guidance manual quality control testing of...

Guidance Manual-Quality control testing of Streptokinase of 42

NIB/EHL/GM/01

Guidance Manual

“Quality Control Testing of Streptokinase”

NATIONAL INSTITUTE OF BIOLOGICALS (Ministry of Health & Family Welfare)

Government of India A-32, SECTOR-62,

NOIDA (Uttar Pradesh) PIN: 201307

Web site: www.nib.gov.in, e-mail: [email protected]

http://www.nib.gov.in/


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ABBREVIATIONS USED

AMC Annual maintenance contact

ASO Anti Streptolysin-O

BP British Pharmacopoeia

BSA Bovine Serum Albumin

CDSCO Central Drugs Standards and Control Organization

CoA Certificate of analysis

CPB Citrophosphate Buffer

CRS Certified Reference standard

CSE Control standard endotoxin

DCA Drugs and Cosmetic Act

DCG(I) Drugs Controller General of India

DID Double immune diffusion

EHL Enzymes and Hormones laboratory

EP European Pharmacopoeia

EU Endotoxin unit

FSH Follicle stimulating hormone

FTIR Fourier transform Infrared spectrophotometer

HBsAg Hepatitis B surface antigen

hCG Human Chorionic Gonadotropin

HCV Hepatitis C virus

HIV Human Immuno deficiency virus

HMG Human Menopausal Gonadotropin

HPLC High performance liquid chromatography

HSA Human Serum Albumin

IAEC Institutional Animal Ethics Committee

IEC International electro technical commission

IP Indian Pharmacopoeia

IPC Indian Pharmacopoeia Commission


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IRS International reference standard

ISO International organization for standardization

IU International Unit

LAL Limulus Amoebocyte Lysate

LH Luteinizing hormone

LRW LAL Reagent Water

MVD Maximum valid dilution

NABL National Accreditation Board for Testing and Calibration Laboratories

NCL National control laboratory

NIB National Institute of Biologicals

NIBSC National Institute of Biological Standards and Control

NPC Negative product control

NWC Negative water control

PEG Poly ethylene glycol

PPC Product positive control

PWC Positive water control

QA Quality Assurance

QC Quality Control

RBC Red Blood Cell

RT Room temperature

rDNA Recombinant DNA

SOP Standard Operating Procedure

SR & RDU Sample Receipt and Report Dispatch Unit

TTI Transfusion transmitted infection

USP United States Pharmacopoeia

WRP Working reference preparation


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Contributors:

Dr. J. P. Prasad Scientist Grade II, Laboratory Head [email protected]

Mrs. Sudha V. Gopinath Scientist Grade-III [email protected]

Mr. N. Nanda Gopal Junior Scientist [email protected]

Mr. Subhash Kumar Laboratory Technician [email protected]

Mr. Rajeev Shrivastava Laboratory Assistant [email protected]


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ACKNOWLEDGEMENT

I take this opportunity to express my gratitude to all the scientific staff of Enzymes and

Hormones laboratory Ms. Sudha V. Gopinath, Mr. N. Nanda Gopal and help provided by

Mr. Subhash Kumar and Mr. Rajeev kumar Shrivastava in preparing the “Guidance

Manual on Quality Control testing of Streptokinase”.

I would like to show my greatest appreciation to Dr. Surinder Singh, Director (i/c),

National Institute of Biologicals, who has motivated and encouraged every time, without

his encouragement and guidance this document would not have materialized.

This document has taken a shape with the past experience in establishing the laboratory

and taken a way to get an Accreditation for ISO/IEC 17025. I hope this document will

provide guidance to manufacturers / importers /personnel engaged with Quality Control

testing of Streptokinase in establishing quality system.

The guidance and support received from all the members who contributed was vital for the

success. I am grateful for their constant support and help.

Any suggestion in improving the document is highly appreciable.

I wish all success who has contributed in bringing the guidance document.

Dr. J. P. Prasad

Head,

Enzymes & Hormones Laboratory.


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Table of Contents

S.No Contents Page No

1. Purpose 7

2. Scope 7

3. Introduction to Healthcare and Biotherapeutics 8

4. Introduction to Streptokinase 9

5. Quality control test parameters of Streptokinase 11

6. Sample receiving, transfer & testing plan 12

7. Inter laboratory testing 12

8. Data analysis and preparation of Certificate of Analysis 13-16

9. Infrastructure of the laboratory 16-17

10. Reference standards 17-20

11. Biosafety procedures & biomedical waste disposal 21-23

12. Description of test procedures 23-36

13. Documents required for protocol scrutiny 37

14. Quantity of vials required for evaluation 37

15. Training 37

16. Annexures 38-41

17. References 42


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The National Institute of Biologicals (NIB) is an autonomous institute under Ministry of

Health & Family Welfare, Government of India. The mandate of the Institute is to test

Biological products, both manufactured indigenously and imported as per Pharmacopoeial

specifications, collaborate with Indian Pharmacopoeial Commission in finalizing the

specifications, train personnel in the public and private sectors, prepare National reference

standards, collaborate with other scientific institutions in upgrading technologies and

keeping abreast of scientific advances made in the field of quality assessment of Biological

products. The Institute also provides technical expertise to the Central Drugs Standards

and Control Organization (CDSCO) and participates in regulatory visits to manufacturing

facilities on the request of CDSCO.

Purpose

The purpose of this guidance manual is to assist the manufacturers of Streptokinase

including laboratories in developing a quality assurance (QA) program and quality control

(QC). This will also guide in establishing tests related to biophysicochemical and safety

test parameters given in this manual are intended to help the manufacturers / labs to

strengthen the quality of Streptokinase.

Scope:

This document will provide a guidance to manufacturers / personnel working in a quality

control testing Laboratory of Biotherapeutics like enzymes (Streptokinase) to put the

quality system in place while establish a testing laboratory. This gives a systematic

approach to establish as per the regulation, data standards and to maintain good

laboratory practices that will provide reliable and reproducible results. The approach will

ensure the quality of the test procedures carried out and the information which is

generated to provide robust evidence.


