international journal of innovative pharmaceutical …and incubate the plates at 20 - 25 c for 14...

REVIEW ARTICLE Jinna Swathi et.al / IJIPSR / 3 (1), 2015, 150-163

Department of Pharmaceutical Analysis ISSN (online) 2347-2154

Available online: www.ijipsr.com January Issue 150

STERILITY AND QUALITY CONTROL TESTS FOR

BIOLOGICAL PRODUCTS: REVIEW

1Jinna Swathi*,

2Ch.Shankar,

3Kranti Sri Mulpuri

1,2Department of Pharmaceutical Analysis & Quality Assurance, Vikas College of Pharmaceutical

Sciences, Rayanigudem, Suryapet, Nalgonda, INDIA 3 Research Scholar, Emergent laboratories, Ameerpet, Hyderabad, INDIA

Corresponding Author

Jinna Swathi

Department of Pharmaceutical Analysis & Quality Assurance

Vikas College of Pharmaceutical Sciences,

Rayanigudem, Suryapet,

Nalgonda, INDIA

Email: [email protected]

Mobile: +91 9010582656

International Journal of Innovative

Pharmaceutical Sciences and Research www.ijipsr.com

Abstract

Biological products/biopharmaceuticals are the medicinal products that are manufactured or extracted

from various biological sources which are different from chemically synthesized pharmaceutical

products. As we use these products as a medication for various diseases or infections through the

parenteral route preparation, packaging and storage of the biological products has to be done under very

sterile and aseptic conditions. The contamination in the environment for production is measured through

the confined number of particles present in the air. All the types of sterility and quality control tests that

have to be followed for various types of biological products is elucidated in this review.

Keywords: Biological Products, Aseptic, Contamination, Sterility

mailto:[email protected]

http://en.wikipedia.org/wiki/Biology

http://en.wikipedia.org/wiki/Chemistry




INTRODUCTION

Definition

Biological products are the substances those are produced by or extracted from a biological source

(living cells) and that needs for its characterization and determination of its quality by a

combination of physico-chemical-biological testing, together with the production process and its

control. These are applicable to the prevention, treatment or cure of a disease or condition of

human -beings which are used for a wide range of diseases and conditions, including serious and

life threatening conditions [1, 2].

Biological products include [3]

Whole Blood and its Components

Blood Derivatives

Proteins

Cellular & Gene Therapies

Xeno-transplantation Products

Vaccines (preventive and therapeutic use)

Human Tissues

Allergenic Extracts

Classification of biological products

1. Immunological medicinal products: [4]

Any medicinal product consisting of vaccines, toxins, serums or allergen products:

a) Vaccines, toxins and serums including:

Agents used to produce active immunity [5]

Agents used to produce passive immunity

b) Allergen product: Any medicinal product which is intended to identify or induce a specific

acquired alteration in the immunological response to an allergizing agent.

2. Medicinal products derived from human blood and human plasma

3. Medicinal products developed by means of one of the following biotechnological processes:

Recombinant DNA technology

Controlled expression of genes coding for biologically active proteins in prokaryotes

and eukaryotes including transformed mammalian cells




Hybridoma and monoclonal antibody methods

4. Other miscellaneous group of products:

Produced by or extracted from biological origin (except plants) including hormones, and

larger peptides etc.

Properties of Biological products:

Made with/from live cells/organisms

Inherent & contamination risk

Generally high molecular weight

Many critical process steps

Less easily characterized

Structure may or may not be completely defined or known

Heterogeneous mixtures

May include variants

Often Immunogenic

Sources for biological [3]

Mammalian cells - culture

Avian cell - culture

Mice

Bacteria

Humans

Plant cell - culture

Insect cell- culture

Transgenics

Yeast.

Sources of Microbial Contamination in biological

Microorganisms are ubiquitous in nature. Microorganisms can adapt and survive under a variety

of conditions and can pose a significant risk to biologic products. An understanding of the

microbial entry points and implementation of measures to prevent microbial contamination is

critical for manufacture of safe, pure and potent biologic products [6]. Microorganisms can gain

entry into a production process stream from several sources as shown in Fig.1. All sources of




microbial contamination should be considered when developing a microbial control strategy and

performing an investigation for a microbial contamination deviation [6].

Fig. 1: Sources of Microbial Contamination

GENERAL TESTS

A. Sterility tests for biological products

B. Pyrogen test

A. STERILITY TESTS FOR BIOLOGICAL PRODUCTS

Biological products manufactured under GMP conditions require sterility testing performed

under GMP guidelines.

