
Aliasger K. Salem, Professor the United States during 2016 with a death rate of approximately 850
Division of Pharmaceutics and Translational Therapeutics, College of Pharmacy per 100,000 population.1 For many types of cancer, therapies approved
University of Iowa by the United States Food and Drug Administration thus far have not
significantly reduced mortality among cancer patients but instead,
at best, have slowed the progression of the disease and/or extended
patient survival.2 Chemotherapy, one of the commonly administered
conventional cancer therapies, is associated with major limitations
since it is a non-specific approach and limited in effectiveness due to
a steep dose-response relationship and narrow therapeutic window.3
Therefore, there is a dire demand for more effective therapies to meet
the urgent medical needs for cancer patients. Adaptive immune
cancer therapy through the administration of vaccine formulations has
met with undulating fortunes over the past century, where promising
preclinical findings have generally not translated effectively into the
clinic.4 A major reason for the lack of success in cancer patients is the
highly evolved immunosuppressive properties of the tumor, creating
a tumor microenvironment inhospitable to antitumor immune
effector cells. However, with the recent promising clinical outcomes
for cancer patients treated with immune checkpoint inhibitors (ICI),
such as pembrolizumab (αPD-1) and ipilimumab (αCTLA-4), there is
justified optimism that the cancer patient’s own immune system can
play a significant role in combatting cancer; and that an appropriately
designed cancer vaccine can act in synergy when combined with ICI.5-7
Whilst the development of cancer vaccines has progressed promisingly
in preclinical settings,8-10 strategies to further enhance their efficacy

32 | | April 2019
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are still needed. One strategy that holds

promise in the war on cancer involves the
employment of technology to fabricate
nano- and micro-sized particle-based vectors
formulated as cancer vaccines capable
of generating robust antitumor immune
responses.11,12 Specifically, polymeric particle-
based cancer vaccines have shown immense
promise in preclinical studies, possessing the
potential to promote appropriate immune
responses against tumor cells without
damaging healthy tissue.12 Many of the
distinctive traits possessed by polymeric
particles make them strong candidates for
the vector component of cancer vaccine
formulations, and many preclinical studies
over recent years have highlighted the
advantages of using polymeric particles
as cancer vaccine delivery systems.13,14 In
this review, we describe the attributes that
polymeric particle-based cancer vaccines
possess in terms of improving cancer vaccine
efficacy. This review also recapitulates the
most recent advances in using polymeric
particles in cancer vaccine applications and
new approaches to using unique features of Figure 1. Basic steps in the development of a tumor-specific cytotoxic T lymphocyte
polymeric particle-based delivery systems to immune response.
enhance cancer vaccine efficacy.
The major fundamental component of most to be taken up by professional antigen- tumor antigen in the context of a surface
putative cancer vaccine formulations is the presenting cells known as dendritic cells. For protein (known as MHC class I) that will favor
tumor antigen which may be provided as a sufficient quantities of antigen to be taken the activation of effector T lymphocytes,
purified protein(s), whole tumor cells (e.g. up by dendritic cells, the residence time of commonly referred to as cytotoxic T cells,
irradiated tumor cells) or fractions thereof the antigen is of paramount importance. which upon activation have the potential
(e.g. tumor cell lysates) or nucleic acids Encapsulation of tumor antigen into particles to specifically kill tumor cells (Figure 1).25
encoding tumor antigen(s).15-18 There are, can improve the stability of the tumor antigen Soluble antigen, on the other hand, will tend
however, a number of considerations that by protecting it from proteases. The notion to be processed and presented differently;
need to be taken into account that often of protein encapsulation is well-established in the form of MHC class II, thus favoring an
delineate from a vaccination approach that and various preclinical studies have antibody-mediated response. Generating
is designed to protect against invasion demonstrated that tumor antigen loaded antigen-specific antibody responses,
by a highly immunogenic pathogen (e.g. into polymeric particles promotes stronger whilst sufficient for vaccines against many
smallpox virus) versus one designed as a antitumor immune responses compared pathogens, is generally insufficient when
therapeutic against tumor cells which are to soluble tumor antigen.19-21 In addition to generating a tumor-specific immune
derived from the host and are therefore susceptibility to premature enzymatic and response. This is primarily due to the fact
inherently non-immunogenic; the degree proteolytic degradation, soluble antigens that most tumor antigens are not expressed
of difficulty is inordinately higher when have low uptake rates by dendritic cells. in their native form on the cell surface and
attempting a therapeutic vaccine as Polymeric particles can significantly boost thus cannot be accessed by antibodies.22,26,27
compared to a prophylactic one. The first the uptake efficiency, and the entrapment of Also, when considering tumor antigens, due
of these considerations is protection of the tumor antigen into particles substantially primarily to tolerance mechanisms, most
the antigen from exposure to the harsh enhances the phagocytic efficiency by are not sufficiently immunogenic per se
degradative extracellular environment upon dendritic cells.22-24 Another advantage to adequately stimulate potent antitumor
administration. It is known that the tumor of providing antigen in association with immune responses. Therefore, cancer
antigen of interest, in order to commence particles versus in soluble form is that, upon vaccines often require an immunostimulatory
the process of inducing a tumor antigen- uptake by dendritic cells, particles will favor adjuvant capable of promoting dendritic cell
specific immune response, ideally needs the processing and presentation of the activation and enhancing the ability of the

34 | | April 2019
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Exploring ways clinical supplies and quality Amicus Therapeutics
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changed?
» FORMULATION AND DEVELOPMENT »

vaccine to promote tumor antigen-specific the local draining lymph node, smaller which include: emulsification, such as single
cytotoxic T lymphocyte responses.28,29 The particles (< 100-200 nm) can potentially and double emulsion solvent-evaporation
most widely investigated adjuvants are traffic independently to the draining lymph methods, and nanoprecipitation (also
Toll-like receptor (TLR) ligands and include; node where they can be taken up by the known as solvent displacement method)
polyinosinic:polycytidylic acid (a TLR3 resident dendritic cells, which are present (Figure 3). Adjusting the parameters of
agonist), monophosphoryl lipid A (a TLR4 at high densities and may more efficiently the fabrication process can determine the
agonist), pentaerythritol lipid A (a TLR4 process and present antigen than migrating properties of particles, such as size, size
agonist), and CpG oligodeoxynucleotides dendritic cells.34,35 The same researchers also distribution, encapsulation efficiency, and
(CpG ODN: a TLR9 agonist). These ligands, also investigated the effect of surface charge and the yield. There are a variety of polymers
known as pathogen-associated molecular hydrophobicity on the capacity of particles that can be used to fabricate particles,
patterns (PAMPs), are capable of stimulating to drain independently to the lymph node however those polymers that offer desirable
dendritic cell maturation by binding to TLRs and found an inverse correlation between characteristics when considering them for
and initiating a signaling cascade resulting in hydrophobicity and lymph node targeting; use as cancer vaccine delivery systems are
up-regulation of surface antigens (e.g. CD80 and a direct relationship between anionic preferred. Specifically, polymers should be
and CD86) and cytokines (e.g. interleukin-12) charge density and lymph node targeting biodegradable and biocompatible where
that promote effector T lymphocyte of particles.34 the degraded by-products are readily
responses. Polymeric particles possess the
Several techniques have been used to metabolized and non-toxic.36 Examples of
capability to efficiently encapsulate and
fabricate polymeric particles, examples of such polymers are poly(lactic-co-glycolic
deliver the tumor antigen and immunological
adjuvant (either co-loaded or separately
encapsulated) to dendritic cells.19-21 Finally,
the ability of polymeric particles to be tuned
or functionalized provides the opportunity to
target the particles to dendritic cells, thereby
promoting more efficient uptake and this is
discussed in more detail below.
Aside from the many advantages listed
above, polymeric delivery systems are
considered to be promising candidates
for vaccine delivery because they possess
pathogen-mimicking properties in terms Figure 2. Electron photomicrographs of polymeric particles. (A) PLGA particles; (B) PA
particles; and (C) PSN particles. Scale bar represents 1 μm.
of shape, size and morphology (Figure
2); and they can deliver the antigen in a
similar fashion to how it would be delivered
during a natural infection.30,31 The size of the
particles delivering vaccine components
is considered to be a determining factor in
terms of the endocytic pathway used by
dendritic cells for uptake. In general, larger
particles are engulfed by phagocytosis
while smaller particles are taken up by
pinocytosis.32 A comparison of antigen-
loaded particles of different sizes (300, 1000,
7000, and 17000 nm diameter) showed
that smaller-sized particles were readily
taken up by bone-marrow derived dendritic
cells and consequently they were more
efficient at stimulating antigen-specific
effector immune responses in vivo in a
murine model.33 Other groups have shown
that, while larger particles (> 100-200 nm
Figure 3. Fabrication process of polymeric particles (emulsification and
diameter) generally remain at the site of
nanoprecipitation techniques).
vaccination and require uptake by migratory
dendritic cells in order to be delivered to