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Indian Health care & biotherapeutics:

India‟s growing population, demand for quality drug products and innovative drugs to

combat diseases are leading to increased demand for biotechnology drugs and products.

New types of diseases and demand for improved drugs are also leading to greater

research and development (R&D) activities. A category of such biopharmaceutical

promising to combat diseases and disorders includes therapeutic enzymes.

The concept of the therapeutic enzyme has been around for at least 40 years. In 1987, the

first recombinant enzyme drug, Activase®. (Alteplase; recombinant human tissue

plasminogen activator), was approved by the Food and Drug Administration (FDA). This

„clot-buster‟ enzyme is used for the treatment of heart attacks caused by the blockage of a

coronary artery by a clot formation. This was the second recombinant protein drug to be

marketed.

Biotechnology innovations have made significant contributions in the field of health care

with more than 200 biologic medicines and vaccines benefiting millions of patients

worldwide and further a large number of biotech products are in pipeline.

Advances in biotechnology over the past 20 years have allowed pharmaceutical

companies to produce safer, cheaper enzymes with enhanced potency and specificity.

One such kind of drugs includes r-Streptokinase, r-Follicle stimulating hormone,

r-Somatropin were taken up for evaluation at enzymes and hormones laboratory.


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1.1. Introduction to Streptokinase:

Streptokinase is a 47-kDa (414-amino-acid) protein from pathogenic strains of the

Streptococcus. To dissolve a blood clot, streptokinase forms a 1:1 molar complex with

plasminogen, by converting plasminogen to plasmin with a half-life of 30 min.

The extracellular enzyme streptokinase is produced by various strains of h-hemolytic

streptococci. The enzyme is a single-chain polypeptide that exerts its fibrinolytic action

indirectly by activating the circulatory plasminogen. The complete amino acid sequence of

streptokinase was first established by Jackson and Tang. The protein exhibits its

maximum activity at a pH of approximately 7.5 and its isoelectric pH is 4.7 .The protein

does not contain cystine, cysteine, phosphorous, conjugated carbohydrates and lipids.

Streptokinase produced by different groups of streptococci bacteria, differ considerably in

their structure. Consequently, research has focused on structurally modifying

streptokinase to extend half-life, reduce or eliminate immunogenicity, and improve

plasminogen activation. Structurally modified streptokinases have been produced in

several ways including genetic mutation, recombinant DNA technology and chemical or

enzymatic modification of the native streptokinase.

Mutant streptokinases with improved stability have been prepared. Two of the major sites

of the proteolytic action of plasmin on streptokinase are Lys59 and Lys386. This

knowledge has been used to engineer variants of streptokinase that are resistant to

plasmin and, therefore, have a longer functional half-life. The plasmin-resistant forms of

streptokinase have been found to be as active as the native streptokinase. Recombinant

streptokinase also produced in other vectors like yeast Pichia pastoris is glycosylated, and

this appears to enhance its resistance to proteolysis.

Following a similar path, attempts have been made to extend the half-life of native non-

glycosylated streptokinase by complexing it with polymers such as polyethylene glycol

(PEG). Plasmin-resistant, long-life variants of protein-engineered streptokinase have been


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produced in a protease-deficient recombinant Bacillus subtilis WB600. It appears that the

streptokinase domains responsible for activity, stability and immunogenicity have

considerable overlap. Therefore, a future therapeutically optimal streptokinase will not be

necessarily the most active nor the longest lived.

Streptokinase variants with one or more of the normal amino acids residues replaced by

others have been prepared in attempts to enhance plasminogen activation. Some of the

modified variants displayed an enhanced stability. The preferred variant had Lys59

replaced with a glutamic acid residue. Streptokinase derivatives having platelet

glycoprotein-binding domains have been reported. These derivatives produced higher

local concentrations of plasmin in vivo when compared to unmodified streptokinase.

Commercial production of streptokinase requires special attention to biosafety

considerations because the protein is potentially immunogenic to process workers.

In addition, care is necessary if streptokinase is being produced using natural strains of

streptococci because all streptokinase producing streptococci are potentially pathogenic.

Streptokinase is immunogenic and can cause drug resistance, fever, and allergic

reactions. Streptokinase is certainly more cost-effective; however, its use is not risk free.

Streptokinase mechanism


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Natural Streptokinase amino acid sequence

Chemical

Formula C2100H3278N566O669S4

Chemical

Structure

Streptokinase

IAGPEWLLDRPSVNNSQLVVSVAGTVEGTNQDISLKFFEIDLTSRPAHGGKTEQGLSPKS

KPFATDSGAMSHKLEKADLLKAIQEQLIANVHSNDDYFEVIDFASDATITDRNGKVYFAD

KDGSVTLPTQPVQEFLLSGHVRVRPYKEKPIQNQAKSVDVEYTVQFTPLNPDDDFRPGLK

DTKLLKTLAIGDTITSQELLAQAQSILNKNHPGYTIYERDSSIVTHDNDIFRTILPMDQE

FTYRVKNREQAYRINKKSGLNEEINNTDLISEKYYVLKKGEKPYDPFDRSHLKLFTIKYV

DVDTNELLKSEQLLTASERNLDFRDLYDPRDKAKLLYNNLDAFGIMDYTLTGKVEDNHDD

TNRIITVYMGKRPEGENASYHLAYDKDRYTEEEREVYSYLRYTGTPIPDNPNDK

1.2. Quality control testing procedures:

Sample receiving procedures:

Lab Personnel receives the coded samples along with the Sample receiving register and

entries done by Sample receiving unit are verified by the analyst and acknowledged.

Physical verification of the code of sample on all the vials/bott les, total number of

vials/bottles received with cold chain and entered in to the lab register with an individual

identification number of the lab to be given to each batch by the analyst, submitted to the

laboratory head for information and signatures. In case of non receipt of samples,

mentioning specific reason for not receiving the samples in the register and acknowledged.

Table: 1.1

S.No SRRDU Code No.

Assigned Lab ID No.

Name of the product and dose/ Strength

No. of vials received for

testing


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Sample receiving, transfer and testing plan

Verifying the vials received for QC testing were then allocated as per testing plan at

Enzymes and Hormones Laboratory to conduct specific test parameter as T1, T2…as

follows. The tests which are performed by central facility and other testing laboratories,

were segregated and sumitted to concern laboratory along with particular proforma for inter

laboratory testing.

