Validation and Verification of Analytical Method:

Approach and Continuous Improvement

Hanslibrery Simanjuntak
Research and Development – PT Kalbio Global Medika
Outline
• Analysis of Biopharmaceutical Products
• Sterility
• Antimicrobial Efficiency Test
• Re-validation
Product Analysis – What to Test

Typically, formula of biologics drug product consists of:

• Active pharmaceutical ingredient (API)
• Stabilizer (surfactant or ionic stabilizer)
• Preservative
• Buffer
• Adjuvant (for vaccine)
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Analysis of Biologics Product

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Analysis of Biologics Product

We usually categorize the type of biological analysis into 3:

• Biochemistry test
Ex: In-vitro or In-vivo activity test, PCR test, etc.

• Physico-chemistry test
Ex: HPLC, GC, Capillary Electrophoresis, etc.

• Microbiology test
Ex: Endotoxin, Mycoplasma, Sterility test, etc.

Common microbiology test for Biopharmaceutical Products

(a) Sterility Test
(b) Endotoxin Test, Antimicrobial
(c) Efficiency Test (AET)
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Sterility

The test is applied to substances, preparations or articles which, according to the

Pharmacopoeia, are required to be sterile. However, a satisfactory result only indicates that
no contaminating micro-organism has been found in the sample examined in the conditions of
the test.

The test for sterility is carried out under aseptic

conditions. In order to achieve such conditions, the
test environment has to be adapted to the way in
which the sterility test is performed. The
precautions taken to avoid contamination are such
that they do not affect any micro-organisms which
are to be revealed in the test. The working
conditions in which the tests are performed are
monitored regularly by appropriate sampling of the
working area and by carrying out appropriate
controls.

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Sterility – What to test?

1. Drug Product

2. Primary Packaging (Container and Closure)

3. Ready to Fill product

4. Other materials as per usage requirement

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Sterility – How to Validate?

We follow 2 type of test, depending of the samples that currently handled:

1. Membrane filtration test
Used for liquid sample such as drug product and ready-to-fill bulk

2. Direct Inoculation test

Used for solid type sample such as vial, syringe, cartridge, or rubber stopper

Validation of Sterility test (usually called as method suitability test) is performed:

a) when the test for sterility has to be carried out on a new product, ensuring compatibility of
test item and test procedure;
b) whenever there is a change in the experimental conditions of the test.

The method suitability test may be performed simultaneously with the test for sterility of the
product to be examined.

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Sterility – How to Validate?

Membrane Filtration Test

Typical test is performed following this

sequential steps:

• Membrane pre-wetted with fluid A

2x50mL → Flowed out
• Product per container is transferred to
canister (volume of product? Number of
container?) → Flowed out
• Rinse with 100mL fluid A → Frequency?
• Flow out the fluid A
• Transfer 100 mL of media, FTM or TSB
• Incubate, FTM in 32.5±2.5°C for 14
days and TSB 22.5°C±2.5°C for 14 days

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Sterility – How to Validate?

Direct Inoculation Test

Typical test is performed following this

sequential steps:

• Prepare liquid media in a glass bottle

• Place the item to test inside the glass
bottle (number of item?)
• Top up the media until it covers all
surface of the item
• Incubate, FTM in 32.5±2.5°C for 14
days and TSB 22.5°C±2.5°C for 14 days

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Sterility – How to Validate?

During the validation, prepare:

• Negative control media. Ensuring media is completely absent of contaminant to avoid bias of
validation result.
Incubate the media inside the cannister for 14 days
• Positive control media. Similar as growth promotion test (without product). For each
microorganism target use:
o FTM : S. aureus, P. aeruginosa, C. sporogenens, incubated for 3 days
o TSB : B. spizizenii, C. albicans, A. brasilliensis, incubated for 5 days
Ensure the bacterial number is <100 CFU/ mL
• Negative control product. Ensuring product is completely absent of contaminant to avoid
bias of validation result.
• Positive control product. Main item for validation. Similar as positive control media, but this
time use cannister and product to test

Following procedure previously mentioned

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Sterility – What to consider?

Volume of Product per Container to Transfer

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Sterility – What to consider?

Number of sample per Cannister

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Sterility – When to re-validate?

We re-validate sterility method following 2 cases:

a) Every 2 years, with of without changes;
b) whenever there is a change in the experimental conditions of the test

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Sterility – Unique Cases

Sterility Test of 50 mL Vial

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Sterility – Unique Cases

Sterility Test of Monoclonal Antibody

If we were to follow and use the table Instead,
suggested by pharmacopeia, we will use • we can use the total amount of vial
half container of liquid, and 20 container (split for 2 cannister)
per cannister • We can reduce the number of product
per cannister

2
20

Total amount of product required is Total requirement : ~38 vials.

around 146 vials → Expensive In exchange to more cannister per
routine analysis
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Antimicrobial Efficiency Test (AET)

Typically, formula of biologics drug product consists of:

• Active pharmaceutical ingredient (API)
• Stabilizer (surfactant or ionic stabilizer)
• Preservative
• Buffer
• Adjuvant (for vaccine)
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Antimicrobial Efficiency Test (AET)