36 | | April 2019

acid) (PLGA), poly(anhydrides) (PA), poly(sulfenamides) (PSN), and
poly(phosphazenes) (PPZ). These polymers have been well-studied
and are the most widely explored polymers for particle fabrication and
use in vaccine applications. Particle-based formulations fabricated
with certain types of polymer can function not only as delivery vehicles,
but also as immunological adjuvants. PA and PPZ particles have
been reported to have an adjuvant effect in that they can stimulate
dendritic cells through binding to TLRs.22,37,38 Another important
characteristic of many polymer-based systems is that the degradation
rate of the particles can be tailored according to polymer composition.
To explain, varying the molar ratio of PA monomers during the
copolymerization process can tune the degradation rate and therefore
control the release kinetics of the cargo from polymeric particles
(Figure 4).22,38,39 A number of studies have indicated that varying the
molar composition of copolymers can also have a significant effect on
the properties of particles.38 One major factor is hydrophobicity which
plays a key role in the opsonization and cellular uptake of particles.
For example, increasing the molar ratio of the carboxyphenoxy
hexane (CPH) monomer in a PA copolymer composition resulted in
a significant increase in the hydrophobicity of particles and, in turn,
stimulated more potent antitumor immune responses and improved
their in vivo performance.38 The effect of hydrophobicity, however, may
vary depending on the size of the particles used.34
One of the critical factors affecting cancer vaccine efficacy is the number
and timing of vaccinations. Many vaccines are required to be given as a
prime-boost involving multiple doses to achieve desirable outcomes.
Reducing the number of vaccinations required to achieve an optimal
immune response would be desirable from the perspective of patient
compliance. Interestingly, preclinical studies showed that vaccination
with a single subcutaneous dose of PA-based particles encapsulating
a tumor model antigen was found to induce enduring tumor antigen-
specific cytotoxic T lymphocytes and generate long-term protection
against lethal tumor challenge, and this was as effective as a prime-
boost regimen.22 Other novel immunization strategies involving
polymeric particles are currently being studied. For example, in situ
vaccination, is a promising strategy where particles are injected
Figure 4. Cumulative in vitro release profile of a model tumor
antigen (ovalbumin, OVA) from polyanhydride particles of
different molar ratio compositions (50:50 and 20:80 CPTEG:CPH).
Data points are plotted as mean ± standard deviation.
« FORMULATION AND DEVELOPMENT »
intratumorally to manipulate the tumor microenvironment. A recently
reported example is in situ immunization against B cell lymphoma
with PLGA particles co-encapsulating CpG ODN and doxorubicin, a
chemotherapeutic compound inducing immunogenic apoptosis. The
principle of this strategy is that dying tumor cells provide a source of
antigen for dendritic cells in the tumor microenvironment while CpG
ODN functions as an immunological adjuvant to enhance dendritic
cell maturation and therefore antitumor immune responses.40,41 Also,
heterologous (diversified) prime-boost vaccination strategies where
different versions of cancer vaccine formulations are administered
hold the promise of inducing more effective cytotoxic T lymphocyte
immune responses.8 This could be of particular importance for
immunogen-encoded viral vectors where homologous prime-boost
vaccinations could be problematic due to high immunogenicity
and limited effectiveness owing to the production of viral-specific
neutralizing antibodies.42 One such example to address this issue is the
diversified prime-boost vaccination using antigen-loaded polymeric
particles (as a prime vaccine) and attenuated adenoviral vector
encoding antigen (as a booster).43
Another attractive characteristic of polymeric particles is that their
surface is amenable to chemical modifications, allowing researchers
to improve targeting and refine the ability of the delivery system to
interact specifically with the host’s immune system, hence, generating
robust tumor-specific immune responses.44 The surface chemistry
of polymeric particles is of crucial importance in determining their
cellular uptake, biodistribution, targeting, and therapeutic effects.
Although polymeric particles are generally easily recognized and
ingested by dendritic cells (Figure 5), appropriate surface modulation
can afford them an enhanced capacity to bind to specific targets or
penetrate through biological barriers; therefore, further enhancing
the performance of the cancer vaccine formulation.45 For example,
mannose and carbohydrate grafted onto polymeric particles
were found to enhance active targeting to dendritic cells and
macrophages.46,47 In addition, coupling of polymeric particle surfaces
to specific proteins such as antibodies offers the opportunity for active
immunological targeting.24,48 As an example, a recent study used a
novel targeting approach involving ICI-functionalized particles to
Figure 5. Bone-marrow derived dendritic cell uptake of
polyanhydride particles loaded with coumarin-6 (a fluorescent
compound). (A) untreated cells; and (B) cells treated with
coumarin-6 loaded polyanhydride particles. Scale bar
represents 20 μm.
www.americanpharmaceuticalreview.com | | 37
» FORMULATION AND DEVELOPMENT »
target PD-1 on dendritic cells and demonstrated that these polymeric
particles could enhance the efficacy of ICI.49 The surface charge of
particles also plays a crucial role in determining the fate of particles
within the cell. Once particles have been taken up, or endocytosed, by
dendritic cells, they need to escape the endosome in order to deliver
their cargo to the cytoplasm. Switching the surface charge of particles
from negative to positive by using cationic polymers or dendrimers
such as chitosan, poly(amidoamine), and poly(propyleneimine) can
promote endosomal escape through a mechanism known as the
proton sponge effect.36,50 The ease of designing polymeric particles
with multifunctional and versatile platforms has made them attractive
candidates for cancer vaccine delivery. Collectively, many of the
favorable traits of polymeric particle-based platforms discussed above
have culminated into the successful implementation preclinically, and
many of them are under active and intense investigation for cancer
vaccine applications.
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9. Rosalia RA, Cruz LJ, van Duikeren S, et al. CD40-targeted dendritic cell delivery of PLGA-
nanoparticle vaccines induce potent anti-tumor responses. Biomaterials. 2015;40:88-97.
10. Chen Q, Xu L, Liang C, Wang C, Peng R, Liu Z. Photothermal therapy with immune-adjuvant
nanoparticles together with checkpoint blockade for effective cancer immunotherapy. Nat
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11. Ahmed KK, Geary SM, Salem AK. Applying biodegradable particles to enhance cancer
vaccine efficacy. Immunol Res. 2014;59(1-3):220-228.
12. Salem AK. Nanoparticles in vaccine delivery. AAPS J. 2015;17(2):289-291.
13. Geary SM, Salem AK. Exploiting the tumor phenotype using biodegradable submicron
carriers of chemotherapeutic drugs. Crit Rev Oncog. 2014;19(3-4):269-280.
14. Joshi VB, Geary SM, Salem AK. Biodegradable particles as vaccine antigen delivery systems
for stimulating cellular immune responses. Hum Vaccin Immunother. 2013;9(12):2584-
2590.
15. Gross BP, Wongrakpanich A, Francis MB, Salem AK, Norian LA. A therapeutic microparticle-
based tumor lysate vaccine reduces spontaneous metastases in murine breast cancer. AAPS
J. 2014;16(6):1194-1203.
16. Joshi VB, Geary SM, Gross BP, Wongrakpanich A, Norian LA, Salem AK. Tumor lysate-loaded
biodegradable microparticles as cancer vaccines. Expert Rev Vaccines. 2014;13(1):9-15.
17. Geary SM, Lemke CD, Lubaroff DM, Salem AK. Proposed mechanisms of action for prostate
cancer vaccines. Nat Rev Urol. 2013;10(3):149-160.
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18. Abbas AO, Donovan MD, Salem AK. Formulating poly(lactide-co-glycolide) particles for
plasmid DNA delivery. J Pharm Sci. 2008;97(7):2448-2461.
19. Geary SM, Hu Q, Joshi VB, Bowden NB, Salem AK. Diaminosulfide based polymer microparticles
as cancer vaccine delivery systems. J Control Release. 2015;220(Pt B):682-690.
20. Joshi VB, Geary SM, Carrillo-Conde BR, Narasimhan B, Salem AK. Characterizing the
antitumor response in mice treated with antigen-loaded polyanhydride microparticles.
Acta Biomater. 2013;9(3):5583-5589.
21. Ahmed KK, Geary SM, Salem AK. Development and Evaluation of Biodegradable Particles
Coloaded With Antigen and the Toll-Like Receptor Agonist, Pentaerythritol Lipid A, as a
Cancer Vaccine. J Pharm Sci. 2016;105(3):1173-1179.
22. Wafa EI, Geary SM, Ross KA, Goodman JT, Narasimhan B, Salem AK. Single dose of
polyanhydride particle-based vaccine generates potent antigen-specific antitumor
immune responses. J Pharmacol Exp Ther. 2018.
23. D’Mello SR, Yoo J, Bowden NB, Salem AK. Microparticles prepared from sulfenamide-based
polymers. J Microencapsul. 2014;31(2):137-146.
24. Walk RM, Elliott ST, Blanco FC, et al. T-cell activation is enhanced by targeting IL-10
cytokine production in toll-like receptor-stimulated macrophages. Immunotargets Ther.
2012;1:13-23.
25. Song C, Noh YW, Lim YT. Polymer nanoparticles for cross-presentation of exogenous
antigens and enhanced cytotoxic T-lymphocyte immune response. Int J Nanomedicine.
2016;11:3753-3764.
26. Blum JS, Wearsch PA, Cresswell P. Pathways of antigen processing. Annu Rev Immunol.
2013;31:443-473.
27. Andersen MH, Schrama D, Thor Straten P, Becker JC. Cytotoxic T cells. J Invest Dermatol.
2006;126(1):32-41.
28. de Barros CM, Wafa EI, Chitphet K, Ahmed K, Geary SM, Salem AK. Production of
Adjuvant-Loaded Biodegradable Particles for Use in Cancer Vaccines. Methods Mol Biol.
2017;1494:201-213.
29. Goforth R, Salem AK, Zhu X, et al. Immune stimulatory antigen loaded particles combined
with depletion of regulatory T-cells induce potent tumor specific immunity in a mouse
model of melanoma. Cancer Immunol Immunother. 2009;58(4):517-530.
30. Gregory AE, Titball R, Williamson D. Vaccine delivery using nanoparticles. Front Cell Infect
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31. Ulery BD, Petersen LK, Phanse Y, et al. Rational design of pathogen-mimicking amphiphilic
materials as nanoadjuvants. Sci Rep. 2011;1:198.
32. Champion JA, Walker A, Mitragotri S. Role of particle size in phagocytosis of polymeric
microspheres. Pharm Res. 2008;25(8):1815-1821.
33. Joshi VB, Geary SM, Salem AK. Biodegradable particles as vaccine delivery systems: size
matters. AAPS J. 2013;15(1):85-94.
34. Rao DA, Forrest ML, Alani AW, Kwon GS, Robinson JR. Biodegradable PLGA based nanoparticles
for sustained regional lymphatic drug delivery. J Pharm Sci. 2010;99(4):2018-2031.
35. Reddy ST, van der Vlies AJ, Simeoni E, et al. Exploiting lymphatic transport and complement
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complex biological barriers. Journal of Controlled Release. 2015;219:548-559.
37. Andrianov AK, Marin A, Fuerst TR. Molecular-Level Interactions of Polyphosphazene
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Biomacromolecules. 2016;17(11):3732-3742.
38. Wafa EI, Geary SM, Goodman JT, Narasimhan B, Salem AK. The effect of polyanhydride
chemistry in particle-based cancer vaccines on the magnitude of the anti-tumor immune
response. Acta Biomater. 2017;50:417-427.
39. Torres MP, Vogel BM, Narasimhan B, Mallapragada SK. Synthesis and characterization
of novel polyanhydrides with tailored erosion mechanisms. J Biomed Mater Res A.
2006;76(1):102-110.
40. Makkouk A, Joshi VB, Lemke CD, et al. Three steps to breaking immune tolerance to
lymphoma: a microparticle approach. Cancer Immunol Res. 2015;3(4):389-398.
41. Makkouk A, Joshi VB, Wongrakpanich A, et al. Biodegradable microparticles loaded
with doxorubicin and CpG ODN for in situ immunization against cancer. AAPS J.
2015;17(1):184-193.