PROFORMA FOR INTER LABORATORY SAMPLE TRANSFER

Ref. No: Date:

Test to be conducted for:

Method to be followed:

SAMPLE DETAILS:

S. No SRRDU Code No.

Lab ID No.

Name of the product No. of vials submitted

Status of vials on receipt Cold chain maintained YES / NO

Seal intact YES / NO

Remarks:

Received by Name & signature with date.

…………………………. …………………….

Signature of Lab Head/ I/c Signature of Lab Head

E&H Laboratory


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Data compilation and Preparation of Certificate of Analysis:

After completing preliminary tests, compilation of data, analyzed and a Certificate of

Analysis (CoA) for decoding the sample is prepared and submitted to SRRDU alongwith

with the raw data and formats. The SRRDU decodes the sample and submits all the

documents received from the manufacturer / importer to the laboratory. Up on receiving the

documents, the laboratory scrutinize the documents and comments on summary of

protocol is prepared. The analyst prepares final Certificate of Analysis and the file is

submitted to the Director for approal before relaese of test report.

Table. 1.2.

S.No Documents required Submitted by

manufacturer / Importer

Yes/No

Comments Remarks

1. Certificate of analysis of the

finished product with name of pharmacopoeia complied

2. Letter of DCG(I)/ADC/ Port office/CDSCO

/Manufacturer‟s Letter

3. Manufacturing protocol

4. Batch Release Certificate from NCL/Manufacturer of country of

Origin

5. Copy of test/ import / manufacturing license

6. Non-withdrawal certi ficate

7. Product insert


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Test parameters conducted on Streptokinase for evaluation as follows.

Streptokinase bulk:

Table: 1.3.

S.No Test Parameter Specification Pharmacopoeia followed

1. Description Complies with the monograph Streptokinase IP2010.

2. Identification Method: A Clot forms and lyses with in 30mins in with citrated human plasma and lyses does not occur with in 1 hour with citrated bovine plasma.

IP2010.

3. pH 6.8 to 7.5 in 5000 IU/ml

IP2010.

4. Loss on drying Not more than 4.0% on 1g by drying over Phosphorus pentoxide at a pressure not exceeding 2.7 kPa for 24 hours.

IP2010.

5. Streptodornase activity Not more than10 IU of Streptodornase activity/ 1, 50,000 IU of Streptokinase activity per ml. (A3+A4)-(A1+A2)< (A5+A6+A7+A8) / 2- (A3+A4) at 260 nm

IP2010.

6. Streptolysin activity Not more than 1.5 times absorbance obtained by the reference standard at 550 nm.

IP2010.

7. Assay The estimated potency is not less than 90 per cent and not more than 111 per cent of the stated potency. The confidence limits (P = 0.95) of the estimated potency are not less than 80 per cent and not more than 125 per cent of the stated potency.

IP2010.

8. Bacterial endotoxin NMT 0.02 EU/ 100 IU of Streptokinase activity. Complies with test for BET.

IP2010.

9. Abnormal toxicity Complies with test for Abnormal toxicity IP2010.

10. Sterility Complies with test for sterility IP2010.


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Test parameters of Streptokinase injection:

Table: 1.4.

S.No Test parameter Specification Pharmacopoeia followed

1. Clarity Clear, no visible residues found on visual observation IP 2010

2. Particulate matter Free from particles on visual inspection IP 2010

3. Identification Method: A Clot forms and lyses occurs with in 30mins in with citrated human plasma and lyses does not occur up to 1 hour with citrated bovine plasma.

IP 2010

4. pH 6.8 to 7.5 in 5000 IU/ml

IP 2010

5. Streptodornase Activity A1-A2< 0.5 (A3+A4) - A2 at 260 nm IP 2010

6. Streptolysin Activity Not more than 1.5 times absorbance obtained by the reference standard at 550 nm.

IP 2010


IP 2010

8. Bacterial Endotoxin NMT 23.33 EU/ 10000 IU of Streptokinase per ml. Complies with test for BET

IP 2010

9. Sterility Complies with test for sterility IP 2010


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Test parameters of r-Streptokinase injection:

Table: 1.5.

1.3. Infrastructure:

The laboratory is equipped with instruments like High Performance Liquid

Chromatography (HPLC), UV-Visible spectrophotometer, Micro plate Reader, Micro plate

washer, Vertical Electrophoresis System, pH meter, Kjeldahl apparatus, Vacuum Oven,

Refrigerated centrifuge, Digital Water bath with external temperature recorder, Incubator,

Under Counter Freezer, Electronic Weighing Balance and other basic facilities like, Bio-

safety cabinets, Fume hood etc. Major equipment are placed under annual maintenance

contract (AMC) and calibrated annually by an NABL accredited laboratory or equipment

specific vendors as per preventive maintenance schedule.

Intermediate Performance checks: To give an accurate and reproducible result the

intermediate checks for equipment is performed by the lab personnel as per the schedule

according to the concern equipment SOP. Any deviation from acceptance criteria of

equipment during the check is intimated to the vendor for rectification.

S.No Test parameter Specification Pharmacopoeia followed

1. Clarity Clear, no visible residues found on visual observation IP 2010

2. Particulate matter Free from particles on visual inspection IP 2010

3. Identification Method: A Clot forms and lyses occurs with in 30mins in with citrated human plasma and lyses does not occur up to 1 hour with citrated bovine plasma.

IP 2010

4. pH 6.8 to 7.5 in 5000 IU/ml

IP 2010


IP 2010

6. Bacterial Endotoxin NMT 23.33 EU/ 10000 IU of Streptokinase per ml. Complies with test for BET

IP 2010

7. Sterility Complies with test for sterility IP 2010


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List of equipment in Enzymes and Hormones Lab:

Table: 1.6.