• The most commonly used preservative in biopharmaceutical products are:

o Meta-cresol
o Benzyl alcohol
o Phenol

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Antimicrobial Efficiency Test (AET)

Consideration for selection:

a) Antimicrobial Effectiveness
b) Compatibility of microbial preservative with
protein product

Continuous Improvement for a Better Pharma & Biopharma Laboratory

Antimicrobial Efficiency Test (AET)

Thus it become crucial to

determine:
• The type of preservative to use
• The concentration of
preservative in the product
• The level of effectiveness in
product

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Antimicrobial Efficiency Test (AET)

• Antimicrobial Effectiveness Testing (AET) is a microbial challenge methodology to

determine the antimicrobial efficacy in products to assure the antimicrobial compound in
the product is sufficient to inhibit the introduction and proliferation of microorganisms.

• AET is performed to help determine the safety of sterile multi-use product subjected to
repeated withdrawal of dosage.

• Repeated usage of products can potentially introduce microorganisms and make the
products harmful to use in humans.

• Antimicrobial preservatives must not be used as a substitute for good manufacturing

practice.

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Antimicrobial Efficiency Test (AET)

• The efficacy of an antimicrobial preservative may be enhanced or diminished by the

active constituent of the preparation or by the formulation in which it is incorporated or
by the container and closure used.

• The antimicrobial activity of the preparation in its final container is investigated over the
shelf life to ensure that such activity has not been impaired by storage.

• Such investigations may be carried out on samples removed from the final container
immediately prior to testing.

• The test is not intended to be used for routine control purposes

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AET – How to validate?

Preparation of Inoculum Stock

• The target concentration of inoculum stock is more than 108 CFU/mL.
To aid during the process, turbidity can be used as initial measurement of the culture
concentration, but the final number of microorganism must still be determined by plate
count.

• List of challenge microorganism

ATCC Growth Incubation Incubation

Organism
No. Medium Temperature Time
Pseudomonas aeruginosa PA 9027 TSA or TSA+LP 30-35°C 18-24 hours
Use 9 g/L NaCl as
Staphylococcus aureus SA 6538 TSA or TSA+LP 30-35°C 18-24 hours
Candica albicans CA 10231 SDA 20-25°C 2-3 days*
diluent
Aspergillus brasiliesnsis AB 16404 SDA 20-25°C 5-7 days* Add 0.5 g/L PS-80
in the 9 g/L NaCL

• It is recommended to add contaminant that might likely represent the contaminant of

preparation
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AET – How to validate?

Conducting AET Test tube containing 108 challenge microbial

PA SA CA AB

Sample Drug Product Positive Control

Spike the test item with ≤1% volume of
each microbial test – target microbial
concentration is 105 – 106 CFU/mL

Store all sample preparation in 20 – 25 oC. Perform microbial count as per incubation per
time point by removing ≤ 1mL of product each time from every container

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AET – Test Evaluation?

• Do not forget to take T0 sample

• Remove the sample per time point, take 1
mL of sample and return it back to
incubation location
• The 1 mL sample taken, must be diluted
before starting the microbial count to
avoid TMTC result

The A criteria express the recommended efficacy to be achieved. Log Reduction calculation
10 Log [T0 count] – 10 Log [Time point count)
In justified cases where the A criteria cannot be attained, for example
for reasons of an increased risk of adverse reactions, the B criteria Example:
must be satisfied. T0 = 108
T24h = 104
Log Reduction = 4

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Antimicrobial – Consideration

• Benzyl Alcohol is the preferred antimicrobial in most of European Countries

• In practice, there are 2 concentration of Benzyl Alcohol preservative available in the market:
0.9% and 1.1%

• Lower concentration of Benzyl alcohol is preferable, however when EP Criteria A is difficult to

achieve

• Justification is required to comply

to criteria B instead of A

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AET – Usual difficulties?

Preparation of Inoculum Stock

When preparing inoculum stock, microbial concentration of 108 CFU/mL can no be

achieved.

Recommendation

• Use Petri Dish to cultivate instead of slanted agar in test tube to obtained more culture .

• Prepare more than 1 Petri Dish per challenge microorganism

• If the number can not be achieved in 1 subculture, do not prolong the incubation. Re-
subculture instead

• Use smaller volume of 9 g/L NaCl. Even if we perform the test in duplicates for 50 mL
product per container, we will only require 8 mL of 108 CFU/mL culture in total → Reduce
this number when applicable

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AET – Usual difficulties?

Test Evaluation

Difficulties to quantify the bacterial count after incubation.

Recommendation

• Use membrane filtration technique instead of plate count.

• Wash the filtered culture using sterile phosphate buffer before incubation to eliminate
residual antimicrobial activity

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AET – When to re-validate?

• It is important to determine if the preservative chosen for a product is compatible with the
formulation soon after manufacture

• It best if a second challenge test is determined to ensure that, as the product ages, the
preservative system is not breaking down and becoming less effective.

• It is also necessary to re-evaluate preservative effectiveness in your product whenever the

formulation, an associated manufacturing process, or product packaging changes occur

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