42. Siemens DR, Elzey BD, Lubaroff DM, et al. Cutting edge: restoration of the ability to
generate CTL in mice immune to adenovirus by delivery of virus in a collagen-based matrix.
J Immunol. 2001;166(2):731-735.
43. Lemke CD, Geary SM, Joshi VB, Salem AK. Antigen-coated poly alpha-hydroxy acid based
microparticles for heterologous prime-boost adenovirus based vaccinations. Biomaterials.
2013;34(10):2524-2529.
44. Morris AS, Wongrakpanich A, Geary SM, Salem AK. Chapter Nine - Microparticles and
Nanoparticles for Cancer-Targeting Vaccines. In: Skwarczynski M, Toth I, eds. Micro and
Nanotechnology in Vaccine Development. William Andrew Publishing; 2017:171-183.
45. Morris AS, Salem AK. Surface Engineered Nanoparticles: Considerations for Biomedical
Applications. Advanced Engineering Materials. 2017;19(11):1700302.
46. Kim N, Jiang D, Jacobi AM, et al. Synthesis and characterization of mannosylated pegylated
polyethylenimine as a carrier for siRNA. Int J Pharm. 2012;427(1):123-133.
47. Vela-Ramirez JE, Goodman JT, Boggiatto PM, et al. Safety and biocompatibility of
carbohydrate-functionalized polyanhydride nanoparticles. AAPS J. 2015;17(1):256-267.
48. Nobs L, Buchegger F, Gurny R, Allemann E. Poly(lactic acid) nanoparticles labeled
with biologically active Neutravidin for active targeting. Eur J Pharm Biopharm.
2004;58(3):483-490.
49. Ordikhani F, Uehara M, Kasinath V, et al. Targeting antigen-presenting cells by anti-PD-1
nanoparticles augments antitumor immunity. JCI Insight. 2018;3(20).
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About the Authors
Suhaila O. Suliman is a graduate student in the Ph.D.
program in Pharmaceutics at the University of Iowa. As
part of her Ph.D. studies, she focuses on enhancing cancer
vaccine efficacy and stimulating robust tumor-specific
immune responses using particle-based formulations. She is also
« FORMULATION AND DEVELOPMENT »
interested in studying biodistribution and pharmacokinetics of polymeric
nanoparticles for drug delivery.
Emad I. Wafa is currently a graduate student in the Ph.D.
program at the College of Pharmacy, University of Iowa.
Prior to joining the University of Iowa, Emad earned
his bachelor’s degree in Pharmaceutical Sciences from
Misurata University, Libya. His research primarily deals with designing
and developing cancer vaccines using biodegradable polymeric particles.
He is also interested in improving transfection of antigen-presenting cells
using polymeric nanovectors.
Dr. Sean Geary is an assistant research scientist who
obtained his PhD in Tumor Immunology from the University
of Adelaide, Australia. For the past decade, he has been
intensively involved in the development and testing of a
range of particle based and viral cancer vaccines in preclinical settings,
resulting in many publications in peer-reviewed journals.
Aliasger K. Salem is the Bighley Chair and Professor of
Pharmaceutical Sciences and Head of the Division of
Pharmaceutics and Translational Therapeutics at the
University of Iowa College of Pharmacy. Aliasger Salem
is also currently the leader of the Experimental Therapeutics program
at the Holden Comprehensive Cancer Center and co-director of the
Nanotoxicology Core at the Environmental Health Sciences Research
Center. Prior to joining the University of Iowa in 2004, he was a postdoctoral
fellow at the Johns Hopkins School of Medicine and completed his PhD at
the School of Pharmacy and Pharmaceutical Sciences at the University of
Nottingham in the UK.
www.americanpharmaceuticalreview.com | | 39
» BIOPHARM DEVELOPMENT »

The Application of Risk Assessments for the Design and

Development of Devices for Biological Products
Manfred Maeder, PhD
Global Head Device Development & Commercialization
Global Drug Development/Technical Research & Development
Novartis

and Mitigation Strategy) program if it was determined necessary to

Introduction ensure the benefits outweigh the risks. This type of REMS could be
required pre- or post-approval.
Within the pharmaceutical industry the share of combination
On the contrary considering the device constituent part, ISO 14971,
products has increased significantly during recent years. This is linked
the standard for the application of risk management for medical
especially to the rise of biologics products. For the US, during fiscal
devices, was harmonized and applied as of June 1993. Within the
year 2017, for the first time FDA received more biologic applications
European Union Directive 93/42/EEC also known as the Medical
compared to small molecule applications.
Device Directive (MDD) defines these requirements. As of May 2020,
Typically, combination products consist of a drug and a device the Medical Device Regulation (MDR) will finally replace the MDD with
constituent part. In most cases we see either a drug/device or a a complete set of additional, more strict expectations. This standard
biologic/device combination. The typical case for pharmaceutical
defines the minimum expectations for medical devices and device
companies is to have combination products with a Primary Mode of
constituent parts of combination products. The most recent revision
Action (PMOA) of the drug or the biologic constituent part. For these,
was during 2012.
the device part is used as an application tool, only in some cases
however as very sophisticated ones. For manufacturers of medical
devices, combination products with a PMOA of the device are more
relevant (e.g. drug coated stent), where the primary mode of action How Do We Manage Risk?
is the device functionality and the drug has a supporting function.
We try to achieve the optimum risk reduction for all stakeholders. This
In Europe these products are considered as DDCs (Drug Device
does not automatically indicate that all risks are minimized under all
Combinations). Since a Combination Product (CP) or DDC consists
of two constituent parts with quite different regulatory and GMP circumstances. Reducing every risk as far as technically possible in one
expectations both sets of regulation have to be considered. area might introduce risks in other areas and/or lead to very complex
devices or devices with poor usability. The target is to develop devices
For companies producing, assembling, handling, and distributing
and therapies where the risks are balanced well and that are proven
combination products this would require fulfilling the expectations
effective. Risk management can be a very valuable tool to identify
of CFR 210/211 as well as for CFR 820. However, following the
critical design, usability or manufacturing aspects and to optimize
streamlined approach of CFR Part 4 for combination products, most
speed of iteration of existing systems.
pharmaceutical companies decided to fulfill the CFR 210/ 211 plus the
relevant parts of CFR 820 in order to meet minimum expectations. The most important guidance for risk management for devices and
device constituent parts is the ISO 14971. Figure 1 describes the
Compared to device regulation the application of risk management
iterative process to be applied.
for pharmaceuticals is fairly new. For example, the ICH guidance for
industry “Q9 Quality risk management” was introduced in 2006. ISO 14971 requires that devices must be safe when used as intended
Following that, the FDA Amendments Act of 2007 authorized FDA to by the manufacturer. Additionally, risks have to be outweighed by the
require sponsors to develop and comply with a REMS (Risk Evaluation benefits of the treatment.

40 | | April 2019
 « BIOPHARM DEVELOPMENT »

for reviewing and monitoring during development, production and

post-production.

The main objectives of risk management are to investigate the risk and
the underlying root cause to minimize risk and to finally determine the
impact of the risk management initiatives taken.

For risk management all interactions of the user with the device need
to be evaluated in depth.

Topics like:

• Normal, intended use

• Reasonably foreseeable misuse need to be taken into

consideration and evaluated.

Design factors for use errors could be:

• Constricted visibility, audibility

• Confusing control system

Figure 1. Risk management process as defined in ISO 14971 • Ambiguous, unclear device status

• Displayed information unclear

Use factors, such as user unable to perform task, or user completes

To achieve that the standards provide a framework for risk analysis, task with difficulties or incompletely, as well as environmental factors
evaluation, control, and management, and also specify a procedure like noise, heat, and lighting conditions need to be considered.

