Good Laboratory Practices

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- Introduction
- Requirements of GLP
- Stepwise GLP in QC
- Audit Findings
- Issues due to not following GLP
- Future Trends in GLP

Contents pharmauptoday@gmail.com
Introduction
 Introduction

Good Laboratory Practice (GLP) deals with the organization, process and conditions
under which laboratory studies are planned, performed, monitored, recorded and
reported.

GLP data are intended to promote the quality and validity of test data.

Weller gave an excellent practical explanation on what is expected from working in a

regulated environment:

If experimental work is conducted in compliance with GLP, with or without the aid of
computer, it should be possible for an inspector, maybe four or five years hence, to look
at the records of the work and determine easily why, how and by whom the work was
done, who was in control, what equipment was used, the results obtained, any problems
that were encountered and how they were overcome.
 Introduction

Good laboratory practice requires testing normal and abnormal controls for each test
atleast daily to monitor the analytical process.

For the quality of a medicine sample to be correctly assessed:

The submission of a sample of an API, excipient or pharmaceutical product or a

suspected counterfeit material to the laboratory, selected in accordance with national
requirements, should be accompanied by a statement of the reason why the analysis
has been requested.

The analysis should be correctly planned and meticulously executed.

The results should be competently evaluated to determine whether the sample complies
with the specifications or other relevant criteria.
 Introduction

Regulatory requirements, inspection and enforcement practices are quite dynamic.

What is appropriate today may not need to be appropriate tomorrow. Regulations
change but more often it is the inspection practices that change.

In the early 90s the focus of inspections was on basic requirements of GLP and GMP,
but then it changed to equipment hardware and later on to software and computer
systems.

Today, the clear focus is on Quality control, Microbiology, Laboratory controls, data
security, traceability and integrity of electronic records, driven mainly but not only by
FDAs regulation 21 CFR Part 11.
Requirements of GLP
Requirements of GLP
Requirements of GLP
Requirements of GLP
Stepwise GLP in QC
 Stepwise GLP in QC.

Lab Reagents ,
Chemicals Glassware Computers Sampling Microbiology Stability
Entry etc

Document Chromato
Lab Safety Instruments Standards Weighing Investigations
ation graphy

Lab Upkeep
Lab
Procedures
Log Measurin
Sample
Preparati
Lab
Behavior Books g Volume
on Exit
 Activity wise GLP in QC

People

QC ACTIVITIES
Sampling
LAB MANAGEMENT

RESOURCE MANAGEMENT
Lab entry& exit
Lab safety Procedures Weighing
Reagents, Measuring volume
Lab upkeep
Chemicals & Sample preparation
Lab behavior Solvents Chromatography
Standards Microbiology
Glassware Stability
Instruments Documentation
Log Books Investigations
Computers
Laboratory Entry
 Laboratory Entry

There should be Standard Operating Procedure (SOP) for laboratory entry.

Laboratory entry should be restricted to authorised persons only.

Before entering the Lab ensure

Dress properly during a laboratory activity
Long hair, dangling jewelry, and loose or baggy clothing are a hazard in the laboratory. Long
hair must be tied back, and dangling jewelry and baggy clothing must be secured.
Cut Nails, Shave your beard (for Gents)
Wash hands with soap solution
Wear proper gowning (Lab coat / Apron)
Wear Safety shoe & Shoes must completely cover the foot.
No sandals allowed in Laboratory.
Wear Goggles.
 Laboratory Entry

The entry procedure may vary depending on the nature of the laboratory (e.g.
Microbiology / QC laboratory of sterile plant / QC Laboratory of Solid dosages).

Laboratory should have complete laboratory layout at the entrance.

Do not allow any eatables / drinkables in to the laboratory.

Do not enter the laboratory in case of sickness.

Contact lenses may be not be worn in the laboratory.

Laboratory Health & Safety
 Laboratory Health & Safety

All the persons who work in laboratory must undergo regular health checkups.

All the persons must be trained on health hazards associated with chemicals and
protective measures.

Exposure to various chemicals / Drugs could cause a serious allergic reaction asthma,
and other respiratory problems.

These MSDSs on Infectious Agents are organized to contain health hazard information
such as infectious dose, viability (including decontamination), medical information,
laboratory hazard, recommended precautions, handling information and spill procedures.
 Laboratory Health & Safety

Before entering the laboratory it is mandatory to know the lab safety procedures
thoroughly.

All individuals must be trained on handling PPEs (Personal Protective

Equipments) and Lab Safety.

All must know the availability & usage of

Emergency Exit & Assembling point
Fire extinguishers & Fire Alarm
Emergency showers
Eye washers
First Aid kit etc.
Report any accident (spill, breakage, etc.) or injury (cut, burn, etc.) to the supervisor
immediately, no matter how trivial it seems.
 Laboratory Health & Safety

If you or your lab partner is hurt, immediately (and loudly) yell out the others
attention. Do not panic.

If a chemical splash in your eye(s) or on your skin, immediately flush with running water
for at least 20 minutes.

Avoid using electric extension cords whenever possible. If you must use one, obtain a
heavy- duty one that is electrically grounded, with its own fuse, and install it safely.
Extension cords should not go under doors, across aisles, be hung from the ceiling, or
plugged into other extension cords.

When using compressed air, use only approved nozzles and never direct the air towards
any person.

Exercise care when working with or near hydraulically- or pneumatically-driven

equipment. Sudden or unexpected motion can inflict serious injury.
 Laboratory Health & Safety
Treat every chemical as if it is hazardous.