Sterility Method Suitability for various Biological:

It is important to determine if the test article that will be tested for sterility contains elements that

will interfere with the growth of microorganisms within the growth media used for the assay.

[This testing is commonly referred to as the Bacteriostasis/Fungistasis (B/F) test or sterility test

validation, qualification or verification [10].

The method suitability test is typically required only once for a given sample type, provided that

no additional changes to the product, formulation or manufacturing process occurred. However,

suitability testing could be performed on a periodic basis to confirm that no significant changes

have occurred to the product or process that may affect the sterility assay results.

There are two common types of sterility test methods: [7, 8, 9]




1. Immersion test: (Direct Inoculation)

For bacteria - Using Petri dishes 9 to 10 cm in diameter. A mixture of 1 ml of the

pretreated preparation and about 15 ml of a liquefied Soya bean casein digest medium, at

NMT 45 °C is added to each dish.

After solidification, alternatively spread the preparation on the surface of the medium.

Prepare at least two such Petri dishes using the same dilution and incubate at 30 - 35 °C for

14 days. Count the number of colonies that are formed.

For fungi - proceed as described in the test for bacteria but use Fluid thioglycollate medium

and incubate the plates at 20 - 25 °C for 14 days.

2. Membrane filtration

Use membrane filters which are 50 mm in diameter with nominal pore size of 0.45 µm.

Transfer 10 ml or a quantity of each dilution containing 1 g of the test preparation that has

to be examined to each of two membrane filters and filter immediately.

The filter is then rinsed and then the membrane is transferred into the appropriate medium.

(Soyabean Casein digests medium for bacteria and Fluid thioglycollate medium for fungi).

Then the membrane is incubated for 14 days in the test medium.

Interpretation:

If there is no evidence of microbial growth then the preparation under examination passes the test.

Test is repeated if:

(a) The data of the microbial monitoring of the sterility test facility show a fault

(b) A review of the testing procedure used during the test in question reveals a fault

(c) Microbial growth is found in negative controls

(d) After determination of the identity of the microorganisms isolated from the test, the growth of

this species or these species may be ascribed unequivocally to faults with respect to the

material and/or technique used in conducting the sterility test procedure.

The majority of biological samples will be tested using the immersion method.

However, for test articles with large volumes in which the entire contents must be tested or if

there is a substance within the test article that is known or determined to be bacteriostatic or

fungistatic (e.g., cell culture containing methotrexate), the membrane filtration method may be

required.




Sample requirements based on the type of biological product:

Cell & Virus Bank Testing:

Cell and virus banks manufactured under cGMP conditions require sterility testing has to be

performed as per the appropriate test methods outlined in the pharmacopeias and also in the Code

Of Federal Regulations (21 CFR 610.12). Sampling requirements for cell and virus banks are not

provided within USP, EP or 21 CFR 610.12 guidelines. However, ICH Q5D “Derivation and

Characterization of Cell Substrates Used for the Production of Biotechnological/Biological

Products” states that sterility testing will be performed on individual containers. It also states that

1% of the bank or NLT 2 containers will be tested. This recommendation has become a standard

industry practice. However, since many cell and virus banks are produced under GMP conditions

some organizations use the USP/EP/JP Final Product testing guidelines. Either sampling plan has

been accepted by worldwide regulatory authorities.

Unprocessed Bulk Testing (For Protein and virus products)

Within the biotherapeutics industry “Unprocessed Bulk” has many other descriptions, including

cell/viral harvest, clarified cell/viral harvest, end of production cells or cells at the limit of in -

vitro cell age. Although the pharmacopeias and 21 CFR 610.12 do not reference or provide

sterility guidelines for these sample types, the FDA documents “Points to Consider in the

Characterization of Cell lines Used to Produce Biologicals” (1993) and “Points to Consider in the

Manufacture and Testing of Monoclonal Antibody Products for Human Use” (1997) do reference

the need for sterility or bioburden testing on each unprocessed bulk lot. These two documents

reference 21 CFR 610.12 for guidelines on appropriate sterility test methods to use. 10 mL/media

(for a total of 20 mL) is recommended for sterility testing of unprocessed bulk material.

Bulk Drug Substance (BDS) Testing

Sterility testing is required on each manufactured BDS lot. Sampling requirements for Bulk Drug

Substance is defined in 21 CFR 610.12, which states that no less than 10 mL will be tested. It is

not clear if that is meant for each media or if the amount is to be split into the two test media.

Industry practice has taken a conservative approach and 10 mL of BDS is tested in each media.