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www.americanpharmaceuticalreview.com | | 41
» BIOPHARM DEVELOPMENT »
Current guidelines do not prescribe which tools to use. In most cases
FMEA (Failure Mode and Effects Analysis) is being used for evaluation
of medical devices and combination products. This tool is being used
to evaluate the design, usability, process (e.g. assembly, packaging,
shipping), and biocompatibility.
Which Information Needs to Go into
the Risk Management File?
For all marketing applications and marketed products risk
management needs to be documented properly. For the US this will
be part of the Design History File (DHF) and for EU it will be part of the
Technical File (TF) following the requirements of the medical device
regulation (MDR) Annex I.
The following topics need to covered and documented accordingly
during the risk evaluation:
• Risk Management Plan
• Hazard Identification
• Use related Risk Analysis
• Design Risk Analysis
• Process Risk Analyses
• Risk Management Report
Following this main structure, a significant number of additional
evaluations need to take place.
Typically, the risk management plan describes the roles and respon-
sibilities of contributing team members. It contains the risk accep-
tance matrix and lists all definitions related to the product evaluated.
For example, during the hazard identification step the potential haz-
ards themselves as well as hazardous situations need to be evaluated
and discussed. The sequence of events and potential harm to the user
need to be taken into consideration.
During the use related risk analysis, all use steps have to be
evaluated thoroughly using tools of human factors or usability. Of
course, the instructions for use are at the center of this evaluation.
Also, reasonably foreseeable misuse has to be evaluated in depth.
For all topics above a well thought through ergonomic design will
help to alleviate a significant number of the problems. Especially for
combination products, during this step it is of utmost importance
to consider the indication of the drug product or biologic. Based on
the indication the patient population will be defined. For example,
when developing a treatment for arthritic patients the ergonomic
design might have to be completely different compared to the
needs of young adults. Starting use related risk analyses early in
the development process allows designers to address use failures
by optimizing the device design which is the most effective risk
mitigation.
During the design risk analysis, the ISO 10993 standard needs to be
applied additionally, in order to evaluate biocompatibility. In most
cases, for devices with skin contact (intact skin or damaged skin)
42 | | April 2019
this at least involves sensitivity, cytotoxicity, and skin irritation. Risk
management tools are used to evaluate the overall impact considering
the cumulated exposure time, location of exposure, release of potential
toxins, etc.
Process risk analysis is evaluating the impact of all process steps on
the quality of the device. During this step not only the device, but
the combination product itself needs to be evaluated. All process
steps need to be considered such as filling of the drug product or
biologics into primary packaging, assembly of the drug constituent
part with the device constituent part(s). Filling of drug products is
fairly straightforward, if state of the art methods for aseptic filling are
being used. In case the drug constituent part is biologic, this might
complicate the matter. For example, a number of pump types can’t
be used because of shear forces they create. A significant portion
of large biologic products are susceptible to this problem. For this
reason, the correct type of pumps has to be chosen or filling speeds
have to be reduced to an acceptable level. This property of biologics,
susceptibility to shear forces, also needs to be considered in the
design of the device, as very thin needles, which clearly is a current
trend, would also exhibit this problem. Furthermore, packaging
and shipping need to be evaluated thoroughly, as they typically
bear some surprises. During all processing steps the heat exposure
of any biologics have to be controlled. This is especially true for
the compounding, filling, and packaging step. To consider and
implement a temperature control during compounding and filling
is very straightforward, however during packaging the heat-sealing
process is easily forgotten.
Finally, the risk management report should summarize the overall risk/
benefit analysis and also point out the residual risks.
Why are We Implementing Risk
Management Tools for Combination
Product Development?
Clearly, the proper implementation of a risk management process can
be a win-win for both, the patient and the company.
It is an expectation that the patient has a product in his hand, which
is easy to use and has been designed properly. It is important to
realize for combination products, at least for the device constituent
part, there is a similarly high expectation as for consumer products.
As a patient, I can’t make a judgement on the quality of the drug or
biologic, but I can draw a lot of conclusions on the functionality and
design of the device.
In turn, for any company being able to create inspiring, reliable devices
easing the treatment for the patient there is a huge competitive
advantage especially in the field of generics and biosimilars, where
there is by definition no difference in the drug part.
 June 3-6, 2019
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» ROUNDTABLE »
Contamination Control
Roundtable
In general, what are some current issues or
problems pharm/biopharma companies are dealing
with in regards to controlling contamination in
their facilities?
Tony Cundell, PhD, Principal Consultant, Microbiological Consulting,
LLC: The usual suspects are aging facilities, the incomplete implementa-
tion of barrier technologies, sterile products being aseptically filled when
they are candidates for bioburden-based terminal sterilization, poor prod-
uct failure investigations and corrective actions, and the general lack of
manufacturing excellence.
Paula Peacos, Senior Consultant, ValSource, Inc.:
1. Lack of specific regulatory requirements and guidance
for many of the emerging technologies that are neither
traditional aseptic processing operations nor true
nonsterile operations is a problem. This basically put the
onus on the firm to determine the requirements, and some
are more proficient in that than others.
2. In my opinion, the industry in general is still learning how
to appropriately apply QRM to their operations. QRM
is quickly being incorporated, but not all programs are
well-designed or sufficiently robust. For example, some
risk assessments are still justifying current practice as
opposed to identifying potential contamination risks. This
is a particular problem where specific regulatory guidance
or requirements are lacking.
3. Lack of new microbiological monitoring methods.
Detecting/recovering microbial contamination is still done
using antiquated methods that are known to be relatively
inefficient, highly variable, and extremely limited. A very
well designed and robust EM and trending program helps
44 | | April 2019
Tony Cundell, PhD
Principal Consultant
Microbiological Consulting, LLC
Paula Peacos
Senior Consultant
ValSource, Inc
Tim Sandle, PhD
Head of Microbiology and Sterility Assurance
Bio Products Laboratory Limited
Jeanne Moldenhauer
Vice President
Excellent Pharma Consulting
to offset that, but we are still working with estimates
of what might be actually present. There are many
developments in technologies allowing faster turnaround
times to obtain results, but most of these still utilize the
old sample collection methods (i.e., contact plates, settle
plates, and viable air sampling methods).
Tim Sandle, PhD, Head of Microbiology and Sterility Assurance,
Bio Products Laboratory Limited: The problems will vary between
different facilities, and these will center on the different sources of
contamination in relation to people, air, water, transfer of items,
equipment cleanliness, and bioburden of starting materials. The most
difficult challenges are invariably around people: how personnel
behave in cleanrooms, how they are gowned, and whether they follow
the correct procedures. Although the regulations around personnel
have largely remained unchanged, its noticeable that the number
of warning letters and other regulatory citations have increased. The
reason for this must rest with training, knowledge and with time (in
terms of allowing operators sufficient time to carry out their duties and
to clean and disinfect effectively).
Keeping track of data is also a challenge. With large facilities in particular,
assessing microbial and particulate trends remains important so
that appropriate actions can be taken promptly. Furthermore, it is
important to understand when the process is leaning out of control,
to enable personnel to be alerted to a potential change in the process.
To aid microbiologists, there are a number of powerful databases on
the market which provide graphical illustration of trends and which
meet data integrity requirements.
Jeanne Moldenhauer, Vice-President, Excellent Pharma Consulting:
1. Some of the control problems result from the establishment
of unrealistic limits for types of contamination, e.g., setting a
zero-tolerance level for molds.

2. Another concern with contamination control is the
reluctance of companies to use/implement newer
technologies that actually prevent microbial growth.
Looking at the latest in contamination control
products and solutions, can you tell us about
some new technologies and/or processes that are
available now that you are excited about?
Cundell: With all aseptic processes, removing human interventions
is a key design and operation strategy for improving contamination
control. Automation will continue to be emphasized. Equipment like
depyrogenation tunnels, multiple-container filling and stoppering
stations, vision systems for defect detection, online container-closure
integrity testing, and robotic lyophilizer loading and unloading are
now commonplace in sterile product manufacturing facilities.
The ability to exclude people from aseptic processing has a hierarchy of
risk: laminar flow hood > biological safety cabinets > restricted assess
barrier systems > open isolator systems > closed isolator systems with
gloves > gloveless isolator systems. Robotic systems are uniquely
suitable for inclusion in barrier systems like gloveless isolators.
Remember, robotic systems will automate standard manipulations,
exclude people from critical aseptic processing areas, and do not look
or move like humans.
Peacos:
1. I am a huge fan of single use/disposable technologies
that minimize the amount of product exposure to human
intervention because they are very effective and relatively
easy and inexpensive to implement. A firm can greatly
reduce their risk of contamination in a short amount of
time through the use of these technologies as opposed to
having to do new construction or retrofitting. Examples
include the use of sampling apparatus that can be sterilized
in place, pre-assembled and/or pre-sterilized parts and
components, welded connections etc. These can go a long
way to aid in contamination control for small firms that
simply don’t have the capital to convert their operations to,
for example, isolator technologies.
2. Technologies such as the Growth Direct, which decrease
the turnaround time for obtaining microbiological
data (colony counts). Environmental excursions and
contamination risks are often not detected until 3-7
days after sample collection using traditional processing
methods. Meanwhile, manufacturing operations are
continuing. Furthermore, this method is nondestructive,
meaning microbiologists can culture and identify the
organisms to allow for appropriate investigation. Some
rapid detection methods destroy the organisms or do not
allow for recovery, which in my opinion significantly lowers
their value.
Sandle: With aseptic processing, ideal technologies are those that
separate people from products, such as isolators, and which can
« ROUNDTABLE »
replace the need for humans for carry out operations, as with the use
of robotics. An advantage with automation is that processes can be
stopped if there has been a shift in environmental conditions, such as
an increase in particle counts.
Technologies which help to show that tasks are being conducted
correctly are important, such as glove stations which can indicate how
often and for how long hands have been sanitized for, and tracking
technologies that indicate that cleanrooms are being disinfected
for the appropriate time periods are useful for helping to address
personnel related contamination control issues.
Other technologies of interest are the spectrophotometric particle
counters, which can be used to differentiate between inert particles
and those which are biologic. Although there are still some factors to
overcome in terms of screening for false positives, taking samples over
a sufficiently long time period provides useful data for benchmarking.
Such data can then be used to assess changes in a facility, such as
the impact of maintenance work or to assess the impact of increased
numbers of personnel in an area.
A further area that’s receiving increased coverage is with air
decontamination units with HEPA filters. These offer an additional
technology to destroy a a range of airborne microorganisms.
Moldenhauer:
1. There are many new water-based technologies that are
antimicrobials, e.g., increased oxygens (ozonated) and
electrostatically charged hydrogens. These products have
sporicidal properties without leaving residues and without
damage to stainless steel surfaces or employees.
2. There is also an increased focus on products that prevent
growth, both in building materials, coatings, paints and the
like. For example, nanotechnology infused products.
In the past few years have regulatory expectations
from the FDA and other global agencies resulted
in more scrutiny placed on contamination control
efforts? Can you elaborate? Do inconsistent or
different global regulations place a regulatory
strain on pharmaceutical companies?
Cundell: Contamination control during aseptic processing has always
been scrutinized by regulatory agencies. From 2014 through 2017,
48% of the U.S. sterile drug recalls were for visible particles, 31% for
lack of sterility assurance, 8% for labeling issues, 3% for container
defects and 10% for other reasons (Johns et al, 2018). The biggest
disruption presently is the publication of the revision to the EU Good
Manufacturing Practice, Annex 1 that contains rules that differs from
current industry practice and the 2004 FDA Sterile Drug Products
Produced by Aseptic Processing — Current Good Manufacturing
Practice. Gap analysis will reveal many procedural changes and capital
improvements that may be necessary when the revision becomes
official. This will consume many resources.
www.americanpharmaceuticalreview.com | | 45
» ROUNDTABLE »
Peacos:
1. Inspectors in general are increasing the depth of their
knowledge of microbiology, spending more time in the
labs and delving deeper into contamination control
procedures and programs. We see the evidence of this
in the citations that are issued. Site microbiologists are
addressing technical questions that they have not been
asked before. I think this is appropriate given some of the
findings that have come out in recent years, some truly
egregious, that could impact patient safety.
2. Inconsistent or ambiguous regulations are a big problem
in my opinion. When the regulations are ambiguous,
they are subject to interpretation. Sometimes the
firm’s interpretation is not in lock step with that of the
inspectorate, particularly when it concerns the finer details.
It is this room for interpretation that can make it difficult for
a firm to ensure compliance.
Sandle: Inspectors are asking more questions about contamination
control activities and this is reflected in the content of warning letters
and the number of items raised in warning letters. One area that
stands out is with cleaning and disinfection. Inspectors are asking for
evidence that disinfectants have been adequately qualified, against
a range of different surfaces, for example, and that sporicides are
being used where appropriate. A related area of importance is with
demonstrating the absence of residues that might interfere with
disinfectant products.
Regulators are also coming down heavily on evidence of poor
disinfection practices, such as the continued recovery of fungi or spore
forming bacteria, which signal ineffective practices, such as linking with
cleaning and disinfection frequencies or poor application techniques.
A unified approach to contamination control would be very useful to
the pharmaceutical community, especially when receiving inspections
from different inspectorates. The forthcoming changes to EU GMP
Annex 1 could signal that; however, there are now variances between
some of the FDA guidance and that from Europe. An example is with
the design of aseptic process simulations and the acceptance criteria.
Moldenhauer:
1. One of the biggest changes is the draft of Annex 1
which has a complete section on the requirements for a
Contamination Control Strategy/Program.
2. There are no clear differences in programs across
regulatory agencies today.
What advice would you give to a pharmaceutical
company struggling with their contamination
control efforts? Is there a top 5 list of items you
would suggest to these companies to help them?
Cundell: Concentrate on the knitting! My top 5 items to improve
contamination control are as follows:
46 | | April 2019
1. Hire and retain experienced microbiologists who are as
home in the manufacturing areas as in the laboratory.
2. Apply the tools of Quality Risk Management to formulation
and packaging development, manufacturing process
design, and routine aseptic processing.
3. Embrace single-use processing equipment, barrier systems,
robotics, and continuous bioburden, environmental and
water monitoring.
4. Develop skills in expert systems, machine learning, and big
data management to better control your processes.
5. Look for the best opportunities for the implementation of
rapid microbiological methods.
Peacos:
1. Minimize human intervention wherever possible. Invest
in single use/disposable technologies and barrier/closed
system technologies etc. While this can be expensive to
implement, it must be weighed against the risk to the
patient as well as the capital, labor and logistical costs
of rejected batches. Even small reduction in human
interventions can have a big impact.
2. Good trending involves more than just reporting data
from quarter to quarter. It involves data analysis and
interpretation of that analysis which leads to better,
science-based decision making. I would strongly
recommend they invest in a good trending software that
allows them to quickly and effectively analyze a large
amount of data. The ability to do this is critical to good,
reliable contamination control.
3. The entire staff, including quality, facilities and
manufacturing floor staff, should be trained in basic,
practical microbiology. This training should focus on the
way microbes enter and move through a facility, and
more importantly, how each individual has a role in an
effective contamination control program, and how their
performance of their roles impact its success. If the staff
doesn’t not have the knowledge to recognize a problem
that is right in front of them, the most robust, well-
designed program will likely fail.
4. The Micro staff should thoroughly understand the
manufacturing processes they support. It will improve the
quality and efficacy of the contamination control program,
as well as root cause analyses etc. They cannot effectively
investigate something they don’t really understand. The
Micro staff also needs to spend time on the shop floor as
this will enable them to make appropriate adjustments
to the elements of the contamination control program
programs (EM, trending, etc.) to maximize value.
5. The Contamination Control Program needs to be a living,
dynamic program. It needs to be flexible and able to
adapt quickly and easily to changes in the environment or
processes etc. to maintain effective control. To that end,