Make sure all chemicals are clearly and correctly labeled with the substance
name, concentration, date, and validity.

Never return chemicals to reagent bottles.

Never allow a solvent to come in contact with your skin. Always use gloves.

Never pipette by mouth; use a bulb.

Clean up spills immediately.

Heated glassware remain very hot for a long time. They should be set aside in a
designated place to cool, and picked up with caution. Use tongs or heat
protective gloves if necessary.
 Laboratory Health & Safety

Do not work alone in the laboratory.

Do not touch the reagents / solvents / standards / drugs with bare hands.

Do not smell any reagent / solvent / standard / drug.

Do not run / rush in the laboratory.

Do not keep any article which blocks the passage / emergency exit.

Do not lift the weights / heavy bottles suddenly

Do not leave an on-going experiment unattended.

Do not handle broken glass with your bare hands. Use a brush and dustpan to clean up
broken glass. Place broken glass in the designated glass disposal container.
 Laboratory Health & Safety

Stop your work, or that of your colleagues when you are concerned or
suspicious of danger or unnecessary risk.

Keep the poisonous chemicals and reagents under lock & key and usage record
shall be maintained.

As per USP General Notices & Requirements:

Laboratory Upkeep
 Laboratory Upkeep

Keep your lab space clean and organized.

Maintain unobstructed access to all exits, fire extinguishers, electrical panels, emergency
showers, and eye washes.

Do not use corridors for storage or work areas.

Do not store heavy items above table height. Any overhead storage of supplies on top of
cabinets should be limited to lightweight items only. Also, remember that a 36" diameter
area around all fire sprinkler heads must be kept clear at all times.

Areas containing lasers, biohazards, radioisotopes, and carcinogens should be posted

accordingly. However, do not post areas unnecessarily and be sure that the labels are
removed when the hazards are no longer present.

Clean your lab bench and equipment.

 Laboratory Upkeep

Know the locations and operating procedures of all

safety equipment.
Clean up spills immediately.
Use volatile and flammable compounds only in a fume
hood. Procedures that produce aerosols should be
performed in a hood to prevent inhalation of hazardous
material.
Make sure all chemicals are clearly and currently labeled
with the substance name, concentration, date, and name
of the individual responsible.
Guards on machinery must be in place during operation.
Make sure that acids and bases are not stored together.
If there were to be a spill, chemical reactions could occur
if they are stored in the same storage area.
 Laboratory Upkeep

Make sure you check the MSDS of the acid or base to

determine you are not storing incompatible chemicals
together.

Be aware that all containers MUST be properly labeled so

that identification can be achieved as easy as possible.

Pay close attention to expiration dates, making sure that the

acid or base is disposed of on or before the expiration date.

Acids and bases should be stored in a Corrosive Cabinet.

Obtain and read the most current Materials Safety Data

Sheet (MSDS) for the specific acids and other chemicals
that you will be using.
 Laboratory Upkeep

Chemicals should not be stored in the lab haphazardly. Many chemicals are incompatible
with others and improper storage can be quite dangerous.

Organic compounds and inorganic compounds should be stored separately as well.

Many compounds have specific storage requirements and these are often listed on the
label.

The most common source of mercury in a lab is a broken thermometer. Once a

thermometer has been broken, quick and thorough clean up is required. Mercury is quite
volatile and the health effects are not trivial.

Corrosive liquids (e.g. mineral acids, alkali solutions and some oxidizers) represent a
very significant hazard because skin or eye contact can readily occur from splashes and
their effect on human tissue generally takes place very rapidly. Bromine, sodium
hydroxide, sulfuric acid and hydrogen peroxide are examples of highly corrosive liquids.
 Laboratory Upkeep

Liquid corrosives should be stored below eye level.

Corrosive gases and vapors are hazardous to all parts of the body; certain organs (e.g.
the eyes and the respiratory tract) are particularly sensitive. The magnitude of the effect
is related to the solubility of the material in the body fluids. Highly soluble gases (e.g.
ammonia, hydrogen chloride) cause severe nose and throat irritation, while substances
of lower solubility (e.g. nitrogen dioxide, phosgene, sulfur dioxide) can penetrate deep
into the lungs.

Regulators and valves should be closed when the cylinder is not in use and flushed with
dry air or nitrogen after use.

When corrosive gases are to be discharged into a liquid, a trap, check valve, or vacuum
break device should be employed to prevent dangerous reverse flow.
 Laboratory Upkeep

Corrosive solids, such as sodium hydroxide and phenol, can

cause burns to the skin and eyes. Dust from corrosive solids can
be inhaled and cause irritation or burns to the respiratory tract.
Many corrosive solids, such as potassium hydroxide and sodium
hydroxide, can produce considerable heat when dissolved in
water.

Wear gloves and eye protection when handling corrosive solids.

When mixing with water, always slowly add the corrosive solid to
water, stirring continuously. Cooling may be necessary.

If there is a possibility of generating a significant amount of dust,

conduct work in a fume hood.

Use bottle top dispensers wherever required.

 Laboratory Upkeep

Adequate lighting is necessary in the Laboratory to perform the activities.

Light sensitive drugs can be affected by sunlight (ultraviolet light) or by artificial light (like
fluorescent light). Proper precautions shall be taken to store, handle the Light sensitive
products.

The Room Temperature & the Humidity shall be controlled and monitored in the
laboratory.

The area of the sample storage in the laboratory shall be mapped for temperature and
documented appropriately.