Thus a total of 20 mL is recommended for sterility testing.

Final Drug Product (FDP)Testing

Sterility testing is required on each manufactured FDP lot. Sampling requirements for final drug

product are provided in the pharmacopeias. The following tables outline the requirements as

stated in the current USP.




B.PYROGEN TEST [11]

The test involves measurement of the rise in body temperature of rabbits following the

intravenous injection of a sterile solution of the substance under examination.

Test animals: Healthy, adult rabbits of either sex, each weighing not less than 1.5 kg fed on a

balanced diet are used for the test.

Dose: NLT 0.5 ml/kg and not more than 10 ml/kg of the body weight.

Method: Insert a clinical thermometer into the rectum of each rabbit and normal readings of

body temperature are taken prior to the injection of test solution. Two such

readings are taken at an interval of 30 minutes and the mean is calculated. This

reading is taken as initial temperature of the rabbit. The test solution is injected

into the ear vein of each rabbit. Record the temperature of each rabbit at an interval

of 30 minutes for 3 hours after the injection. The difference between the maximum

temperature and initial temperature is taken as response.

Interpretation:

If the first test fails then repeat the test on additional 5 rabbits

QUALITY CONTROL OF BIOLOGICALS: It includes operational techniques and individual

activities that focus on controlling or regulating processes and materials to fulfill requirements for

quality. The focus is on preventing defective products or services from being passed on. [12]

A) Vaccines:

Vaccines are microbial preparations of killed or modified microorganisms that can stimulate an

immune response in the body to prevent future infection with similar microorganisms.

Batch Size

Minimum Number

to be Tested in Each

Media

100 or less 10% or 4 containers,

whichever is greater

101-500 10 containers

>500 2% or 20 containers,

whichever is less

Bulk – Up to 4 containers Each container

5- 50 20% or 4 containers,

whichever is greater

>50 2% or 10, whichever is

greater

Volume / Container

Minimum Number

to be Tested in Each

Media

Less than 1 mL The entire contents of

each container

1 – 40 mL

Half the contents of

each container,

but not less than 1 mL

41 – 100 mL 20 mL

>100 mL

10% of the contents of

the container,

but not less than 20 mL




Quality control tests:

1. Staining test:

Approximately 10 mL of the test sample is centrifuged in a pointed centrifuge tube at

approximately 2,000 xg for 30 minutes. The sediment or the bottom portion is spread on a slide

glass, dried and heat-fixed over a flame. The smear is then stained by the Gram’s Method and,

unless otherwise specified, examined microscopically at an approximately 1,000-fold

magnification.

Criterion for judgment:

No bacterial shall be observed other than those defined in the individual monographs.

2. Inactivation test:

Each purified bulk material shall be tested in mice for effective inactivation of the virus

before the addition of preservative and other substances.

The test should be performed with undiluted purified bulk material injected intra-

cerebrally into at least 20 mice, each weighing between 15 and 20 g. these mice shall be

observed for 14 days. Any symptoms caused by the virus shall be confirmed by

Immuno-florescence assay. At the end of the observation period, no cytopathetic effects

should be observed.

3. Freedom from abnormal toxicity:

Test in mice Test in guinea pigs

Take 5 healthy mice weighing 17-22g Take 2 healthy guinea pigs (250-350 g)

Inject one human dose NMT 1 ml Inject one human dose NMT 5 ml

Intra-peritoneally Intra-peritoneally

Observe the mice for 7 days Observe the guinea pigs for 7days

If more than one animal dies If more than one animal dies / ill health

preparation fails the test health preparation fails the test

If one animal dies repeat the test If one animal dies / ill health repeat the test




Preparation passes the test if no Preparation passes the test if no animal

animal dies in the second group. dies/ill health in the second group.

4. Sterility test and Pyrogen test

B) Immune sera

It contain antibodies to specific bacteria or viruses and it is of 2 types [13]

1. Antitoxin: It contains an antibody capable of destroying microorganisms including viruses

and bacteria.

2. Antivenom: It contains an antibody that is active against the venom of a snake, spider, or

other venomous animal or insect.


1. Immunoglobulin test

The cellulose acetate membrane electrophoretic test is used to analyze the protein

constituents in a test sample by differences in mobility of the protein solutions in electric

fields.

Dilute the test sample with diethylbarbiturate buffer solution (pH 8.6) to render the protein

solution approximately 5%.

Electrophorise the solution and stain it through the membrane with Ponceau 3R.

Protein constituents and relative concentrations are analyzed by densitometry.

