bioassay

evolution and
lessons learned
Tom Luntz
13.03.15

Agenda

Why a Bioassay?
Bioassay Evolution
Data trending
examples
Lessons Learned:
Basic tools to prepare
your bioassay for QC
Readiness

Why a Bioassay?
Well-Characterized Biopharmaceutical Product
Identity

Size

Charge

1/2/3/4 Structure

post-translational modifications

Quantity
Purity/Impurities

Degradation Pathways (Storage in Buffer)

Potency

Correlate with In Vivo Characteristics

Pharmicokinetics
Metabolism
Toxicology/Immunogenicity

Why a Bioassay?
Well-Characterized Biopharmaceutical Product
Each of these aspects will have a different
combination of analytical techniques to describe it.

To avoid the potential gaps inherent in any particular

analytical technique, the molecule characterization
should be based upon complementary or orthogonal
testing systems/modalities.

Why a Bioassay?
Analytical Techniques
Physicochemical Analyses

Characterize the size, charge and structure of the molecule.

Able to characterize the purity/heterogeneity of a given preparation.

naturally occurring variants

For biocomparability, highly effective in establishing the degree to

which the compounds being tested have the same chemical structure
and identity

Physicochemical analyses are not able to assess product potency.

Why a Bioassay?
Analytical Techniques
POTENCY:
Potency is a measure of the ability of a material to elicit its
function.

If that ability is to induce a biological response, then a potency assay

should be a bioassay.

For some products (e.g. monoclonal antibody therapeutics), binding

assays may suffice as a potency assay, especially if binding to its
target is that molecules chief known mechanism of action.

BIOASSAY:

WHO/NIBSC, J. Immunol. Methods (1998), 216, 103-116.

International consensus, Dev. Biol. Standard. (1999) vol 97:

"A bioassay is defined as an analytical procedure measuring a

biological activity of a test substance based on a specific, functional,
biological response of a test system"

Why a Bioassay?
Physicochemical Assays vs. Bioassays
Physicochemical assays (e.g., HPLC) define and quantify the
protein content

Protein Concentration

Type and number of peaks that define product

Will determine clinical dosing

Values are in terms of weight/volume

Bioassays/Potency Assays define and quantify protein

appropriateness

Quantification of fitness for use

Will not be used to determine dosing

Rather, is the content, used for dosing, valid

Values are in terms of activity/weight or activity relative to a

standard

Bioassay Evolution
Why does it evolve at all?
Bioassay Development
Reduce Variability
Bioassay Qualification (Characterization)

Accuracy
Investigation

Precision

Range
Linearity

Specificity

Criteria Failed

Specifications
Defined
&
More
Regulatory
Guidances

Bioassay Validation
All Criteria Met

Implementation

Technical Transfer

Bioassay Evolution
Basic Timeline
Preclinical
Basic
Research

Development

Clinical: Phases I, II, III

Evaluation

Qualification

Post - Filing
Validation

Commercial
Release

Qualification (some call it Characterization)

After Development, not a regulated event but requires a Protocol,
QA involvement and Report
Prospective no expectations, set criterion after assessing results
Should be done for GMP assays before releasing product to clinic
Validation
Regulated Study Protocol & Reportno surprises!
Confirmation of bioassay performance with specified acceptance
criteria
QA Reviewed

Bioassay Evolution
Validation

ICH Q2(R1) is Showing its Age

Formerly ICH Q2A/Q2B, Parent
Guideline 1993/1994
Complementary Guideline on
Methodology November
1995/1996
Current Step 4 Version
incorporated Validation
Methodology in November 2005
Pre-dated QbD and equivalence
testing

Bioassay Evolution
USP Guidance
USP <1032> Design and Development of Biological Assays
USP <1033> Biological Assay Validation
USP <1034> Analysis of Biological Assays
Captured advances, improvements, and new concepts in
bioassay testing which had developed in the industry in the
absence of guidance from ICH requirements
Implementing <1032>, <1033>, and <1034> leads to the
evolutionary process described earlier.