S. No Name of the Equipment S. No Name of the Equipment

1. U.V/Visible

Spectrophotometer

8 pH meter

2 Electronic Balance 9 Refrigerated Centrifuge

3 Microplate Reader 10 Water bath (Digital)

4 Micro centrifuge 11 Bio Safety cabinet

5 Refrigerator 20 To 80C 12 Fume hood

6 BOD Incubator 13 Vortex mixer

7 Hot air oven 14 Microplate washer

1.4. Chemicals, reagents and labware

An indenting process involves identification of suitable/ probable vendors along with

the track record of items supplied for its quality, timeliness and satisfactory remarks. With

all these information the chemicals, reagents and labware were purchased based on

vendor evaluation. This will then undergo technical suitability and appropriate quantity

requirement and justification in the indent and proceed to the process of procurement as

per the policy.

1.5. Reference standards

The laboratory maintains repository of various International Reference Standards with

metrological traceability. These are procured from National Institute of Biologicals Standard

and Control (NIBSC) U.K., and stored in the Central Reference Standards Laboratory at

-860C. These standards are used for estimation of potency assays and other test

parameters (Streptodornase activity and Streptolysin activity in case of Streptokinase) as

per the requirement for the evaluation of samples.


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List of Reference standards available

Table: 1.7.

Reconstitution of reference standards to prepare working reference preparation:

(Streptokinase reference standard 1030 IU/Amp)

All the activities related to reconstitution, aliquoting should be performed in

biosafety cabinet. Allow the lyophilized ampoule to attain room temperature , tap the

ampoule gently to collect the material to the bottom. Take care that no material is lost from

the ampoule and no glass should falls into the ampoule. Add 1030 µl of Water for injection

to vial containing the reference material to give 1000 IU/ml working standard and vortex

gently.

Aliquoting of reconstituted Streptokinase reference preparation:

Take out 15 µl from reconstituted working reference preparation (1000 IU/ml)

ampoule to 0.5 ml microcentrifuge tubes and capped tightly. The microcentrifuge tubes

are numbered as follows (Eg.STK/Ref/1/60,STK/Ref/2/60etc.,) and store the entire vials in

–860C deep freezer. These vials were used in the test and prepared as per the dilution. Fill

the details of vials in Reference standard stock register and fi le. The micropipette tips and

ampoules are discarded as per biosafety procedures.

S. No Name of the Standard Catalogue No. Source

1. Streptokinase 00/464 NIBSC, U.K.

2. Streptokinase and Streptodornase 62/007 NIBSC, U.K.

3. Anti-Streptolysin-O 97/6620 NIBSC, U.K.


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Transport and use of working reference preparation:

Take out the reference material from the -860C ultra freezer for use and keep it in a

temperature controlled container / mini-cooler. The reference material is used as soon as

possible by diluting with tris buffer solution.

Preparation of Working Reference preparation:

Take one micro-centrifuge vial (eg.STK/Ref/1/60), pipette out 10 µl of 1000 IU/ ml and

add to vial containing 990 µl of Tris buffer to give 10 IU/ml. Table for dilution of working

reference standard for potency testing of Streptokinase as follows.

Table: 1.8.

S. No IU/ml Volume of 10/ml Volume of Buffer Total volume

1 0.5 IU/ml 50 µl 950 µl 1000

2 1.0 IU/ml 100µl 900µl 1000

3 2.0 IU/ml 200µl 800µl 1000

4 4.0 IU/ml 400 µl 600µl 1000


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NIBSC 3rd INTERNATIONAL REFERENCE STANDARD

STREPTOKINASE (1030 IU).

Catalogue No: 00/464

Aliquoted on : ________________

Description: Added 1030 µl of WFI and Dispensed 15 µl to each vial, designated as STK

from 1/60 to 60/60, Store at -860C, make an entry with date at the box

provided against the use.

Scheme of vials used:

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

51. 52. 53. 54. 55. 56. 57. 58. 59. 60.

Remarks:


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1.6. Central facility laboratories (Inter laboratory testing)

The test parameters conducted at central facility laboratories includes abnormal

toxicity and Sterility. The samples are forwarded to testing lab along with inter-laboratory

testing format with duly signed by the laboratory head indicating the tests to be performed

on samples along with the product insert.

Animal related tests were conducted in animal facility with the pre-approval of

protocols by the Institutional Animal Ethics Committee (IAEC), test procedures were

carried up on animals as per regulatory guidelines.

Sterility test is conducted as per the method recommended by the monograph.

1.7. QC test parameters conducted at EHL:

Bio-physico-chemical properties and safety of biotherapeutics are tested /conducted

as per the monographs of Indian Pharmacopoeia method followed by the manufacturer or

any other Pharmacopoeia followed. Written SOPs should be followed to conduct the test

parameters, equipment use and documentation. Bench protocols, formats for test results,

and statistical software for calculating potency of Streptokinase for evaluation are followed

as per ISO/ IEC 17025 guidelines.

1.8. Bio-safety procedures to be followed in laboratory for safe disposal of

biomedical waste:

Bio-safety, a key component of total quality control programme. There is a potential risk

of infection to the laboratory staff who handle samples of body fluids/tissues, infected

waste and infected specimens etc..


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There are several ways that workers can acquire blood borne infection from a donor or

from his/her specimen either by:

1. Direct contact with blood / body fluids

2. Accidental inoculation of infected blood/body fluids

3. Accidental cuts with contaminated sharps

4. Indirect contact with contaminated equipment or any other inanimate infected

object.

In a laboratory, all bio-safety measures should be ensured and workers must take all

precautionary measures to protect themselves from accidental injury while handling the

blood.

Bio-safety procedures

1. All laboratory staff shall be vaccinated against Hepatitis B and immunization

records be maintained.

2. Only authorized persons enter the laboratory working areas.

3. Keep laboratory doors closed.

4. Keep children out of laboratory areas.

5. Do not wear open toed footwear /street shoes in laboratories- change footwear

before entering /leaving.

6. Wear protective clothing when working in the laboratory and remove before leaving

the laboratory.

7. Store used protective laboratory clothing in separate lockers or cupboards.

8. Eating, drinking, smoking, applying cosmetics and handling contact lenses is

prohibited in the laboratory.

9. Storing foods or drinks for consumption anywhere in the laboratory areas is

prohibited.