Acceptance criteria:

NLT 95% of the total proteins shall be immunoglobulin.

2. Test for residual proteolytic enzymes

When measured by a suitable method for the detection of proteolytic enzyme activity, the test

material shall be practically free from residual proteolytic enzyme activity.

3. Test for antitoxin/ antivenom content.

In order to determine the antitoxin/antivenom content of the preparation under

examination its potency is determined with respect to the standard antitoxin preparation of

the test preparation by carrying out the assay.




The antitoxin/ antivenom content of each test sample shall be determined by statistical

analysis of assay results. The final product shall contain antitoxin/ antivenom at no less

than the value stated on the label.

C) Toxoids

A toxin that has been treated, as with chemicals or heat, so as to eliminate the toxic qualities

while retaining the antigenic properties.


1. Purity Test

Each bulk material shall be tested for

Protein nitrogen content

Toxoid content.

Protein nitrogen content test

The protein nitrogen content test is a method used to determine protein content by

measuring nitrogen in heated trichloroacetic acid-precipitable protein in the test sample by

the micro-Kjeldahl method. The criterion for judgment shall be given in the individual

monographs.

Dilute the sample if necessary with water and transfer to centrifuge tube.

Add 1/10th

volume of 50% w/v trichloroacetic acid solution to render trichloroacetic acid

concentration 4.5% w/v or higher.

Heat the mixture at 100 oC for 15 minutes and then cool it to room temperature.

Centrifuge the mixture at greater than 1400 xg for 10 minutes.

Add appropriate amount of 5% w/v of trichloroacetic acid solution to the precipitate,

shake and centrifuge it again.

Measure the nitrogen content in the precipitate using an appropriate method such as the

micro-Kjeldahl method.

Calculation of protein content

The protein content is calculated from the nitrogen content by the formula:

1 mg protein nitrogen (N) = 6.25 mg protein

Toxoid content test

The test shall be conducted by the flocculation test. The bulk material shall contain no less than

1,500 Lf toxoid of per mg protein nitrogen.




2. Detoxification test

The test shall be conducted on two kinds of the sample: the one shall be prepared by diluting the

test sample with 0.017 mol/L phosphate-buffered sodium chloride solution (pH 7.0) to the

concentration of x Lf/ml, and the other to a concentration higher than that of the final bulk not

exceeding y Lf/ml. The latter sample shall be preserved at 37 °C for 20 days prior to the test.

Following test shall be conducted on the samples with and without preservation at 37 °C for 20

days. Concentration of the diluted samples depends upon on the type of the toxoid and is given in

the monograph of that particular toxoid.

Procedure

Each sample shall be given by subcutaneous injection at a dose of 5 mL into at least 4 guinea pigs

weighing 300−400 g. The injected animals shall be observed for at least 21 days. No animal shall

die due to intoxication, or show specific symptoms of intoxication or other abnormal signs during

the observation period.

3. Sterility test – common for all the biological products

D) Blood products [14]

Human plasma protein fraction

Freeze dried human fibrinogen

Platelet concentrate

Whole human blood

Concentrated red blood cells

Human albumin

Dried human serum

Human normal immunoglobulin


1. Identification tests

Precipitation tests with specific antisera are used to show that only human serum proteins

are present.

The characteristic mobilities of blood proteins in an electrophoretic field are a sensitive

means of identifying fibrinogen, immunoglobulin, and the plasma protein fraction.

Proteins can also be identified by their sedimentation rate in an ultra-centrifuge; this

method is suitable for identifying and quantifying the different types of gamma globulins.




2. Sterility and Pyrogen test

All blood products must comply with the official tests for sterility, and those preparations (i.e.,

immunoglobulin and the plasma protein fractions) that are exposed to special risk of

contamination with pyrogens due to lengthy processing must also pass the pyrogen test.

3. Solubility

Complete solubility in an appropriate volume of the usual solvent, sometimes a specified time is

required for all solid preparations except fibrin foam. This indicates that the protein constituents

have not deteriorated.

4. Assay

For whole blood and concentrated red cells the assay is a determination of the hemoglobin

value. For the remaining products, except fibrin foam and thrombin, the protein content is

determined chemically.