Bioassay Evolution
Beyond Validation
Guidance for Industry
Analytical Procedures and Methods
Validation for Drugs and Biologics
Draft, February 2014
Section VIII.
Life Cycle Management of Analytical
Procedures
Recommends periodic evaluation of the
appropriateness of a products analytical
methods and to consider new or
alternative methods.
May require revalidation or analytical
method comparability studies

From DDW Online

Process Control Charts / Assay Trending

Average Potency = 99.9%

SD = 6.0%

With n = 50, 3.8 SD, 22.6%

Bioassay Performance TrackingAssay 1

Assay 1
Historical values from 4PL fits used to set acceptance
intervals
Track reference values (A, D, B) and Sample/Reference
ratios to detect drift over time.

A (Reference)
0.8000

0.6000

0.4000

0.2000
22-May-14

22-Apr-14

22-Mar-14

22-Feb-14

22-Jan-14

22-Dec-13

22-Nov-13

22-Oct-13

22-Sep-13

22-Aug-13

22-Jul-13

22-Jun-13

22-May-13

22-Apr-13

22-Mar-13

22-Feb-13

22-Jan-13

22-Dec-12

22-Nov-12

22-Oct-12

22-Sep-12

22-Aug-12

22-Jul-12

22-Jun-12

22-May-12

22-Apr-12

22-Mar-12

22-Feb-12

22-Jan-12

22-Dec-11

22-Nov-11

A(Sample)/A (Reference) Ratio

Bioassay Performance TrackingAssay 1

1.200

1.100

1.000

0.900

0.800

D(Sample)/D(Reference) Ratio

Bioassay Performance TrackingAssay 1

1.200
1.100
1.000
0.900
0.800

2.5000

D (Reference)

2.0000
1.5000
1.0000
0.5000

B (Reference)
2.2500

1.7500

1.2500

0.7500
22-May-14

22-Apr-14

22-Mar-14

22-Feb-14

22-Jan-14

22-Dec-13

22-Nov-13

22-Oct-13

22-Sep-13

22-Aug-13

22-Jul-13

22-Jun-13

22-May-13

22-Apr-13

22-Mar-13

22-Feb-13

22-Jan-13

22-Dec-12

22-Nov-12

22-Oct-12

22-Sep-12

22-Aug-12

22-Jul-12

22-Jun-12

22-May-12

22-Apr-12

22-Mar-12

22-Feb-12

22-Jan-12

22-Dec-11

22-Nov-11

B(Sample)/B(Reference) Ratio

Bioassay Performance TrackingAssay 1

1.2

1.1

0.9
1

0.8

Bioassay Performance TrackingAssay 2

Bioassay produces ELISA for readout

Assay encountered periods of high failure rate
Solving failure rate was reflected in greater reproducibility in assay
performance

Bioassay Performance TrackingAssay 2

Reference EC50
8

Concentration

7
6
5
4
3
2
1
0
-1

Bioassay Performance TrackingAssay 3

Bioassay currently in active repair mode

Most significant problem is assay failure, usually does not meet
requirement for amplitude of response, i.e., D/A
Analysis of historical data highlighted the most common symptom,
which will be a focus of investigationA values are too high.

Bioassay Performance Assay TrackingAssay 3

16
14
12
10
8
6
4
2
0

7000.0
6000.0
5000.0
4000.0
3000.0
2000.0
1000.0
0.0

Bioassay Performance TrackingAssay 4

Well-behaved system, so deviations over time should be relatively easy

to detect.