10. Wear protective gloves and personnel should disinfect their hands before and after

using gloves.

11. Gloves must be removed and discarded after completion of bench work.


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Decontamination:

Work bench practices

I) Used microtips are discarded in 1% sodium hypochlorite solution.

II) Test devices are discarded in 1.0% Sodium Hypochlorite solution after the test .

III) Reagents/ controls from kits (Positive Control, Negative Control) are emptied into

1% Sodium Hypochlorite and discarded directly into zip lock bags.

IV) Used gloves, filter paper sheets spread on workbench and alcohol / Hypo swabs

are also discarded into a punched zip lock bags.

V) The collected waste is allowed to stand for overnight in contact with 1% Hypo and

emptied into punched zip lock bag.

VI) The punched zip lock bags containing all kinds of wastes are collected into larger

punched biohazard bags.

VII) Two or three such bags are placed in the decontamination autoclave and

autoclaved at 1210C , 15 psi for 40 minutes.

viii) All instrument touch points, single channel pipettes, marker pens, forceps,

refrigerator are swabbed with 70% alcohol after use.

1.9. Description of test parameters applicable for Streptokinase bulk, injection

Identification by Clot lyses method:

Procedure: Place 0.5 ml of citrated human plasma in a hemolysis tube maintained in a

water-bath at 37°C. Add 0.1 ml of a dilution of the preparation under examination

containing 10 000 IU of streptokinase activity per milliliter in phosphate buffer pH 7.2 and

0.1 ml of a solution of human thrombin containing 20 IU/ml in phosphate buffer pH 7.2. Mix

immediately.

Acceptance criteria: A clot forms and lyses within 30 min in tube containing Streptokinase

and citrated human plasma. Repeat the procedure using citrated bovine plasma. The clot

does not lyse within 60 minutes.

Results proforma: See annexure-I

Reference: Indian Pharmacopoeia 2010.


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pH Determination:

Procedure: Determine the pH of solution prepared by diluting the streptokinase in water

for injection to obtain a solution containing 5000 IU of streptokinase activity per milli liter.

Acceptance criteria: 6.8 to 7.5


Test for pH

EQUIPMENT DETAILS:

pH meter:

Equipment ID:

Calibrated on:

Details of Reference buffer(s) used in calibration

Product name/Sample code:

Manufacturer:

Batch No: Mfg. date: Exp. date:

Reference Buffer Source Date of Expiry Readings

4.01

7.0

9.0

Name of product/

sample code

Reading-1 Reading-2 Reading-3 Average Comply/ Not

comply


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Results:

Streptodornase activity:

Reconstitution of Streptokinase – International Reference Standard for Streptodornase

(2400 IU) & Streptokinase 3100 IU – NIBSC (62/007) – Add 1 ml of water for injection to

the ampoule containing Streptokinase (3100 IU) and Streptodornase (2400 IU) and vortex.

Aliquoted 10 µl of reconstituted reference preparation into sterile microcentrifuge tubes

(1.5ml), designated as STDR (Streptodornase), numbered accordingly (1/STDR,

2/STDR…,) & immediately stored at -860C.

Preparation of working preference reparation of Streptodornase:

Take out one microcentrifuge tube of 1.5ml containing 10 µl of aliquot, add 1190 µl of

Imidazole buffer pH 6.5 to achieve streptodornase activity of 20 IU/ml, i.e. (120 time‟s

dilution).

Procedure:

Add 0.5 ml of a 0.1 % w/v solution of sodium deoxyribonucleate in imidazole buffer pH

6.5 into each of eight centrifuge tubes. In each of the first two tubes add 0.25ml of

imidazole buffer of pH 6.5 and 0.25ml of solution of the Streptokinase (150,000 IU / ml) in

imidazole buffer pH 6.5 followed immediately by 3.0 ml of 0.25M Perchloric acid. Mix the

contents of each tube. Centrifuge for 5 minutes at 3000 rpm and measure the absorbance

of the each of the supernatant liquids at about 260 nm using as the blank a mixture of 1.0

ml of imidazole buffer pH 6.5 and 3.0 ml of 0.25M perchloric acid. Calculate two

absorbencies as A1 and A2 respectively for first and second tubes. In each of the

remaining six tubes (numbered as 3 to 8) add, respectively 0.25, 0.25, 0.125, 0.125, 0 and

0 ml of imidazole buffer pH 6.5 followed by 0.25 ml solution of the Streptokinase (150,000

IU / ml) and finally 0, 0, 0.125, 0.125, 0.25 and 0.25 ml respectively of a solution of the

working Standard Preparation containing 20 Units of Streptodornase activity/ ml in

imidazole buffer pH 6.5. Mix the contents of each tube, incubate at 37o C for 15 minutes

and add to each tube 3.0 ml of 0.25M Perchloric acid. Mix the contents of each tube,


NIB/EHL/GM/01

centrifuge and measure the absorbencies as A3 to A8 of each of the supernatant liquids at

260 nm, using as the blank the mixture specified above. Calculation is performed by using

the formula: (A3+A4)-(A1+A2) < (A5+A6+A7+A8) / 2- (A3+A4).

Limit: Not more than 10 IU of Streptodornase activity per 100 000 IU of streptokinase

activity for streptokinase bulk.

Acceptance criteria: (A3+A4)-(A1+A2) < (A5+A6+A7+A8)/2- (A3+A4) for Streptokinase

Bulk solution and (A1 -A2) is less than 0.5 (A3+A4)-A2 for Streptokinase injection and

Streptokinase as per IP 2010.

Results proforma: See annexure-II


Streptolysin activity:

Preparation of working reference of Anti-Streptolysin-O, Human- NIBSC Reagent 97/662:

Add 50 µl of 10 IU/ml Anti Streptolysin-O, Human reference reagent to tube containing 50

µl of buffer (9 volumes of 0.9% saline and Citrophosphate buffer of pH 7.2) to prepare 5

IU/ml working reference reagent.

Preparation of 1.6% Rabbit erythrocyte Suspension:

Collect 5ml of blood in 3.2% w/v EDTA from Rabbit.