The samples which are supposed to store at other temperature (Refrigerated / Freezer)
shall be stored appropriately.
 Laboratory Upkeep - 5 S principle

Follow 5 S principle in the laboratory. Benefits of 5S

Safety
The 5S system is a tool that supports an
operating philosophy that emphasizes Employee involvement
discipline, efficiency, and attention to detail Organization/Efficiency
Cleanliness
1.Sort
Space Savings
2.Set in Order Lower inventories
3.Shine More consistent experiments
Faster on-boarding of new employees
4.Standardize
Timely delivery of supplies
5.Sustain Additional funding
 Laboratory Upkeep - 5 S principle

1. Sort
Document baseline work area before condition (pictures, audits, etc.)
Perform the initial cleaning of the work area
Separate the necessary items from unnecessary items
Remove unnecessary items from the work area
 Laboratory Upkeep - 5 S principle

1. Sort

Sorting Methodology:

Establish a holding area for items (Red tag area)

Identify items not required at the

current location, if at all

Team members evaluate items in Red Tag area

Dispose of items with team approval

 Laboratory Upkeep - 5 S principle

2. Set in Order
Identify necessary tools, equipment, supplies, etc.
Determine location for necessary items
Designate & outline permanent locations for items
 Laboratory Upkeep - 5 S principle

2. Set in Order

How to set in order

Define the major processes performed

For each process, draw a map of the

locations of each activity

Identify sources of waste and corrective

measures

Position items where they are needed

based on frequency
 Laboratory Upkeep - 5 S principle

3. Shine

Clean area thoroughly Make it shine!

Clean & inspect areas daily

 Laboratory Upkeep - 5 S principle

3. Shine

Machines & Equipment

Fix leaks & other problems

Prevent contamination

Inspect daily

Fix potential issues before they become

major problems
 Laboratory Upkeep - 5 S principle

4. Standardise
Develop SOPs

Document equipment & processes

Develop audit schedule and assign tasks

to individuals

Establish a methodology to ensure

consistent strategy for 5S implementation
 Laboratory Upkeep - 5 S principle

5. Sustain

Establish procedures on 5 S.

Maintain established procedures.

Ensure 5 S is a long term, laboratory-

wide goal.

Analyze results, hold team meetings and

train scientists on the importance of 5 S.
Laboratory Behavior
 Laboratory Behavior

Scientists shall behave professionally in the laboratory.

Observe keenly each step of the experiment.

If errors are obvious, such as the spilling of a sample solution or the incomplete transfer
of a sample composite, the analyst should immediately document what happened.

Scientists should not knowingly continue an analysis they expect to invalidate at a later
time for an assignable cause (i.e., analyses should not be completed for the sole
purpose of seeing what results can be obtained when obvious errors are known).

Analysts should check the data for compliance with test specifications before discarding
test preparations or standard preparations.

Do not shake hands while working.

Do not use mobile phones in the laboratory.

People
 People

People should undergo periodic health check-ups.

Be able to confirm that employees are qualified for their positions by having a current CV
and Job description on file.

All employees should update periodically (annually, biannually, etc.) and as

responsibilities change (for example, after a promotion).

Have accurate training records on file that are easily retrieved

Conduct frequent refresher training on quality and safety topics

Instruments
 Instruments

All the critical instruments (new & modified) must be Validated (IQ/OQ/PQ) and
Calibrated.

Written procedures must be established to operate / Calibrate the instruments.

21 CFR 211.160 (b) (4) :

 Instruments

All instruments must be numbered appropriately for easy identification.

All instruments must be labeled with Calibration status & due date.

Maintain schedules for Preventive maintenance & Calibration activities of instruments.

All the instruments must have usage and maintenance log books.

Schedules and procedures (including assignment of responsibility) should be established

for the preventative maintenance of equipment.
 Instruments

Equipment calibrations should be performed using standards traceable to certified

standards, if existing.

Records of these calibrations should be maintained.

Instruments that do not meet calibration criteria should not be used.

Deviations from approved standards of calibration on critical instruments should be

investigated to determine if these could have had an impact on the quality analysed using
this equipment since the last successful calibration.
Reagents, Chemicals & Solvents
 Reagents, Chemicals & Solvents

All reagents and solutions in the laboratory areas shall be labeled to indicate
identity, titer or concentration, storage requirements, and expiration date.

Deteriorated or outdated reagents and solutions should not be used.

If reagents and solutions used for non-regulated work are stored in the same
room as reagents for regulated studies, all reagents must be labeled.

Reagents that are not adequately labeled, even if not intended for use in
regulated studies, may have an adverse effect on regulated laboratory work. It is
also good practice to include the Date opened. This can be critical for some
chemicals such as ether.
 Reagents, Chemicals & Solvents

The expiration date depends on the nature of the chemical. E.g. Sodium chloride has
practically no expiration date. In these cases it might be acceptable to indicate NONE or
Not applicable (N/A) on the label for expiration date.

The laboratory must be prepared to justify this designation. Formal studies are not
required to justify assigned expiration dates.

It is sufficient to assign expiration dates based on literature references and/or laboratory

experience.

The label should indicate special environmental conditions, for example Refrigerate or
Protect from light.

Do not mix sulphuric acid with water inside a cylinder.

Check the conductivity / pH of the water before use.

Glassware
 Glassware

Use Class A Glassware only for measurement.

Validate the glassware / vial cleaning procedure.

Do not use broken glassware.

Verify & Preserve the Certificate provided by the

vendor for glassware.