Relative potency (%)

140
120
100
80
60
40
20
0
20100730 TOL
20100803 TOL
20100813 TMS
20100817 TOL
20100827 TOL
20100903 TMS
20100914 TMS
20100924 TMS
20100928 TMS
20101005 TMS
20101015 JAS
20101022 JWP
20101102 JAS
20101105 TOL
20101116 JWP
20101203 JAS
20101210 JAS
20110118 JAS
20110121 TOL
20110208 JAS
20110211 JAS
20110311 JAS
20110311 JAS
20110315 JAS
20110318 JAS
20110318 JWP
20110322 JAS
20110322 JAS
20110329 JAS
20110401 JAS
20110405 JAS
20110405 JAS
20110405 JWP
20110408 JAS
20110412 JAS
20110415 JWP
20110419 JWP
20110510 JWP
20110513 JWP
20110520 JAS
20110607 JWP
20110610 JWP
20110722 JWP
20110722 JWP
20110719 JAS
20110913 JAS
20110916 JWP
20111014 JWP
20111014 JAS
20111021 JAS
20111209 JWP
20111213 JAS
20111216 JWP
20120207 JAS
20120214 JAS
20120309 JWP
20120316 JAS
20120320 JWP
20120323 JWP
20120427 JWP
20120501 JAS
20120508 JAS
20120605 JAS
20120612 JAS
20120615 JAS
20121204 JAS
20121207 JAS
20130122 MLS
20130305 LS
20130308 JAS
20130611 JAS
20130614 MLS
20130806 JAS
20130816 JAS
20131022 JAS
20131029 ADW
20131108 ADW
20131115 ADW
20131119 JAS
20131224 ANP
20140204 ANP
20140210 ADW
20140314 ANP
20140321 ADW
20140325 ANP
20140718 ADW
20140829 ADW
20140905 ANP
20141007 ANP

Doubling Time (Days)

Bioassay Performance TrackingAssay 4

2.5

2.0

1.5

1.0

0.5

0.0

Assay Number

QC sample
Mean - 2STD

Assay

Mean + 2STD
Grand Mean

Lessons Learned
Basic tools that need to be controlled

Analyst Training
Documentation
Equipment
Critical Reagent Care
Cells
All sources of variability

Preparation

Implementation

Review

Analyst Training
Use a Training SOP to document number of bioassay
runs and results expectations.
Start with simple assays using the Reference Standard or
Training Samples.
Training Samples can be made from previously tested lots.

Train up to a standard/routine bioassay run.

What is the expectation regarding the size of a
standard/routine bioassay run?

Label as Training Samples with newly coded identification

numbers.

Three plates with Reference Standard, Controls and six samples?

Train as you will perform with fully burdened bioassay runs that
include the Reference Standard and previously tested controls.

Does the training data fall within the range described in the
Training SOP?

Analyst Training
Additional considerations for cell-based bioassays
Specific cell culture SOP training
Culture examples/ pictures, know/ understand pitfalls
What do good and bad cultures look like?
Master the cell culture methods prior to performing the
bioassay
Practice seeding cells in plates with multichannel pipets and/
or conduct specific training on liquid handling machines

Analyst Training
Maintenance Assays
How are the potentially long periods between lot
release, stability sample assays addressed?
Does each analyst performs periodic (monthly)
Maintenance Assay with RS and one sample
from a Proficiency Panel?

Not just the 100% sample or RS; dig out those

Training Samples.

Analysts may alternate months.

Maximum sample numbers periodically.

Documentation
Is there a method from the R&D lab?
Does an SOP exist?
Start your development process with laboratory
notebookswrite everything downand establish an
SOP as you gain confidence in the method.
Once you have a draft SOPstart filling in the details:
Very inclusive list of reference SOPs
Specifics at every time point 30 min
Specifics for every liquid handling step

0.025 ml transfer with a 0.1 ml pipette and a 0.1 ml tip

1.0 ml transfer with a 1.0 ml pipette and a 1.0 ml tip

Vs. a 1.0 ml pipet or 5 ml pipet, or 10 ml pipet

Documentation
Write a Training Protocol

No guidance on specific number of bioassays

Include transfer acceptance criteriabe prepared to

move the assay

Specify raw materials and consumables

Define methods

List equipment

Timing difficult task vs. meeting manufacturing

timeline

Equipment

Certified, qualified, or mapped?