Centrifuge at 2000 rpm for 10min and discard the plasma

Take 0.5 ml settled erythrocyte and make up to 25ml with 0.9% saline solution, in centrifuge tube

Centrifuge at 3000 rpm for 20 minutes and after centrifugation discard the supernatant

Wash four times until a clear supernatant

Dilute 0.8ml of the suspension of erythrocytes to 50ml (1.6 %) with a mixture of

1 volume of citrophosphate buffer pH 7.2 & 9 volumes of a 0.9% w/v solution of sodium chloride.

Prepared suspension may be used up to one month, stored at 20 to 80 C:


NIB/EHL/GM/01

Procedure:

Take Streptokinase bulk in 500,000 Units in 0.5 ml in a mixture of 90 volumes of

saline solution and 10 volumes of Citrophosphate buffer pH 7.2 in a sterile glass tube. Add

0.4 ml of a 2.3% w/v solution of sodium thioglycollate and incubate in water bath at 37oC

for 10 minutes.

Add 0.1 ml of a solution of a reference reagent of human Antistreptolysin-O (5

IU/ml) for each tube and incubate at 37oC for 5 minutes. Add 1.0 ml of rabbit erythrocyte

suspension; continue the incubation for 30 minutes and centrifuge at about 1000 rpm.

Repeat the above procedure using 0.5 ml blank (mixture of saline solution and

Citrophosphate buffer pH 7.2) in place of Streptokinase solution. Measure the absorbance

of the supernatant of the test and blank at 550 nm.

Acceptance criteria: The absorbance of the test solution is not more than 50 per cent than

that of the reference solution.

Results Proforma: See annexure-III


Loss on drying:

Procedure: Determine on 1g by drying over Phosphorus pentoxide at a pressure not

exceeding 2.7 kPa for 24 hour

Acceptance criteria: Not more than 4.0%



NIB/EHL/GM/01

Assay/Potency by Chromogenic method:

Procedure: The potency of streptokinase is determined by comparing its capacity to

activate plasminogen to form plasmin with the same capacity of a reference preparation of

streptokinase calibrated in International Units; the formation of plasmin is determined using

a suitable chromogenic substrate.

The International Unit is the activity of a stated amount of the International Standard

for streptokinase. The equivalence in International Units of the International Standard is

stated by the regulatory authority.

Reference and test solutions: Prepare two independent series of four dilutions of

each of the Streptokinase sample and of the reference preparation of streptokinase in

tris(hydroxymethyl) aminomethane sodium chloride buffer pH 7.4 , in the range of 0.5, 1.0,

2.0 and 4.0 IU/ml. Prepare and maintain all solutions at 37 °C.

Substrate solution: Mix 1.0 ml of tris (hydroxymethyl) aminomethane buffer pH 7.4

with 1.0 ml of chromophore substrate.

Add 5 µl of a 10 % w/v of Polysorbate /Tween 20, Keep at 37 °C in a water-bath.

Immediately before commencing the activation assay, add 45 µl of a 1 mg/ml solution of

human plasminogen. Analyse each streptokinase dilution, maintained at 37 °C, in

duplicate. Initiate the activation reaction by adding 60 µl of each dilution to 40 µl of

substrate solution. For blank wells, use 60 µl of tris (hydroxymethyl) aminomethane

sodium chloride buffer solution pH 7.4 instead of the reference and test solutions. Allow

the reaction to proceed at 37 °C for 20 min in a water bath and read the absorbance at 405

nm.

Add 50 µl of a 50 per cent V/V solution of glacial acetic acid. Best results are

obtained when the absorbance for the highest streptokinase concentration is between 0.1

and 0.2 (after blank subtraction). If necessary, adjust the time of incubation in order to

reach this range of absorbances.


NIB/EHL/GM/01

Streptokinase bulk solution intend for use in the manufacture of parenteral

preparations without a further appropriate procedure for the removal of Bacterial

endotoxins complies with the following additional requirement.

Acceptance criteria: The estimated potency is not less than 90 per cent and not more

than 111 per cent of the stated potency. The confidence limits (P = 0.95) of the estimated

potency are not less than 80 per cent and not more than 125 per cent of the s tated

potency.

Calculations by Statistical analysis: Calculate the regression of the absorbance on log

concentrations of the solutions of the substance under examination and of the reference

preparation of streptokinase and calculate the potency of the substance under examination

using the usual statistical methods for parallel-line assays.

Results proforma: See annexure-IV


Bacterial endotoxin test by Gel clot method:

This method explains to conduct bacterial endotoxin detection by gel clot method using

LAL test kit.

Method: This method is followed to perform Bacterial endotoxin limit test using Limulus

Amoebocyte Lysate (LAL) kit by gel clot method for Streptokinase injection.

Preparation of LAL, CSE and Sample dilution to MVD:

Depyrogenation: All materials coming in contact with the specimen or test reagents must

be endotoxin free. Perform the test carefully to avoid contamination &depyrogenate all

glassware at 250 °C for 30minutes.

Preparation of LAL Reagent (Lysate): A lyophilized vial of LAL reagent is reconstituted

with LAL Reagent water (LRW) as per the certificate of analysis and do not vortex.


NIB/EHL/GM/01

Preparation of Control standard Endotoxin (CSE):

Reconstitute the vial of CSE with 5ml of LAL reagent water (LRW) or as indicated

in the Certificate of Analysis (CoA) supplied by the manufacturer to get 40 EU/ml, vortex

vigorously for 5 minutes before use. Prepared the dilutions 2λ, 1λ, 1/2λ, 1/4 λ as per the

chart with 0.5 EU/ml or 4λas working endotoxin solution.

CSE Calculations must be done as per manufacturer‟s CoA.

(Example given below)

Control Standard Endotoxin 200EU/Vial (Lyophilized)

Add 5 ml of LRW =50EU/ml (CSE)

Preparation of working control standard Endotoxin (CSE):

Take 100 µl of 50 EU/ml CSE + 900 µl of LRW (Tube 1)= 5 EU/ml

Take 100 µl of above solution (from tube 1) + 900 µl of LRW (Tube 2)= 0.5 EU/ml i.e. 4λ

The CSE 0.5 EU/ml is used to prepare serial dilutions in (Duplicates).

i.e.,0.25EU/ml, 0.125EU/ml, 0.06EU/ml & 0.03EU/ml as shown in the below table.(These

dilutions are made in 16 x 100 mm Endotoxin free or Depyrogenated glass tubes)

Table. 1.9.