Do not heat the glassware that is cracked, chipped or

scratched.
Standards
 Standards
Certified reference standards can be purchased from appropriate
suppliers.
If standards are not available, the recommendation is to take a
lot of your own material, and analyze, certify and use it as the
standard.
Sometimes certified standards are too expensive for day-to-day
routine use. In this case in-house laboratory standards can be
used as working standards. However, they should be made from
high purity material and be compared against the primary
standard to ensure the traceability chain. For the comparison,
validated test methods should be used.
All reference material, either purchased or in-house, should be
subject to a quality control procedure.
This includes regular checks of purity, identity and
concentrations.
 Standards
Standards should be stored in their original stoppered
container away from heat and humidity and protected from As per USP General Notices & Requirements:
light
Store up to the validity of current lot.
USP RS should be stored as per the label directions.
Do not dry reference standards in their original container.
Dry only the required quantity of the standard on a glass
Petridish, whenever specified.
Do not put back any excess quantity of the dispensed
standard back into the stock bottles.
Allow the container of the standard to attain room
temperature before using. Wipe the outer surface of the
container with tissue paper to remove moisture.
Procedures
 Procedures

These procedures describe the methods that will be used to implement and
perform the stated policies.
The procedures define who should perform the specific tasks, when the task
should be done, and where the documentation will be made showing that task was
performed.
The Procedure (Standard Operating Procedures) shall be reviewed periodically.

SOPs should preferably be written in the laboratory close to the instrument, and
not in an office. It should be either written or thoroughly reviewed by the
instruments operators.

SOPs should not be written to explain how procedures are supposed to work, but
how they work.
 Procedures

This ensures that the information is adequate

and that the document invites rather than
discourages routine use.

Content should cover:

SOP unique number and revision number,
Page number and total number of pages,
for equipment testing: performance acceptance
criteria, recommended corrective actions, and a
template for continuous entries of test results and
corrective actions,

printing history.
 Procedures

Copies of SOPs for equipment should be located close to the instruments and
must be easily accessible by operators.

Deviations from SOPs in a study must be authorized and significant changes in

established SOPs must be authorized in writing by management.

Standard operating procedures should be drafted in a language understood in the

workplace.
 Procedures
Have approved procedures in place for
Method validation and transfer requirements.

Instrument qualification, calibration and preventive maintenance.

Instructions for qualification and calibration include the actions to be taken when an instrument fails
calibration.

Out of specification/out of trend results, deviations and investigations include some type of root cause
analysis steps to help identify the cause of the issues and prevent it from occurring in the future.

The use and security of lab software, including data acquisition software and LIMS (Laboratory Information
Management System).

To define how raw data and lab documentation is handled.

To define the process for receiving samples and how samples are managed within the lab.
Log Books
 Log Books

Laboratory equipment log books are important records of machine calibration,

verification checks, cleaning, maintenance and use.

Always record entries legibly, neatly and in permanent ink.

All log books require one signature per line entry.

All entries should note the date and time of the entry / observation.

Never leave blank spaces and never erase or remove material added. Draw lines
through any blank spaces at the same time of making entries.

Never remove pages from the log book.

Entries in the log book shall be in chronological order.

 Log Books

Logbooks should be kept near the instrument so it is easy to make entries or to

check.

Laboratory equipment log books should show the following

Date
Time
Product Name
Batch Number
Initials (Sign) of the performer
Documentation
 Documentation

Policy

The first tier of documentation is the policy

manual.

This is the document that defines what will be

done and why.

A quality policy manual should be written so it is

clear, precise and practical, and easy to
understand. The why can be stated just once
as a quality policy statement. This statement
should be a short, simple definition of the
organizations quality intentions.
 Documentation
Procedures
The second tier of documentation is quality procedures. These procedures describe the methods that will be
used to implement and perform the stated policies. The procedures define who should perform the specific
tasks, when the task should be done, and where the documentation will be made showing that task was
performed.

Work Instructions
Work instructions are usually department, machine, task, or product oriented an spell how a job will be done.
The instructions are the most detailed of the documentation hierarchy. A work instruction may be in the form of
a detailed drawing, recipe, routing sheet, specific job function or simply a sample for comparison or conformity.

Records
Records are a way of documenting that the policies, procedures, and work instructions have been followed.
Records may be forms that are filled out, a stamp of approval on a product, or a signature and date on some
type of document, such as routing sheet. Records are used to provide traceability of actions taken on a specific
product or batch of products. They provide data for corrective actions and a way of recalling products, if
necessary.
 Documentation
Raw Data
Raw data refers to any laboratory worksheets, records, memoranda, notes, or exact copies thereof, that are the
results of original observations and activities of a study. The term covers all data necessary for the
reconstruction of the report of the study. Raw data may include handwritten notes, photographs, microfiche
copies, computer print-outs, magnetic media, dictated observations, and electronically recorded data from
automated instruments. Examples include results of environmental monitoring, instrument calibration records,
and integrator output from analytical equipment. Raw data may also be entries in a worksheet used to read and
note information from the LED display of an analytical instrument..

Laboratory Note book

For raw data entries, it is recommended to use controlled forms or a laboratory notebook for each study. This
should be robust, bound and have the pages numbered. All entries should be made in indelible ink. Scientists
and technicians sometimes record raw data on scraps of paper or even on paper towels. Their intention is to
neatly transcribe the information to official data forms at a later time and to discard the originally recorded data.
This practice should be discouraged, because the scraps of paper are the real raw data, and must be retained.
More recently electronic notebooks are used instead of paper notebooks. For US-FDA GLP/GMP regulated
laboratories the regulation on electronic records and signatures, 21 CFR Part11 applies.
 Documentation
Specifications & Analytical Methods
Use only validated analytical methods for testing.
Use only approved Specifications and testing procedures.