the program itself and supporting risk assessments and
procedures etc. need to be revisited and reassessed on a
relatively frequent basis and updated or supplemented as
needed. This is especially critical for new processes, new
facilities and new technologies which are still evolving.
Sandle:
1. Contamination control requires an understanding
of the microbial entry points and the various routes
that can be taken to arrive where they are undesired.
Hazard identification is key and then assessing how the
contamination might end up impacting on a process. This
involves understanding the sources of contamination
and how contamination can be transferred. The risks
associated with the different sources of contamination
and the potential impact should be assessed, in terms of
the severity of contamination (should it occur) and the
likelihood of a contamination event occurring (probability).
This enables an organization to focus on risk mitigation
and then to put in place appropriate detection systems to
ensure that the contamination risks remain within a level
of control.
2. Extending the principles of quality risk management to
microbiological sampling is also important, to ensure that
the samples taken are meaningful and relate to the primary
contamination risks. The use of HACCP (Hazard Analysis
and Critical Control Points) can be employed to determine
microbiological environmental monitoring locations.
3. There also needs to be a greater focus on the training
of cleanroom personnel and implementation of more
robust training programs defining aseptic technique,
pharmaceutical microbiology and so on.
4. Keeping track of process variations is important. If the
process is as validated, then the standard sample set will be
sufficient. However, if the process is different, such as having
a longer process hold, then additional samples should be
considered to assess bioburden and endotoxin risk.
5. Having less paperwork in cleanrooms sounds simple,
but this minimizes contamination and leads to better
control over processes and samples. With this, electronic
systems offer a number of advantages for environmental
monitoring and for completing batch records
Moldenhauer: The biggest recommendation is to know what is
causing your contamination issues, i.e., identify the contaminants and
learn all you can about these organisms to try and find the root cause.
What do you see as the major industry critical
issues over the next five years in regards to
contamination control?
Cundell: Clearly major issues surround gene and cell therapies and
the following quote illustrates this. “The key to making gene therapy
« ROUNDTABLE »
accessible to patients will be better manufacturing processes”, Peter
Marks, Director of FDA’s Center for Biologics Evaluation and Research
(CBER), told attendees at the Galien Forum in New York City on Oct.
25, 2018, in a panel discussion on gene therapy. “At this point, the
FDA has approved one directly-administered and two cell-based
gene therapies”, Marks said, “but the Agency has received over 700
investigational new drug (IND) applications in the gene therapy field”.
As the FDA points out, there is a large gap between R&D, clinical
supplies, and routine commercial production. Perhaps the biggest
challenge in gene and cell therapies is cell viability, but the best
approaches to microbial contamination monitoring and control have
not been established.
The revisions to 21 CFR 610.12 Sterility to allow for more flexibility
in sterility testing biologics was helpful. The proposed USP general
informational chapter <1071> Microbial contamination detection/
sterility testing of short life products: A risk-based approach may
contribute to the discussion in the industry.
Peacos:
1. I see a general push in the industry for firms to more
quickly incorporate available contamination control
strategies such as the use of isolator technology,
automation, single use/disposable technologies and
closed systems to replace traditional aseptic processing
lines. These technologies reduce the amount of human
intervention required, which is the main source of
microbial contamination. New construction should ideally
have these technologies built in as part of the design. A lot
of firms are still using the old traditional methods where
better technology is available and has been for some time.
2. I also see a shift from “standard industry practice” to risk-
based controls specifically tailored to the product and
processes in question. New technologies are emerging
faster than the regulatory bodies can issue specific
requirements. The current lack of specific guidance for
so-called “low bioburden processes”, which have elements
of both aseptic processing and nonsterile manufacturing is
a good example of this. This basically put the onus on the
industry, and will require firms to have very strong, science-
based and holistic QRM programs.
Sandle: It is interesting that compendia appear to be removing
limits, such as recommended levels of bioburden and endotoxin
for excipients and raw materials, and placing the need for an
assessment with manufacturers to assess the risk in relation to their
own processes. There is a similar expectation with ‘objectionable
microorganisms’; things have shifted from prescribed lists to the need
for pharmaceutical manufacturers to deploy scientific decision making
as to which microorganisms, potentially present in the manufacturing
environment, present a risk to a product (which involves understanding
the physicochemical properties of the product) and to the patient
population for which the product is intended.
Moldenhauer: I see the industry working towards a climate of
prevention of contamination rather than reaction to contamination.
www.americanpharmaceuticalreview.com | | 47
» SPECTROSCOPY »
Low-Frequency Raman Mapping and Multivariate
Image Analysis for Complex Drug Products
Daniel R. Willett*, Huzeyfe Yilmaz,
Anna M. Wokovich, Jason D. Rodriguez
Food and Drug Administration (FDA)/Center for Drug Evaluation and Research (CDER)/Office of
Pharmaceutical Quality (OPQ)/Office of Testing and Research (OTR)/Division of Pharmaceutical
Analysis (DPA), St. Louis, MO
*Corresponding Author
This article reflects the views of the authors and should not be construed
to represent the Food and Drug Administration’s views or policies.
Introduction
Confocal Raman mapping provides a robust means of characterization
that can non-destructively provide both chemical and structural
information for a wide range of pharmaceutical dosage forms.1-3 A
literature review of Raman mapping for pharmaceuticals shows the
spectral ranges used in studies is primarily limited to Stokes signals that
are 200 to 4000 cm-1 away from the excitation laser line. This region is
accessible with most commercial instruments and allows extracting
valuable fingerprint information about molecules such as aromatics,
carbonates, sulphates, silicates, oxides and hydroxides within the 500-
1500 cm-1 range, and hydrogen interactions with carbon, nitrogen and
oxygen at around 3000 cm-1.4 By contrast, the low-frequency Raman
region (<10 cm-1 to 200 cm-1) has not been readily available in the past,
even though Raman in this frequency range provides access to lattice
vibrations of molecular crystals and has the potential to more directly
probe intermolecular interactions in solids such as pharmaceutical
dosage forms. Specifically, low-frequency Raman spectroscopy provides
an avenue to probe polymorph detection of pharmaceutical systems as
well as drug identification and quantitation for crystalline materials,
both of which are critical quality attributes that need to be assessed and
monitored during or after manufacturing.5-6 The low frequency Raman
region for measurements of lattice phonons in pharmaceuticals has
become more accessible in recent years with advances in precise optical
filters and narrow linewidth lasers. Therefore, low-frequency region
investigations in pharmaceutical products has started to transition
from the academic laboratory with customized laboratory set-ups to
pharmaceutical labs and production lines.7-10
48 | | April 2019
This article focuses on low-frequency Raman spectroscopic analysis
using a commercially available low-frequency Raman laser/filter
coupled with a Raman microscope to perform pharmaceutical
mapping measurements. In addition, multivariant statistical analysis
techniques are utilized to highlight the unique and wide range of
information that can be obtained from low-frequency Raman mapping
of pharmaceuticals.
Here we provide three examples of using low-frequency Raman
mapping for analysis and characterization of a wide range of
pharmaceutical drug types. First, we demonstrate the capability of low-
frequency Raman mapping for determining the active pharmaceutical
ingredient (API) distributions and crystal sizes in an over-the-counter
(OTC) oral dosage form. The mapping analysis of the OTC product
Excedrin® was performed on a tablet core. Using multivariate analysis
of Excedrin®, three major components were observed in the low-
frequency spectral region, all of which were similar to the reference
spectra of the three declared APIs in the drug product (i.e., caffeine,
acetaminophen and aspirin). In the second example, low-frequency
Raman mapping was utilized for the identification of polymorph
conversions in transdermal drug delivery systems (TDDSs). A principal
component analysis (PCA) of the Raman map revealed unintended
polymorph formation within the TDDS. Finally, a topical cream
(Zovirax®) was mapped via the low-frequency spectral region. The
multivariate analysis of this Raman map demonstrated an additional
capability of low-frequency Raman spectroscopy; probing crystal
orientation. The spectral variance within the low-frequency Raman
map was used to identify various crystal orientations. Orientation
identifications can be useful, as spectral variances in crystalline APIs
can be misinterpreted as polymorphs. Overall, the potential of low-
frequency Raman mapping combined with multivariate statistical
analysis is demonstrated here through analysis of a variety of drug
products. We show that low-frequency mapping is a complementary