S.No CSE

(volume μL)

LRW

(in μL)

Final CSE con. (EU/ml)

1. 125 µl of 0.5 EU/ml 125 µl 0.25(2 λ)

2. 125 µl of 0.25 EU/ml 125 µl 0.125(1λ)

3. 125 µl of 0.125 EU/ml 125 µl 0.06(1/2 λ)

4. 125 µl of 0.06 EU/ml 125 µl 0.03(1/4 λ)


NIB/EHL/GM/01

Preparation of working solution (sample) from Streptokinase Injection 15 Lakhs/ vial to

10,000 IU/ml for Maximum valid dilution (MVD) as follows:

Preparation of Streptokinase 15, 00,000 IU/Vial dilution to 10,000 IU/ml:

Streptokinase 15, 00,000 IU/Vial

Reconstitute with 5 ml LRW (3, 00,000 IU / ml)

100μL of 3, 00,000 IU/ml (30,000 IU/ ml) +900μL LRW

(1:10) (VORTEX)

300μL of 30,000 IU/ml +600μL LRW

(1:2) (VORTEX)

Streptokinase 10,000 IU/ ml:

(Used to calculate the MVD for BET)

Maximum valid dilution is calculated based on the three values

i. Allowable limit (23.33 EU/10000 per ml of Streptokinase activity)

ii. Sample Concentration (10,000 IU/ml)

iii. Declared sensitivity of the lysate (λ) used in the test.

Based on the above values the MVD is calculations were carried out and the number of

times to be diluted the sample is arrived.

Determination of Maximum valid dilution (MVD) for Streptokinase:

The Maximum Valid Dilution (MVD) is the maximum allowable dilution of a sample at

which the endotoxin limit can be determined using the following formula:

MVD= Endotoxin limit (EU/mg or EU/Unit)x Potency/Concentration of test solution

λ (Labeled Sensitivity) (EU/ml)


NIB/EHL/GM/01

23.33 EU/ml x 10,000 IU/ml

= 0.125 EU/ml

= 187 times to be diluted the Streptokinase 10,000 IU/ml.

MVD= 186 times (Rounded off)

Since adapting 50:50 method the sample dilution must be MVD/2.

MVD = 186 times = 93 times

2 2

Dilution of sample to 90 times:

100 µl 10,000 IU/ml Streptokinase + 930 µl of LRW (Tube 1) = 10 times

100 µl of the above solution + 800 µl of LRW (Tube 2) = 9 times dilution

(Used to conduct the MVD for BET)

Dilutions of CSE, Test, PWC, NWC, PPC & Lysate:

(These dilutions are made in 10 x 75 mm Endotoxin free or Depyrogenated glass tubes)

Table No.1.10.

S.No Lysate Sensitivity

(λ)

LRW (volume μL)

CSE (volume μL)

Test Sample of MVD/2 dilution

(volume μL)

LAL Reagent (volume μL)

1. 0.25 (2 λ) 50 50µl of 4 λ -- 100

2. 0.125 (1λ) 50 50µl of 2 λ -- 100

3. 0.06 (1/2 λ) 50 50µl of λ -- 100

4. 0.03 (1/4 λ)_ 50 50µl of 1/2 λ -- 100

5. PWC 50 50µl of 4 λ -- 100

6. NWC 100 -- -- 100

7. PPC -- 50 µl of 4 λ 50 100

8. NPC (Test) 50 -- 50 100


NIB/EHL/GM/01

Where, CSE: Control endotoxin; PWC: Positive Water Control; NWC : Negative water

control; PPC: Positive Product control, LRW: LAL Reagent Water. NPC. Negative product

control (Sample under examination).

Procedure:

(Check the determination of bacterial endotoxin Interference with the each test

sample): Place the reaction tubes (10x75mm) and designate the tubes as 2λ, 1λ, 1/2λ, 1/4

λ, PWC, NWC, PPC and test. The reagents and sample contents are pipetted in to the test

tubes as per the above table No. 10. Place the reaction tubes in 370C + 10C water bath

for 60 min (+ 2 min).

(Note: - Incubation Timing for reaction must be adhered strictly since the reaction to Lysate

to endotoxin is critical.)

The results are declared as “POSITIVE” or “NEGATIVE”,

Test acceptance criteria: A firm gel capable of maintaining its integrity when the test tube

is inverted at 1800C is considered “POSITIVE”. Absence of gel or formation of viscous

mass which does not hold when the tube is inverted is considered “NEGATIVE”.

Test validity criteria : The positive and negative water controls must exhibit their

respective identity by producing a gel clot in positive water control and a clear liquid state

(without clot) in negative control. The product positive control and test were examined to

its reactiveness and results were tabulated in the BET proforma.

Acceptance criteria for product:

Streptokinase & r-Streptokinase injection: NMT 23.33 EU/ 10000 IU of Streptokinase per ml.

Streptokinase Bulk: Less than 0.02 EU/100 IU of Streptokinase activity.



NIB/EHL/GM/01

BACTERIAL ENDOTOXIN TEST BY GEL CLOT METHOD

Name of the Product:

Batch/SRDU No: Mfg.Date: Exp.Date:

Test Reagents

LAL Reagent:

LAL Test Kit Lot No.: Expiry Date:

Labelled Sensitivity:

LAL Diluent (LRW) Batch No: LAL Reconstitution Date:

Endotoxin Control:

Standard (CSE) Lot No: Expiry Date:

Labelled Potency: LAL diluent (LRW) Batch No:

CSE Reconstitution Date:

Results proforma:

Control

Series

Endotoxin Dilution (EU/ml)

0.25 (2λ) 0.125 ( λ) 0.06 (1/2λ) 0.03 (1/4 λ) End Point EU/ml

NPC (Test)

PPC

PWC

NWC

NPC (Test) = Negative Product control, PPC= Positive Product control, PWC= Positive Water Control,

NWC= Negative Water Control. Results recorded as (+ve)= Firm gel formed; (-ve)= Gel not formed

Remarks:


NIB/EHL/GM/01

Sterility: Complies with the test for Sterility (2.2.11) IP2010

Abnormal toxicity:

Procedure: Inject into each mouse a quantity of the preparation under examination

(diluted, if necessary, with water for injections) containing 50,000 IU of streptokinase

activity in 0.5 ml, the injection lasting 15-20 seconds.