Standard Operating Procedures

There should be standard operating procedure for all critical activities / operation and calibration of instruments.
Follow Standard operating procedures.
Computers
 Computers
Identify each user uniquely
Implement adequate password controls
Establish different user roles / access privileges
Establish and maintain a list of current and
historical users
Control changes to the system
Use only trained staff to operate the system
Understand predicate rules for laboratory records
Define and document e-records for the system
Review the audit trails for each batch set
Back the system up regularly
 Dip tubes
Spatulas for liquids
for solids

Sampling
 Sampling
Sampling tools should be available to the sampler, e.g. to open
containers (knives, hammers,...), material to reclose the packages
(sealing tape), self-adhesive labels to indicate that some of the
contents have been removed, etc...

Sampling tools should be made of inert materials (e.g. polypropylene

or stainless steel; avoid glass) and kept very clean. After use,
thoroughly washed, rinsed with water or suitable solvent, dried and
stored in clean conditions.

Disposable sampling materials can also be used.

Washing facilities should be located in, or close to, the sampling area.

Cleaning procedure should be documented and validated (=

demonstrated efficiency).

Sterile pharmaceutical products should be sampled under aseptic

conditions.
 Sampling
Sampling operations should be supervised and documented => sample collection form => always kept
together with the collected sample.
Sample collection form: written record of the sampling operations, containing: batch number, sampling
date/place, reference to sampling protocol used, description of containers and materials sampled, possible
abnormalities, any relevant observations, name/signature of the sampler...
Store the sample in a properly labelled container: sample type, name of material, identification code, batch
number, code, quantity, date of sampling, storage conditions, handling precautions, container number.

Rooms for sample storage:

Security and adequate storage conditions (light, ventilation, safety requirements, and any special
requirements) should be ensured for the rooms in which samples are stored.

Samples should be stored according to the storage conditions as specified for the respective API, excipient or
drug product.

Packaging materials similar to those in which the bulk is supplied should be used for long-term storage.
Weighing
 Weighing
Selection of the suitable balance based on the definition as well as
considering other balance characteristics such as balance
readability, minimum weight, capacity, accuracy, built-in calibration
and uncertainty values.
Use Qualified & Calibrated analytical balance.
Weighing result, as obtained during an analysis period is influenced
by a series of factors relating to balance and ambient conditions.
However, one should also consider sample features during weighing
process. Samples that are liquids, can undergo process of
evaporation.
A factor reverse to evaporation is absorption of moisture from
ambient air by a sample. it is very important in case of hygroscopic
samples.
The effect of moisture absorption is differences in mass
determination, each measurement will have higher mass readout
than the previous one. For the purpose of proper weighing of such
substances, weighing vessel should be clean and dry.
Measuring Volume
 Measuring Volume
Volume may be measured reliably with a pipette, a burette, or a volumetric
flask.

Volumetric equipment is marked by the manufacturer to indicate not only the

manner of calibration (usually TD for "to deliver" or TC for "to contain") but also
the temperature at which the calibration strictly applies. Pipettes and burettes
are ordinarily calibrated to deliver specified volumes, whereas volumetric flasks
are calibrated on a to-contain basis.

TC pipettes deliver all the volume including the tip, and then must be "blown
out" to get the last drop. Blowing out is done using the associated pipette
pump, NEVER your mouth.

A meniscus is the curved surface of a liquid at its interface with the

atmosphere.
 Measuring Volume
Reading a burette / pipette:
X
The tips of two styles of measuring pipettes.

The Mohr pipette is shown on the left, the
graduation lines stop short of the tip,

The serological pipette on the right, the

graduation lines, pass through the tip.

X
Sample Preparation
 Sample Preparation
Sonication:
Frequently change the solution in the bath. Solutions that become contaminated with
particles that settle on the bottom of the bath will decrease ultrasonic activity.
Do not place sample containers directly on the bottom of the bath. Containers on the
bottom of the bath will decrease cavitation and will damage the transducer because
they reflect the ultrasonic energy back to the transducer.
Containers should be placed in an open mesh basket or in an insert tray or be
suspended in the solution to position the container in the optimal zone of the bath and to
allow the ultrasonic waves to penetrate around the containers.
Degas the solution in the bath by sonicating for 510 min to enhance cavitation prior to
sonicating samples.
Sonication often results in heating of sample solutions. If using volumetric flasks,
sonicated sample solutions should be allowed to cool to room temperature before they
are diluted to volume. Since sonication induces localized areas of high temperature, this
may adversely affect thermally labile compounds.
 Sample Preparation
Eliminating Insoluble Particles:
Centrifugation shall be performed as specified in the validated method for specified
time.
Filter membrane selection based on analyte and solvent properties, so the filters shall
be used as specified in the validated method.
Filters with pore size distribution around 0.45 m are suitable for most applications,
although high efficiency LC columns with particles of < 3 m usually require filtration of
particles of 0.2 m.

It is important that the selected filter is validated during the validation exercise to ensure
that the active or related impurities are not lost to the filter and that no significant
interferences are generated by the selected filter.

As part of this process, one needs to study the effect of wetting the filter prior to sample
collection. Usually the first 5 mL of the eluate are discarded to obtain acceptable and
reproducible recoveries.
Chromatography
 Chromatography
All reagents and solvents should be of the highest
quality. HPLC grade reagents may cost slightly more
than lower grade reagents, but the difference in purity is
marked.