Equipment is Certified using standard setting
equipment which has a known valid relationship to
a recognized national or international standard
Certification is used as an alternative concept to
calibration in ISO standards
Certification is usually performed by the equipment
manufacturer
Examples of certified equipment
pipettes,

pH meters, thermometers,
centrifuges, biosafety cabinets

Equipment
Installation/Operational/Performance
Qualifications (IOPQ)
Plate readers/spectrophotometers, cell counters
Flow cytometer, surface plasmon resonance
system, aggregometer
Data analysis software
Temperature mapped equipment
Incubators; including the room temperature ones
Freezers
Refrigerators
Vapor phase liquid nitrogen storage

Equipment
Do you have enough equipment?

Are analysts waiting in line at the plate reader?

Incubators

Air flow, HEPA filtered?

Humidity level (>90%), supplemental humidification

CO2 control: infrared or thermal conductivity backed up with

Fyrite (classic/absorbing fluid) or portable/electronic

Space to separate cultures from bioassays

Critical Reagent Care

Reference Standard (RS)

Make sure there is enough (originating Dept/Lab retains)

May or may not be well characterized, can be non-GMP

May evolve along with product development

May not be the same formulation as the drug product

Ship from originating lab to recipient lab in batches

Monitor stability freeze/thaw bioassay + 2nd method?

Document stability as part of a protocol

Monitor results with Statistical Process Control (SPC) chart

Qualify new/next RS lot with a protocol

Characterize with all methods (bioassay + analytical)

How many bioassay runs constitute an assignable value?

Critical Reagent Care

Identification of Critical Assay Reagents
FBS
Growth Factors
Viable cell indicators, other assay readout materials
Cell maintenance reagents (cell dissociation, medium
components)
Others?
Reagent preparation/storage/handling
Qualification procedures

Cells
Cell characterization, even from repositories
Cell banks for consistency, preparation documented
Monitoring and trending of viability and growth
characteristics
Cells tend to amplify bioassay variability;
not only during bioassay development,
but while maintaining and transferring
bioassays.

Basic Lessons Learned

Invest in training

Cell culture and general observation

Data analysis
Communication

Know your cells

Document everything
Monitor your equipment
Work with a biostatistician
Establish Process Control Charts

Advanced Lessons Learned

Underperforming bioassays may slow clinical trials and/or
manufacturing timelines.
ICH Q2(R1) guides multi-national production and acceptance and is
showing some age

Does provide guidance for both validation and qualification

Somewhat biased towards requirements of physicochemical analyses

USP <1033> does not provide any true guidance for assay
qualification (some aspects covered in <1032>), but it does
provide very useful and detailed guidance for validation of potency
assays.
Follow both ICH Q2(R1) and USP <1033>

Advanced Lessons Learned

Full and proper Qualification/Characterization of the assay is
essential before late stage ICH- or USP-defined validation is done

In other words, have your critical assay parameters well defined before
you plan a Validation.

Most common strategy is a protocol-driven qualification/validation, with

less detail and looser acceptance criteria than ICH- or USP-defined
validation

Understand the needs and requirements you have for the bioassay,
in relationship to the clinical or commercial phase for which you are
using it
Work with a statistician

Thanks
Current Cell-based Assay Team
Andrew Wallace

Project Management Pillar

Jayne Keifer

Ramsey Connor
Tami Conley

BPS QA Mainstay

Aisha Parker

Traci Brinson

Mayur Gajera
Kelly Mahaffey

BPS Manager

Sarah Wickman

Kristen Mark
Biologics Director
Mike Merges

Contact Information

Tom Luntz, Ph.D.

Group Leader
Biopharmaceutical Support Services
Development & Clinical Services
Catalent Pharma Solutions
RTP
Phone: +1 919-465-8136
Thomas.Luntz@catalent.com