The hemoglobin value is a measurement of concentration and is the amount of

hemoglobin present in a fixed volume of the patient’s blood. It is normally expressed as

grams per deciliter (g/dL) or grams per liter (g/L).

a) Human plasma

i. Inspection: Fresh-frozen Human Plasma, upon visual inspection, shall be free from marked

hemolysis, change in color or other abnormal findings

ii. Coagulation test: Take 0.1 mL of the test material in a test tube & kept in a water-bath at

37 °C, 0.1 mL of thromboplastin solution and 0.1 mL of 0.025 M calcium chloride solution

shall be added, and the time until the formation of a fibrin clot shall be recorded, and it shall

be within 20 seconds.

b) Frozen thawed human blood cells

i. Weight: When weighed by a suitable method, NLT 63g of red cells shall be recovered from

200 mL of source material.

ii. Test for hemoglobin content: When the test for Hemoglobin Content is applied, 1mL of

the final product shall contain NLT 0.24 g of hemoglobin.

c) Platelet concentrate

i. Inspection: Platelet, upon visual inspection, shall be free from marked hemolysis, change in

color or other abnormal findings.

ii. Platelet count test: The final product shall have a platelet count of NLT 0.2×1011

of

Platelet count.




iii. Red blood cell count and white blood cell count tests: The final product shall contain

normal counts of red blood cells and white blood cells.

E) Allergenic products

Allergen extracts are biological products that are administered to humans to diagnose,

prevent and treat allergic diseases [15].


Measurements of the total allergenic activity of individual batches of an allergen extract

should be undertaken preferably by IgE inhibition or by direct IgE-binding or other

immunoassay.

The estimated potency derived from the assay of total allergenic activity should be NLT

50% and NMT 200% of the stated potency.

In-vivo diagnostics for allergenic products:

The most widely used of these are the tuberculin employed of infection to detect

sensitization by mycobacterial proteins and hence the possible presence.

Apart from standardization of potency, which also serves as an identity test, the material

must be checked for sterility and for the absence of viable mycobacteria.

The product is also checked for absence of reactogenicity in unsensitized guinea pigs and

if required by the regulatory authority, for abnormal toxicity.

CONCLUSION

Microbial contaminations have a huge effect on biologicals. These are responsible for the loss of

potency, product variability and an increase in the levels of Bacterial endotoxins. As the

manufacture of biological products is a complex process, contamination of the product from

various sources has to be controlled strictly by using sterile conditions at all the stages of

manufacturing. Apart from this, it is also much important to perform the Sterility and Quality

control tests to ensure the manufacture of "biological product with efficient Quality". This review

provides clear information on the required conditions for the same.

REFERENCES

1. www.ich.org/fileadmin/Public_Web_Site/Training/ASEAN_Q5C_workshop_May_2011/

SESSION_Ia_ICH_Q5C

2. Public Health Service Act, Biological Products; as amended

3. www.fda.gov/ Mantej Chhina, Overview of biological products: june2013.

http://www.fda.gov/%20Mantej%20Chhina,%20Overview




4. www.eda.mohp.gov/biological products.

5. www.biologyexams4u.com/2013/02/difference-between-active-and-passive.html

6. Kalavati Suvarna, Anastasia Lolas, Patricia Hughes, Richard L Friedman, Case Studies

of Microbial Contamination in Biologic Product Manufacturing, American

Pharmaceutical Review;january 2014

7. The International Pharmacopoeia, Fourth Edition2014.

8. European Pharmacopoeia/2.6 Biological Products.2005

9. www.alibaba.com/premium/membrane_filter.html?uptime=20141216&ptsid=1012000055

713559&crea=49818132290&plac=&netw=s&device=c&ptscode=0110202010040001

10. www.wuxiapptec-SterilityTstg-Biologics

11. São Paulo Jan, Brazilian Journal of Microbiology 2004:35;( 1-2)

12. www.slideshare.net/nishathfathima/inprocess-quality-control-tests-for-biological-products

13. www.encyclopedia.com/doc/1G2-3409800039.html

14. www.sfda.gov.sa/ar/drug/resources/DocLib2/Guidelines for Production and Quality

Control of BloodProducts_v1101.

15. www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/10/WC500

004630.

http://www.eda.mohp.gov/biological

http://www.americanpharmaceuticalreview.com/1429-AuthorProfile/1733-Kalavati-Suvarna/

http://www.americanpharmaceuticalreview.com/1429-AuthorProfile/1734-Anastasia-Lolas/

http://www.americanpharmaceuticalreview.com/1429-AuthorProfile/1735-Patricia-Hughes/

http://www.americanpharmaceuticalreview.com/1429-AuthorProfile/1736-Richard-L-Friedman/

http://www.scielo.br/scielo.php?script=sci_serial&pid=1517-8382&lng=en&nrm=iso

http://www.sfda.gov.sa/ar/drug/resources/DocLib2/Guidelines

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