HPLC grade reagents contain no impurities to produce

spurious peaks in a chromatogram baseline.

Always use Purified water / Milli Q water as both

inorganic and organic contaminants are removed.

Do not use RO water / de-ionsed water for HPLC.

Due to existence of impurity, deionized water shows

higher absorption
 Chromatography
Buffers used for HPLC
All the solutions should be clear, homogenous & free from particulate matter.
Buffer solutions must be filtered through 0.45m filter or as specified in the
method by using Solvent Filtration Kit.
Do not keep mobile phase under the suction for more than 4 to 5 minutes as
the composition of mobile phase change especially if highly volatile solvents
are used.
Buffer solutions must not be left in system to avoid crystalisation.
effect on pump -- damage plunger and seal
effect on column -- creation of column voids
effect on flow line -- corrosion of stainless steel lines
Possible bacterial growth especially phosphate buffers -- good medium for
bacterial and fungus growth.
Ideally, solutions should be prepared fresh everyday
 Chromatography
Mobile Phase for HPLC
Always cover the container
To prevent evaporation of solvents
To prevent dust enter the mobile phase
To reduce the vapors in the room
Do not expose the bottle to direct sunlight/wind.
Always use a suction filter to Prevent particulates from reaching pumps

Stationary phase (Column) for HPLC

Avoid pressure shocks on the column.
Pressure shocks lead to channeling in the column, which results in peak
splitting in corresponding chromatogram.
Always keep both ends of the column closed, after usage.
Keep the columns in the designated column cabinets after use.
 Chromatography
The solvent lines should be clean, growth-free and should have no sharp bends or
creases in them.
Solvent reservoirs should be placed as high as possible on or in the instrument
always higher than the pump inlet manifold.
Before attempting any solvent change, ensure that the solvent already in the system
and column is compatible with the new solvent.
The column efficiency, capacity factor etcshould be measured at the start and end
of the clean-up procedure to ensure that it has been performed successfully and has
improved the column performance.
Ensure that no buffers/samples are present on the column and that the solvent used
prior to the clean-up is miscible with the first wash solvent.
After the clean-up, ensure that the test mobile phase is compatible with the last
solvent in the column.
Samples require filtration if they contain suspended solids. This can be performed
on-line using a pre-column filter or as the sample is introduced to the vial.
Microbiology
 Microbiology
Good laboratory practices in a microbiology laboratory consist
of activities that depend on several principles: aseptic
technique, control of media, control of test strains, control of
equipment, diligent recording and evaluation of data, and
training of the laboratory stuff.
Clean spills immediately according to established protocols
and disinfect the area thoroughly
Keep bench top uncluttered
Minimize traffic and unnecessary movements around the work
area (movement can stir up air currents which can carry
contaminants into the work area).
All work with infectious material should be carried out in a
specific area, and the material should not be carried
throughout or out of the lab unless in a closed or capped
container.
Minimize aerosol generation; if unavoidable, activities should
be carried out in a biological safety cabinet
 Microbiology
Use proper aseptic technique for the transfer and handling or microorganisms and instruments
Keep sterile and non-sterile objects separate
Minimize exposure to outside air (i.e. keep lids off sterile containers for as little time as possible)
Avoid contact with non-sterile surfaces and items (i.e. never place lids/caps onto a work surface, hold lids in
an opening-down position), and
Hold open containers at an angle whenever possible to prevent contaminants from falling in.

Media, Reagents Preparation, Storage, Qualification:

Its important to choose the correct media or components in making media based on the use of accepted
sources or references for formulas.
The LAL reagentis critical in Endotoxin testing, and must be well controlled.
When the LAL reagent (lot) changes, the Micro Lab must confirm the labeled LAL reagent sensitivity (EU/ml).
This is to re-calculate the Maximum Valid Dilution (MVD), which is the maximum allowable dilution of a sample
to determine the endotoxin limit.
 Microbiology
Reference Standards:
Microbial cultures are delicate standards. Procedures should specify careful handling
instructions.
Preparation and resuscitation of cultures should follow the instructions of the supplier or
a validated, established method.
USP recommends using the "Seed-Lot" technique for storage of stock cultures, i.e.,
using working cultures and never returning unused passages back to original stock.
In addition, there should be an established maximum number of passages (5 or less),
and maximum storage time for working cultures.
Cultures for use in compendial tests should be acquired from a national culture
collection, in frozen, freeze-dried, on slants, or in ready-to-use forms.
Confirmation of the purity and the identity should be performed prior to its use in quality
control testing.
Ready-to-use cultures may require additional confirmation of inoculums size.
 Microbiology
Laboratory Operations/Housekeeping:

Safety first. The Micro Lab should practice aseptic techniques during testing in general, to avoid microbial
contamination and false positives.

In the Micro Lab, areas where EM, water, or product samples are handled/incubated must be adequately
separated from areas where there are tests that involve live cultures or sub-culturing, microbial ID or
investigations.

Housekeeping must be properly maintained to prevent use of expired or contaminated testing mats.

Verify cleanliness of work stations, cleared of extraneous or previous test residue, prompt removal of refuse,
and clean utensils and equipment
Stability
Stability
 Stability
Establish approved procedures for
Conducting Stability studies
Withdrawal of Stability studies
Investigation of deviations, incidences, OOT and OOS results.

Lay down a properly designed test protocol.

Conduct the stability study as per the protocol.
Make estimate of shelf life from the generated data.