tool to fingerprint region Raman mapping for rapid and sensitive
identification of APIs, polymorphs and other crystal information that
is obtained from lattice phonons only.
Materials and Methods
Pharmaceutical products were characterized by StreamHRTM Raman
imaging using a Renishaw InVia confocal Raman microscope
coupled with a TR-Micro Thz-Raman System from Ondax Inc. which
provided the filter/laser combo needed to access the low-frequency
region down to ~5 cm-1 in both stokes and anti-stokes regions.
An 808 nm laser excitation source from the Ondax system, -70°C
thermoelectrically cooled charge-coupled device (CCD) camera and
a Leica DM2500 microscope were used for the Raman acquisition. A
1200/mm grating was used to disperse the light through a spectral
range of -400 cm-1 to 900 cm-1. The objective and step size used for the
low-frequency Raman mapping varied to best fit the product being
analyzed, but in general an optical montage was first obtained to
allow for a representative region to be selected and mapped. Cosmic
ray removal was done in WiRE 4.4 software using the width of feature
and nearest-neighbor methods. Preprocessing and MCR/PCA analysis
of the data was then carried out in Matlab R2017a using PLS Toolbox +
MIA (Eigenvector Research, Inc., Version 8.6.2).
Sample preparation for the products selected for analysis were as
follows: The Excedrin® tablet was prepared by splitting it in half with
a razor blade and placing it on the microscope stage. The release liner
of the transdermal delivery system (TDDS) was removed and the TDDS
was placed backing side down on a gold coated microscope slide
and placed on the microscope stage. The Zovirax® cream was thinly
spread on a gold coated microscope slide and placed on the
microscope stage.
Result and Discussion
API Mapping
To investigate the feasibility of low-frequency Raman mapping for
characterization of pharmaceutical products, an over-the-counter
(OTC) tablet consisting of three active pharmaceutical ingredients
(APIs) was selected for analysis. This OTC product (Excedrin®),
is a combination drug for the treatment of pain comprised of
acetaminophen (250 mg), aspirin (250 mg), and caffeine (65 mg) as
the active ingredients, all of which have well defined peaks in the low-
frequency Raman region.
The Ondax system with an 808 nm laser/filter set coupled with the
Renishaw InVia Raman microscope was used to obtain the low-
frequency Raman mapping data from the Excedrin® tablet. The strength
of the Raman signal in the low-frequency region from ~ 5 cm-1 to
200 cm-1 allowed for data to be acquired with the fastest (100 millisecond)
acquisition times allowed by the Raman system. Raman signals in
the low-frequency region generally have signal intensities of up to
« SPECTROSCOPY »
~4.5 times those in the fingerprint region, allowing for faster mapping to
be performed to obtain the same signal-to-noise ratios.
The data set obtained on the Excedrin tablet was comprised of
approximately 220k unique spectra taken across the entire tablet
surface (Figure 1A). Traditional, spectra by spectra comparison
across the 220k set of spectra is prohibitively time consuming.
However, multivariant image analysis can be applied to rapidly
interpret the Raman data. In multivariate methods, spectral
features are utilized fully by revealing the variance within a spectral
map and, simultaneously, dimensionally reducing the dataset.
Typically, unsupervised methods such as PCA are used for chemical
imaging and performs the task via eigen decomposition of the
covariance of the spectral dataset. However, eigenvectors that are
used in the dimensionality reduction are not true representatives of
the Raman spectra, rather, they represent the contribution of a mixture
of the Raman signatures (peaks) to the spectral variance. To allow for
easily visualized data sets that can be quickly compared to a library
of reference spectra, a better suited method is Multivariate Curve
Resolution-Alternating Least Squares (MCR-ALS). For the analysis of
the Excedrin® tablet, MCR-ALS was applied to the Raman microscopy
mapping with the assumption of a three-component system due to
the 3 APIs expected in the system. Spectral pre-processing included
standard normal variant (SNV) followed by Savitzky-Golay smoothing
(0 order, 7 point) and then a baseline correction with a Whittaker filter
(asymmetry=0.001, lambda=100). Since the data obtained is resulting
from Raman mapping, the components can not only be extracted,
but also visualized spatially to reveal the location of each individual
component across the tablet surface (Figure 1B).
The MCR-ALS analysis neatly highlights three unique components
which can be compared to reference library spectra (Figure 1C). When
these MCR loadings are overlaid with expected spectra for the APIs that
was obtained from the USP reference standards, all three components
can be readily identified. With component 1 shown in red on the MCR
scores image resulting from the acetaminophen and component 2
shown in green resulting from caffeine in very good agreement with
Figure 1. Low-frequency Raman mapping of Excedrin®
tablet. A: Optical montage (bottom left: photo of tablet
prior to splitting) B: MCR 3 component composite
scores image. C1-3: MCR component loadings alongside
corresponding reference spectra for API of interest.
www.americanpharmaceuticalreview.com | | 49
» SPECTROSCOPY »

the USP reference spectra for those two APIs. Component 3 shown in
blue indicates a good agreement with aspirin, however an additional
shoulder is observed at ~140 cm-1 that isn’t present in the reference
spectra for aspirin. This additional peak is likely due to an excipient
present in the Excedrin® tablet that isn’t included in the model that
was built since the limitations were set to three components. These
labeled excipients include benzoic acid, carnauba wax, FD&C blue No.
1, hydroxypropylcellulose, hypromellose, microcrystalline cellulose,
mineral oil, polysorbate 20, povidone, propylene glycol, simethicone
emulsion, sorbitan monolaurate, stearic acid and titanium dioxide.
However, due to the expected amorphous nature of the majority of
these excipients, they are unlikely to contribute well defined features
in the low-frequency region, as opposed to the forest of spectral
signatures that would be expected in the traditional fingerprint
region. Thus, this helps to highlight the ability of low-frequency
Raman mapping to provide API distributions across the tablet surface
with minimal interference from the excipients present.

Polymorph Identification
In addition to rapidly identifying and mapping API location across
the surface of a pharmaceutical product, low-frequency Raman is
capable of identifying changes that may occur in the API due to
polymorphism or co-crystallization which may negatively affect the
product performance. The low-frequency Raman region is a sensitive
screen for polymorph changes due to the impact of changes in the
lattice vibrations on the low frequency spectra.
Figure 2. Low-frequency Raman mapping of two crystals
observed in a TDDS. A: 20x optical montage (insert: 100x
montage of mapped area). B: PCA scores image for PC1.
C: Average spectra that had positive PC1 scores labeled as
“red” crystal and average spectra corresponding to negative
on PC1 labeled as “blue” crystal along with the reference
spectra for the API used to manufacture the TDDS.

For example, a transdermal delivery system (TDDS) with visual crystal

growth was investigated with low-frequency Raman mapping.
Specifically, an area where two crystals were present side-by-side and
visually had an observable contrast (Figure 2A). The two crystal area
was mapped using an 100x objective with three second accumulations
using three micron step sizes for a data set comprised of ~20k spectra.
For this type of data set, PCA was applied due to the unknown
nature arising from the observed crystals, in contrast to Excedrin®
where MCR was expected to comprehensively resolve the three API
system. Spectral pre-processing entailed standard normal variant
(SNV) followed by Savitzky-Golay smoothing (0 order, 7 point) and
mean centering. Principle component (PC) 1 explained 62.34% of the
variance observed and from the scores image for PC1 (Figure 2B), could
be attributed to the differences between the two crystals resulting in
one having a high score for PC1 (very “red”) and one having a negative
score for PC1 (very “blue”), with the surrounding TDDS matrix having
a score close to zero. To represent this visually, the average spectra for
the “red” crystal was compared with that of the “blue” crystal allowing
for the key spectral differences to be observed. These spectra were
then compared to the reference spectra for the expected API and did
not show good agreement with the spectrum of either crystal that was
formed in the TDDS (Figure 2C).
These preliminary results indicated that either the API had a
polymorphic conversion when the API crystallized out of the TDDS
formulation or that the API co-crystallized out with an excipient used
to prepare the TDDS, such as the adhesive, causing the observed
changes in the expected API spectra. Thus, low-frequency Raman
was able to detect the difference in two crystals approximately 200
microns in size and less than 100 microns apart. To perform this using
traditional approaches for polymorph identification such as X-ray
powder diffraction or differential scanning calorimetry would not be
feasible with the current state of the art for such instrumentation.
Effect of Crystal Orientation
The third system analyzed was Zovirax® cream which has a labeled
acyclovir API concentration of 5%. Optical microscopy of this cream
showed rectangular crystals ranging in size from 10 to 80 microns
present amongst the white cream matrix. A low-frequency Raman
map was acquired on two crystals present side-by-side using a 100x
objective with 1-micron step sizes giving a data set of approximately
~1500 spectra (Figure 3A). Spectral pre-processing entailed SNV
followed by Savitzky-Golay smoothing (0 order, 7 point) and then
a baseline correction with a Whittaker filter (asymmetry=0.001,
lambda=100). A two-component MCR model was used and this
resolved two components that emphasized the differences between
the crystals (red) and the background matrix comprising the white
cream base (blue) (Figure 3B1). The MCR loadings are shown in Figure
3B2 where component 1 (blue) has a broad amorphous peak due
to the excipient matrix while component 2 (red) has well defined
features resulting from the crystals that are in good agreement with
low-frequency Raman for acyclovir reference standard (yellow). When
the MCR model was expanded to 4 components, the model showed