Display and Recording of Set Values

Digital display and recording through chart recorders, data loggers, computer and software
Alternatively or in addition, memory back-up with provision of downloading of data at any time
 Stability
Delays are needed to be provided in alarms to avoid unnecessary notification, door opening
condition is not registered unless the door is open for more than five minutes. In case of fluctuation,
the system allots say around 20 min for system to re-equilibrate before setting an alarm.

The alarm might be cleared only when a user ID and password is entered.

Prepare samples and keep at the selected storage conditions

Withdraw at the prescribed sampling intervals and subject to analysis.

Complete the testing of stability samples within specified time period.

Monitor the temperature & Humidity of the stability chambers. All the fluctuations shall be corrected,
documented and justified.
Investigations
Investigations
 Investigations
Investigations of "Out of Specification (OOS) / Out of Trend (OOT)/ Atypical -results" have to be
done in cases of:
Batch release testing and testing of starting materials.

In-Process Control testing: if data is used for batch calculations/decisions and if in a dossier and on
Certificates of Analysis.

Stability studies on marketed batches of finished products and or active pharmaceutical ingredients, on-
going / follow up stability (no stress tests)

Previous released batch used as reference sample in an OOS investigation showing OOS or suspect
results.

Batches for clinical trials.

All solutions and reagents must be retained until all data has been second person verified as being
within the defined acceptance criteria.
 Investigations
Pharmacopoeia have specific criteria for additional analyses of specific tests (i.e. dissolution level
specification for S1, S2 & S3 testing; Uniformity of dosage units specification for testing of 20
additional units; Sterility Testing).
However if the sample test criteria is usually the first level of testing and a sample has to be tested
to the next level this should be investigated as it is not following the normal trend.
Unexpected results and/or events should be documented and thoroughly investigated.
It is not sufficient to determine that additional training is required for the analyst or glassware wasnt
adequately cleaned. Regardless of the method used, it the investigation steps and results need to
be clearly documented. Include supporting documentation so that anyone reading the investigation
report understands the actions taken and the corresponding results. In the event that the cause
cannot be identified, be sure the investigation report illustrates that adequate measures were taken
to attempt to identify the cause.
Dont stop at identifying the root cause. Put a plan in place to resolve the issue in the current
situation and prevent it from occurring in the future, and then follow through to initiate the plan and
evaluate if it has the desired results.
Laboratory Exit
 Laboratory Exit

There should be a defined procedure for lab exit.

There should be evacuation plan defined in safety

manual.

Before coming out of the Lab ensure

Switch off unnecessary instruments.
Remove / Dispose the apron, Cap and gloves.
Wash hands with soap solution.
Audit Findings
 Canada Audit citations

Insufficient testing of raw material.

Incomplete specifications.

Specifications not approved by QC.

Test methods not validated.

Use of raw material after retest date without proper retesting.

Use of raw material after the expiration date.

Incomplete validation of test methods.

No SOP for conditions of transportation and storage.

 Canada Audit citations

Evidence of falsification or misrepresentation of testing results / forgery of COA.

Non-compliant products made available for sale.

Incomplete/inadequate specifications.

Finished product specifications not approved by QC.

Incomplete testing.

Lack of or insufficient validation of test methods.

Inadequate method transfer for a validated analytical method.

Method validation report does not specify the revision of the analytical method
used at the time of validation.
 US FDA Audit Findings Laboratory Controls
Failure to maintain laboratory control records with complete data derived from all tests conducted
to ensure compliance with established specifications and standards, including examinations and
assays.

Your firm failed to establish laboratory controls that include scientifically sound and appropriate
specifications, standards, sampling plans, and test procedures designed to assure that
components, drug product containers, closures, in-process materials, labeling, and drug products
conform to appropriate standards of identity, strength, quality, and purity (21 CFR 211.160(b)).

Your firm failed to ensure that laboratory records included complete data derived from all tests
necessary to assure compliance with established specifications and standards (21 CFR
211.194(a)).

Your laboratorys written procedure failed to establish proper retesting practices for out-of-
specification results.
 US FDA Audit Findings Laboratory Controls
Your firm failed to thoroughly investigate any unexplained discrepancy or failure of a batch or any
of its components to meet any of its specifications, whether or not the batch has already been
distributed (21 CFR 211.192).
Your firm failed to investigate and document out-of-specification results according to a procedure.
Your laboratorys written procedure failed to establish proper retesting practices for out-of-
specification results.
Recognizing that an Out-of-Specification Test Result was obtained in [REDACTION] specification
of [REDACTION] as per requirement of your Quality Control/Quality Assurance Standard
Operating Procedure for OOS Investigation, SOP Number 2802, Reporting, Investigation and
Disposition of Out of Specification (OOS) Laboratory Results, effective 02/05/09 and previous
revisions, it appears that you avoided to perform an Investigation for the assay test result
of [REDACTION].
The Quality Unit is not initiating investigations and documentation of those investigations in a
timely manner.
 US FDA Audit Findings Laboratory Controls
Your firm does not have adequate written procedures for production and process controls
designed to assure that the drug products you manufacture have the identity, strength, quality, and
purity they purport or are represented to possess [21 C.F.R. 211.100(a)].
Failure to have an API stability program to monitor stability characteristics of your firms APIs, and
failure to set an expiry or retest date for APIs based on the evaluation of data derived from stability
studies.
Your laboratorys written procedure failed to establish proper retesting practices for out-of-
specification results.
Your firm failed to establish and follow an adequate written testing program designed to assess
the stability characteristics of drug products and to use results of such stability testing to
determine appropriate storage conditions and expiration dates (21 CFR 211.166(a)).
Your firms quality control unit failed to review and approve all drug product production and control
records to determine compliance with all established, approved written procedures before a batch
is released or distributed (21 CFR 211.192).
 US FDA Audit Findings Microbiology

Your firm failed to establish and follow appropriate written procedures that are
designed to prevent microbiological contamination of drug products purporting to
be sterile, and that include validation of all aseptic and sterilization processes (21
CFR 211.113(b)).