50 | | April 2019

Figure 3. Low-frequency Raman mapping of two crystals
observed in Zovirax®. A: Optical montage B1: MCR
scores image for 2 component MCR. B2: MCR loadings
for component 1 and 2, and acyclovir reference spectra.
C1: MCR scores image for 4 component MCR (showing
components 1 and 4). C2: MCR loadings for components
1 and 4 colored according to MCR image.
the two crystals with two distinct spectra. The MCR scores image for
components 1 (green) and 4 (blue) show two distinct crystals (Figure
3C1) with the primary differences in the MCR loadings being attributed
to a peak at 37 cm-1 for the “blue” crystal and a shoulder at 90 cm-1 for
the “green” crystal (Figure 3C2).
Upon initial investigation, these changes were attributed to a
polymorphic difference between the two crystals, however further
analysis of multiple crystals revealed that this difference was due to
the orientation of the crystals. Because of the directional and polarized
nature of the laser beam used for excitation and the rectangular shape
of the acyclovir crystals, excitation was being obtained either along
the horizontal or vertical axis resulting in distinct low-frequency
Raman peaks from horizontal or vertical crystal orientation. While
crystal orientation will not change the performance of the API, the
knowledge of the impact of orientation is important to note to avoid
false assumptions of presence of polymorph when none are present.
Importantly, the orientation sensitivity can be avoided entirely if the
laser beam is depolarized prior to being focused on the sample.
Conclusions
In summary, the capabilities of low-frequency Raman mapping in
the characterization of APIs in a variety of pharmaceutical products
was demonstrated in three case studies. By utilizing the strong low-
frequency Raman signatures that arise from crystalline API, rapid
data acquisition and high selectivity of the low-frequency region for
the API over excipients (which tend to be more amorphous in nature
resulting in weaker low-frequency Raman signatures), identification,
« SPECTROSCOPY »
size and distribution determination of crystalline components in
pharmaceutical products was established. Additionally, the low-
frequency spectral region was shown to be particularly sensitive to
polymorphic changes in the API. When coupled with multivariant
analysis techniques such as MCR and PCA, low-frequency Raman
maps allowed changes in the API to be visualized across the product
surface. With this technique, critical quality attributes, such as tablet
homogeneity of API location and form, could be rapidly assessed for a
wide range of drug products.
Acknowledgements
This project was supported, in part, by an appointment (H.Y.) to the
Research Participation Program at the Center for Drug Evaluation
and Research administered by the Oak Ridge Institute for Science
and Education through an interagency agreement between the U.S.
Department of Energy and the U.S. Food and Drug Administration.
We would also like to acknowledge Dr. Anjan Roy from Ondax, Inc.
for his valuable insight in discussions related to this project and Dr.
Ahmed Zidan from FDA/CDER/OPQ/OTR/ Division of Product Quality
Research (DPQR) for his assistance with the transdermal studies.
References
1. Ewing, A. V.; Kazarian, S. G., Recent advances in the applications of vibrational
spectroscopic imaging and mapping to pharmaceutical formulations. Spectrochimica Acta
Part A: Molecular and Biomolecular Spectroscopy 2018, 197, 10-29.
2. Gordon, K. C.; McGoverin, C. M., Raman mapping of pharmaceuticals. International Journal
of Pharmaceutics 2011, 417 (1), 151-162.
3. Smith, G. P. S.; McGoverin, C. M.; Fraser, S. J.; Gordon, K. C., Raman imaging of drug delivery
systems. Advanced Drug Delivery Reviews 2015, 89, 21-41.
4. Socrates, G., Infrared and Raman characteristic group frequencies: tables and charts. John
Wiley & Sons: 2004.
5. Otaki, T.; Tanabe, Y.; Kojima, T.; Miura, M.; Ikeda, Y.; Koide, T.; Fukami, T., In situ monitoring
of cocrystals in formulation development using low-frequency Raman spectroscopy.
International Journal of Pharmaceutics 2018, 542 (1), 56-65.
6. Mah, P. T.; Fraser, S. J.; Reish, M. E.; Rades, T.; Gordon, K. C.; Strachan, C. J., Use of low-
frequency Raman spectroscopy and chemometrics for the quantification of crystallinity in
amorphous griseofulvin tablets. Vibrational Spectroscopy 2015, 77, 10-16.
7. Hisada, H.; Inoue, M.; Koide, T.; Carriere, J.; Heyler, R.; Fukami, T., Direct high-resolution
imaging of crystalline components in pharmaceutical dosage forms using low-frequency
raman spectroscopy. Organic Process Research & Development 2015, 19 (11), 1796-1798.
8. Larkin, P. J.; Dabros, M.; Sarsfield, B.; Chan, E.; Carriere, J. T.; Smith, B. C., Polymorph
characterization of active pharmaceutical ingredients (APIs) using low-frequency raman
spectroscopy. Applied Spectroscopy 2014, 68 (7), 758-776.
9. Walker, G.; Römann, P.; Poller, B.; Löbmann, K.; Grohganz, H.; Rooney, J. S.; Huff, G. S.; Smith,
G. P. S.; Rades, T.; Gordon, K. C.; Strachan, C. J.; Fraser-Miller, S. J., Probing pharmaceutical
mixtures during milling: The potency of low-frequency raman spectroscopy in identifying
disorder. Molecular Pharmaceutics 2017, 14 (12), 4675-4684.
10. Wang, H.; Boraey, M. A.; Williams, L.; Lechuga-Ballesteros, D.; Vehring, R., Low-frequency
shift dispersive Raman spectroscopy for the analysis of respirable dosage forms.
International Journal of Pharmaceutics 2014, 469 (1), 197-205.
www.americanpharmaceuticalreview.com | | 51
» ROUNDTABLE »
Raw Materials and
Functional Excipients
Roundtable
In your opinion what are the most important
properties raw materials/functional excipients can
give to finished pharmaceutical products? Why?
Bikash Chatterjee, President and Chief Science Officer, Pharmatech
Associates: The contribution of functional excipients has greatly in-
creased over the last decade. The key impacts made by functional ex-
cipients in finished products include solubility and range in some cases
from drug efficacy to masking taste and extending product shelf life.
Susan Burke, PhD, Director Process Development - Materials
Science; Jackie Milne, PhD, Principal Scientist; Ron Kelly, PhD,
Principal Scientist; Ting Wang, PhD, Senior Engineer; Patrick
Gammell, PhD, Executive Director Process Development, Amgen:
Amgen is an attribute led, patient-centric organization. We design
for the patient; therefore, our excipients are important for ensuring
we can to deliver to our target product profile. Excipients serve
many important roles in the production of finished pharmaceutical
products. They are selected for the specific functionality they afford
to the formulation such as providing the right osmolality or protection
of the therapeutic molecule from shear stresses. The excipients must
be inert to avoid impact to the stability and the pharmaceutical profile
appropriate for the intended use of the finished drug product. In
addition, compendial compliance ensures that the excipients meet
the quality standards for use in pharmaceutical products. Consistent
performance and quality of the excipients is important for optimal
efficacy at the time of administration of the finished drug product.
Adhering to those standards supports our ability to delivery patient-
centric products to the market.
52 | | April 2019
Bikash Chatterjee Jackie Milne, PhD
President and Chief Science Officer Principal Scientist
Pharmatech Associates Amgen
Susan Burke, PhD Ting Wang, PhD
Director Process Development
Materials Science, Amgen
Senior Engineer
Amgen
Ron Kelly, PhD Mark Mitchell
Principal Scientist
Amgen
Principal Engineer
Pharmatech Associates
Patrick Gammell, PhD
Executive Director Process Development
Amgen
As the pharmaceutical market moves toward
more specialized treatments, what benefits can
raw materials and functional excipients provide to
these new types of drugs?
Chatterjee: One area of innovation we see now involves excipients
that are co-processed as part of the API manufacturing process,
resulting in API formulations that can be more easily manufactured.
One significant area where the impact of functional excipients is seen
is in biologic drug and vaccine development. Biologics are susceptible
to degradation from both storage and processing conditions. Newer
functional excipients can stabilize these products that are prone to
destabilization from a variety of mechanisms.
Burke, Milne, Kelly, Wang, Gamell: Raw materials, both non-
compendial and compendial excipients, will continue serve as
enablers for ensuring stability and delivery of therapies for patients, as
these components are essential for the development of such products.
However, for novel non-compendial materials, a paradigm change is
required to be able to meet the needs of specialized medicines. A shift
to a more attribute-focused approach for materials is warranted. Like
the Quality Target Product Profile (QTPP), a Material Target Attribute
Profile (MTAP) can serve as a framework for capturing information on
raw material performance requirements, relevant material attributes,
impact on process and product performance, target attribute ranges,
and control strategies. This approach provides a better understanding
of the influence of raw material variation for enhanced reliability in
manufacturing and product quality. An attribute-centric approach
to raw materials ensures that the attributes important for the

development of specialized medicines are identified and controlled,
which will support patient-centric outcomes.
What are some important attributes a supplier of
raw materials/functional ingredients must have
in order to meet the product demands of the
marketplace?
Mark Mitchell, Principal Engineer, Pharmatech Associates: As the
pharmaceutical industry embraces Quality by Design, a key element
of process understanding is how raw material variability impacts drug
product quality. Providing a raw material that simply “meets spec” is
not sufficient in this new paradigm. A supplier should have intimate
knowledge of their product (i.e., raw material/functional ingredient)
in terms of its process control and capability relative to its quality
attributes. By providing this data to drug product manufacturers
during product design and development, the criticality of that
raw material/functional excipient to finished drug product can be
appropriately evaluated by either design of experiments or risk
assessment. Knowledge of raw material variability when moving from
small-scale to commercial scale processes allows for more predictable
drug quality as product quality issues due to unexpected changes in
raw materials can be avoided.
Burke, Milne, Kelly, Wang, Gamell: To keep pace with the increasing
demands of the pharmaceutical industry, raw material suppliers must
place emphasis on ensuring they develop strong partnerships with
pharmaceutical manufacturers and actively engage in seeking the
voice of customer. It is imperative they understand the requirements
for raw materials to be used in a regulated environment. To deliver on
the needs of the pharmaceutical industry, suppliers must have a strong
quality management system and a philosophy of transparency as their
foundation. It has become increasingly important for suppliers to
implement robust monitoring programs including statistical process
control strategies and the capability to readily transfer raw material
data to their customers. This must be supported by depth and breadth
of technical knowledge, testing capabilities, and engagement with
their supply chain partners.
As the globalization of the pharmaceutical industry
continues – what must ingredient suppliers do
to ensure the quality of their products and the
availability of same?
Chatterjee: Over the last decade the emerging markets in particular
have stumbled badly in terms of ensuring excipient purity,
consistency, and functionality. To be competitive, investing in process
understanding will become essential, especially as the complexity and
sophistication of new drug delivery forms continues to escalate.
Burke, Milne, Kelly, Wang, Gamell: Supply-chain transparency is
of vital importance in an age of increasing globalization. The parties
representing each link of the supply chain need to be open and
« ROUNDTABLE »
appreciative of their roles. A pharmaceutical company must be
knowledgeable about their raw material supply with traceability
throughout each level of supply chain, as far as is necessary. This is
critical to both ensuring supply continuity in the event of a local
disaster and supply-chain integrity in the event of a deliberate attempt
at adulteration.
Transparency leads to increased predictability. Information about
changes introduced to a material allow for an understanding of the
primary sources of variation, thus enabling us to predict the outcome
of pharmaceutical manufacturing. Communicating such changes
clearly allows for better understanding and control over potential
downstream impacts.
The pharmaceutical industry is adopting best practices, such as
developing robust supplier-relationship and detailed technical
engagement, to enhance raw materials reliability. Another important
element for advancing knowledge of raw materials is the transfer and
analysis of suppliers’ data. A standard guide for electronic data transfer
(ASTM E-3077-17) from supplier to pharmaceutical manufacturers
has been established in 2017. Data analytics can facilitate enhanced
engagement between the pharmaceutical industry and suppliers,
through the sharing of the analysis to suppliers for mutual learning
and improvement. The enhanced collaboration requires improved
standardization in key areas such as terminology, units, SKUs etc.
Looking at the future, what role will suppliers
of raw materials/functional ingredient have as
drug development becomes more complex and
more specialized?
Chatterjee: We will see more drug development companies looking to
partner with key functional excipient suppliers to produce consistent
and ready-to-process APIs. Continuous manufacturing itself is hugely
API dependent and demands a steady and reproducible product to
realize the long-term benefits of such new innovative approaches to
drug production.
Burke, Milne, Kelly, Wang, Gamell: As drug development continues
to evolve, so too must the relationship with raw material suppliers.
Strong partnerships built on trust and a commitment to transparent
communication are a must for keeping pace with advancements in the
pharmaceutical industry. With the globalization of supply chains and
increasing complex distribution channels throughout the world, raw
material suppliers must enhance the resilience of their organizations
by building robust business continuity strategies that take into
consideration the potential for unforeseen challenges such as natural
disasters. In addition, suppliers must continue to strengthen their
supply chains, control strategies, and reliability of their materials. The
increased complexity of drug development a deep understanding
of raw material variation is of great importance. Suppliers that
employ advanced data analytics to monitor their material attributes,
in combination with robust process control strategies, will be well
equipped to support their customers in the pharmaceutical industry.
www.americanpharmaceuticalreview.com | | 53
» »
SECTION TITLE

Pharmaceutical

P .N.
P.I N Suspensions of Cyclosporin A Form 2;

Points
Points
Patent Innovation News
A.V. Gore, P.S. Mohanty, and
E.Q. Farnes; Allergan, Inc., USA;
U.S. Patent # 10,206,971, February 19, 2019.
Cyclosporin A is available in the market as an ophthalmic emulsion
under the brand name Restasis. It is indicated to increase tear
production in the treatment of dry eyes. Due to cyclosporine A’s poor
water solubility, it is currently formulated by dissolving it in oil or by
The purpose of this column is to highlight using high levels of surfactants and solubilizers. The former leads

Article Title
to forming an emulsion and the later forms an aqueous solution
and summarize recent key patents in the
pharmaceutical arena issued by the US
Introduction
of cyclosporine. The inventors of the present invention discloses a
nanosuspension formulation of cyclosporine A using its crystalline
Patent Office in February 2019. Paragrapgh
polymorph.TextThis invention may provide a better dosage form of
cyclosporine A. The inventors claim that the nanosuspension may
have advantages such as higher bioavailability, longer retention
Anvit Vasavada, MS, Sunny Christian, MS (RA), time in eye, improved tolerability etc. over the current formulations.
Neelam Sharma, MS and
Hemant N. Joshi, PhD, MBA*
Tara Innovations, LLC
www.tarainnovations.com
*hemantjoshi@tarainnovations.com

Orally Disintegrating Tablet Formulation

Packaging
Author Name System for Oxygen-
Author Title
Sensitive Drugs;
Author Company
T. Devouassoux, E. Forat, and J.K.
Proctor; Fresenius Kabi Deutschland
GmbH, DE; U.S. Patent # 10,214,338;
February 26, 2019.
Present invention provides a packaging system for
oxygen sensitive drugs in injectable dosage form. It
claims to prevent degradation of drugs by oxidation. In
pharmaceutical development, oxygen sensitive drugs
and their formulations require additional development to
prevent degradation during manufacturing and storage.
The packaging system disclosed in the patent uses a
syringe with a cap to accommodate the oxygen sensitive
drug. The syringe is hermetically sealed in a blister pack
with an oxygen absorber. The inventors claim that the
packaging may realize zero percent oxygen levels in the
blister and syringe for at least a year.
for Enhanced Bioavailability;
J. Djordjevic, M.M. Bommana,
W. Phuapradit, N.H. Shah, and
C.A. Pizzo; Kashiv Pharma, USA;
U.S. Patent # 10,195,150; February 5, 2019.
An orally disintegrating tablet (“ODT”) dosage form has certain
important requirements in addition to the proper disintegration
times. The perception that rapid disintegration leads to rapid
rates of absorption and bioavailability does not hold true with
poorly water-soluble drugs. Therefore, following dosage form
disintegration, it still is necessary for the drug to dissolve before
it can be absorbed. It would be advantageous to simultaneously
provide rapid disintegration and a drug solubility enhancement
in a dosage form. This patent invention gives a formulation
comprising of an amorphous solid dispersion or a mixture of a
poorly water-soluble drug and an ionic polymer, which exhibits fast
disintegration of tablet and enhanced dissolution/bioavailability.
The ionic polymer is present in an amount so as to maintain the
poorly water -soluble drug in a substantially amorphous form and
disintegrate the tablet within 30 seconds.

54 | | April
November/December
2019 2011

Three-Component-Multistage
Malaria Vaccine;
H. Spiegel, A. Boes, G. Edgue, V. Beiss,
M. Sack, A. Reimann, and R. Fischer;
Fraunhofer-Gesellschaft zur Foerderung
der angewandten Forschung, DE;
U.S. Patent # 10,213,501; February 26, 2019.
Most vaccines are produced to target a single antigen only.
Producing a successful vaccine against malarial parasites is
challenging because of its complex and multistage life cycle.
Depending on the developmental stage, the parasite displays
different sets of surface antigens, which exhibit extensive
sequence polymorphism that helps them evade an immune
response. The present invention provides novel and improved
multicomponent, multi-stage vaccines composed of different
recombinant proteins, comprising several different Plasmodium
falciparum antigens from the pre-erythrocytic, the blood, and the
sexual parasite main stages.
Salts and Prodrugs of 1-methyl-
D-tryptophan;
M. Mautino, S. Kumar, F. Jaipuri, J.
Waldo, H. Potturi, and H. Zhuang;
NewLink Generics, USA; U.S. Patent #
10,207,990; February 19, 2019.
Indoximod is 1-methyl D-tryptophan. IDO1 (indoleamine-
2,3-dioxygenase) catalyzes the degradation of tryptophan
(Trp) into kynurenines, which plays an important role in
the regulation of immune responses by triggering anergy
(absence of the normal immune response to a particular
antigen or allergen) on reactive effector T cells and by
modulating differentiation and activation of regulatory T
cells (Treg). Indoximod has been demonstrated to relieve
IDO-mediated immunosuppression in vitro and in vivo.
Indoximod is orally bioavailable and provides satisfactory
pharmacokinetic parameters up to 800 mg/dose, which
produces Cmax value of 15 µM. However, the PK profile is
non-linear above this dose. One needs Cmax value of 20
µM or higher for therapeutic activity. By using the salts and
prodrugs of Indoximod, the linearity of PK profile was shown
to extend to higher doses.
« SECTION TITLE »
Topical Tetracycline Compositions;
D. Tamarkin, E. Gazal, I. Papiashvili, Y.
Hazot, D. Schuz, and R. Keynan; Foamix
Pharmaceutical, USA;
U.S. Patent # 10,213,512; February 26, 2019.
Tetracyclines are broad-spectrum antibiotics, which are used
orally for the treatment of dermatological conditions, such as
acne and rosacea. However, despite their high therapeutic value,
tetracyclines are very unstable and incompatible with many
formulation excipients like water, protic substances and oxidizing
agents. This patent relates to oleaginous gel formulations, foam-
able formulations and foams comprising tetracycline, which are
stable. A topical therapeutic hydrophobic breakable composition
includes a carrier comprising 60%-99% (by weight) of one or
more hydrophobic oil, one or more viscosity-modifying agents
selected from the group of fatty alcohol, fatty acid and wax and a
tetracycline antibiotic, where a part of the tetracycline antibiotic
is suspended in the composition. The viscosity of the composition
is ≈ 30% higher than that of the carrier without the tetracycline
antibiotic. Tetracycline is chemically stable in the composition
for at least six months where > 90% of the tetracycline does not
break down. This composition is packaged as a breakable foam
that breaks easily upon application of shear force.
Dry Powder Compositions with
Magnesium Stearate;
K. Yadidi; Otitopic, USA;
U.S. Patent # 10,195,147; February 5, 2019.
Magnesium stearate (MgST) is the magnesium salt of stearic
acid. It is widely used in the production of nutraceuticals and
pharmaceuticals. MgST is known to catalyze acid-based hydrolysis
often causing incompatabilities with active pharmaceutical
ingredients (APIs) susceptible to acid-based hydrolysis such as
non-steroidal anti-inflammatory drugs (NSAID), e.g., acetylsalicylic
acid (ASA). The present invention discloses a stable dry powder
composition for inhalation, which includes ASA and stearate. This
inhaled route of drug administration provides fast and efficient
treatment for reducing the risk of a thromboembolic event.
Formulation contains dry particles with mass median aerodynamic
diameter in a range of about 1 µm to about 5 µm.
www.americanpharmaceuticalreview.com | | 55
» ADVERTISER'S INDEX »

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Lyophilized Drug Product Development Ogunleye
Wolfe Laboratories

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Company Page # Web Address

Advantapure 27 www.advantapure.com/apr3
Ajinomoto Bio-Pharma Services CV3 www.AjiBio-Pharma.com
Albemarle 21 www.ALBcustom.com
Associates of Cape Cod 5 www.acciusa.com
BIO International Convention 43 convention.bio.org/2019
Biomerieux 29 www.biomerieux-usa.com
Cambrex 19 www.cambrex.com
Catalent CV4 www.catalent.com/art
Charles River 15 www.criver.com/celsis
Clinical Trial Supply West Coast 2019 35 www.arena-international.com/ctswest/programme
Eurofins Lancaster Laboratories 33 www.EurofinsLancasterLabs.com
Excipient World Conference & Expo 23 www.ExcipientWorld.org
JOST Chemical Co 1 www.jostchemical.com
Lonza Pharma & Biotech 7 pharma.lonza.com
Millipore Sigma 9 SigmaAldrich.com/SAFC
PERFEX 13 www.PERFEXONLINE.com
PTI  41 www.ptiusa.com
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The contact directory is for information purposes only. While every effort has been made to ensure it is accurate and complete, any errors or omissions are not the responsibility of the publisher.

56 | | April 2019
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