Your firm failed to establish an adequate system for monitoring environmental

conditions in aseptic processing (21 CFR 211.42(c)(10)(iv)).

Your firm failed to establish maximum holding times for vials used in media fills,
prior to incubation
 US FDA Audit Findings Microbiology

Your firm failed to establish and follow appropriate written procedures that are
designed to prevent microbiological contamination of drug products purporting to
be sterile, and that include validation of all aseptic and sterilization processes (21
CFR 211.113(b)).

Your firm failed to establish an adequate system for monitoring environmental

conditions in aseptic processing (21 CFR 211.42(c)(10)(iv)).

Your firm failed to establish maximum holding times for vials used in media fills,
prior to incubation
Issues due to not following GLP
Issues due to not following GLP

Accidents / Incidents
Lack of Lab Safety Health Hazards

Time delay for searching

Lack of Lab Upkeep Contamination & no clarity of work

Accidents / Incidents
Lack of Lab Behavior Erratic results

Lack of Instrument Time delay for repetitions & instrument failures

maintenance Erratic results

Lack of Reagent Accidents / Incidents

maintenance Erratic results & Contamination
Issues due to not following GLP

Lack of Glassware Erratic volume measurement & wrong results

maintenance Contamination

Lack of Standard Wrong / Erratic results

maintenance Contamination

Non Uniformity of performing activities & results

Lack of Procedures Perform activities wrongly

No control on usage of instrument

Lack of Log Books No evidence of usage of instrument

Lack of Evidence of performing the activity

Documentation To control errors / mistakes
 Issues due to not following GLP

Lack of Computer Loss on data (security)

maintenance Misuse / deletion of data

Not following Spillage can lead to wrong / Erratic results

Sampling instructions Contamination & mix-up

Erratic / wrong results

Not weighing properly Time delay to perform investigations

Lack of correct Erratic volume measurement & wrong results

volume measurement Time delay to perform investigations

Not following sample Erratic / Wrong results

preparation technique Time delay to perform investigations
 Issues due to not following GLP
Instrument error / malfunction / breakdown
Lack of Good Erratic results
Chromatography Cost effective
Time delay for investigations

Lack of Good Wrong / Erratic results

Microbiological practices Contamination & mix-up

Not following proper Erratic / wrong time study

stability practices Wrong establishment of Retest / Expiry date

Inadequate Erratic volume measurement & wrong results

Investigations Repetitive issues due to no or incorrect root cause.
 Issues due to not following GLP

Summary of Issues due to not following GLP in QC laboratories

Accidents / incidents & Health hazards
Wrong / errors in volume or weight measurement leads to erratic / wrong results
Some errors may be identified some may not be identified
Instrument errors / malfunctions
Contamination of Sample, Reagent, Solvent / product
Waste of time due to investigation of unexpected / unknown cause.
No evidence / documental proof of data generated.
Increase in cost due to repetitions & investigations
Due to wrong result generation impacts the quality / purity of the product
All the errors generated knowingly / unknowingly can effect patient safety.
Regulatory agency action results in 483s, Warning letters, Import alerts, Non-Compliance
reports etc.
Future Trends in GLP
 Future Trends in GLP

The Good Laboratory Practices shall be followed strictly in the Quality control laboratories
of all analytical laboratories i.e. in pharmaceutical QC lab, Analytical R &D as well as
contract testing laboratories.

GLP also to be trained & implemented in the contract research and testing lab (CRTO) /
contract research and manufacturing organization (CRMO).

GLP audits should be performed frequently for Vendors and CRTO / CRMO sites.

We can learn GLP from customer / MNCs and external training programs.

We can learn GLP from 483s, warning letters and Non-Compliance reports of other
companies.

Perform GAP analysis to identify gaps in the system and correct and implement GLP.
A to Z of GLP
 There is no end for discussion on Good Laboratory Practices.

Good Laboratory Practices are to be improved continuously to determine the exact

quality of the product.

GLP is required to be followed to ensure Compliance and Data Integrity

(http://www.slideshare.net/skvemula/presentation-on-data-integrity-in-
pharmaceutical-industry) and proper guidance on usage of computers and
Chromatographic Data Systems
(http://www.slideshare.net/skvemula/chromatography-data-system-cds-in-pharma).

GLP helps to understand the importance of each step to minimize errors

(http://www.slideshare.net/skvemula/understand-the-importance-of-each-step-to-
minimise-laboratory-errors) during the analysis and to reduce Laboratory errors
(http://www.slideshare.net/skvemula/laboratory-errors).
 GLP helps to follow best practices and precautions to produce accurate results with the
knowledge of Sample preparation techniques
(http://www.slideshare.net/skvemula/sample-preparation-techniques-of-solid-dosage-
forms) and to follow Good Chromatographic practices
(http://www.slideshare.net/skvemula/good-chromatographic-practices) and guidance on
proper Peak integration (http://www.slideshare.net/skvemula/hplc-peak-integration-for-
chromatography-38032765).

GLP is also required to investigate the atypical results

(http://www.slideshare.net/skvemula/investigating-aberrant-potency-values-in-pharma-
analysis) and to ensure that only quality product should reach to the patient.
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