Other parameters applicable under parenterals of powder for injection

Clarity:

Procedure: Reconstitute the injection as per manufacturer‟s instructions in leaflet. Allow to

dissolve completely. Observe for solid dissolves completely, leaving no visible residue as

undissolved matter. The constituted injection is not significantly less clear than an equal

volume of the water for injection contained in the similar container and examined in the

same manner. Compare water for injection and reconstituted sample vial for observation.

Criteria for acceptance: Clear, colourless liquid.

NIB/EHL/PRO/XV PROFORMA FOR PARTICULATE MATTER BY VISUAL OBSERVATION

Date of testing:

SAMPLE DETAILS:

S.No Name of the product/ SRRDU Code No.

Diluent volume

(ml)

Visual observation after reconstitution

Any other remarks

Tested by …………………………… Verified by……………………….

(Analyst)

Authorized by (Signature of Lab Head)



NIB/EHL/GM/01

Particulate matter:

Procedure: Reconstitute the injection vial as per manufacturer‟s instructions in the leaflet.

Allow to dissolve completely. Observe for the solution is essentially free from particles of

foreign matter that can be seen on visual inspection.

Criteria for acceptance: No visual particles observed.

Results proforma:

NIB/EHL/PRO/XIV

PROFORMA FOR PARTICULATE MATTER BY VISUAL OBSERVATION

Date of testing:

SAMPLE DETAILS:

S.No Name of the product/ SRRDU Code No.

Diluent volume

(ml)

Visual observation after reconstitution

Any other remarks

Tested by …………………………… Verified by……………………….

(Analyst)

Authorized by

(Signature of Lab Head)



NIB/EHL/GM/01

1.9. Documents required under scrutiny of protocols

i. Certificate of analysis of the finished product with name of pharmacopoeia complied

ii. Letter of DCG(I)/ADC/ Port office/CDSCO/Manufacturer‟s Letter

iii. Manufacturing protocol

iv. Batch Release Certificate from NCL/Manufacturer of country of Origin

v. Copy of test/ import / manufacturing license

vi. Non-withdrawal certificate

vii. Product insert

1.10. Number of vials required for batch testing/evaluation

Table.1.11

S.No Product No. of vials required for a

batch

1. Streptokinase bulk 02 Bottles

2. Streptokinase injection 10 vials

3. r-Streptokinase injection 08 Nos

1.11. Training:

Training of scientific/technical personnel is one of the mandates of NIB. Professionals from

industry, academia and government institutions/organizations, who require training on

specific products / tests /quality system may be undertaken.


NIB/EHL/GM/01

Annexures:

Annexure-I

IDENTIFICATION OF STREPTOKINASE BY CLOT LYSIS METHOD

Product Code/Name: Manufacturer: Batch No: Mfg Date: Exp.Date:

S.No

Plasma

(μL)

Streptokinase

In

Citrophosphate

buffer pH 7.2

(10000 IU/ml)

(μL)

Thrombin

(IU/ml)

20 IU/ml

(μL)

Time (in minutes)

3 min 5 min 10 min 15 min 20min 25 min 30 min 60min

1 Blank-500ul

2 Human

Plasma-

500ul

3 Bovine

Plasma-

500ul

Remarks:


NIB/EHL/GM/01

Annexure-II

STREPTODORNASE ACTIVITIY FORMAT

Sample Name: Batch No: Date of Mfg: Date of Expiry:

S.No 0.1% DNA

(ul)

Imidazole

Buffer(ul)

Test solution

(ul)

Reference 20

IU/ml(ul)Vial No.---

Perchlorate

(ul)

O.D at

260 nm

Blank -- 1000 -- --

Immediately

add

3000

Centrifuge at

3000 rpm/ 5 min Take

absorbance of

supernatant.

1 500 250 250 -- 3000

2 500 250 250 -- 3000

1+2 =A1 A1=

3 500 250 250 --

Incubate

at 370C

for 15min in

water bath

3000

4 500 250 250 -- 3000

3+4 =A2 A2=

5 500 125 250 125 3000

6 500 125 250 125 3000

5+6 =A3 A3=

7 500 -- 250 250 3000

8 500 -- 250 250 3000

7+8 =A4 A4=

Streptokinase Injection Calculations (Corrected)= (A2-A1) < 0.5 (A3+A4) -A2

Streptokinase Bulk Calculations= (A3+A4)-(A1+A2)< (A5+A6+A7+A8) / 2- (A3+A4)

Calculations:

Result:


NIB/EHL/GM/01

Annexure-III

ESTIMATION OF STREPTOLYSIN ACTIVITY

Code/Product name:

Manufacturer:

Batch No: Mfg.Date: Exp.Date:

Results:

Tube Streptokinase

5,00,000 Units/

0.5 ml

Buffer 2.3% w/v

Sodium

thioglycollate

Incubate

at 370c

for 10

min

5 Units of

Human

streptolysin-O

Incubate

at 370c

for

5 min

1.6 % Rabbit

Erythrocyte

suspension

Incubate

at 370c

for

30 min

Centrifuge

1000 rpm

for 10 min

Absorbance

At 550 nm

Blank1 -- 0.5 ml 400 ul 50ul of 10 IU +

50ul Buffer

1 ml

Sample

0.5 ml -- 400 ul 50ul of 10 IU +

50ul Buffer

1 ml

Sample

0.5 ml -- 400 ul 50ul of 10 IU +

50ul Buffer

1ml


NIB/EHL/GM/01

POTENCY ESTIMATION OF STREPTOKINSE BY CHROMOGENIC METHOD Annexure-IV

Product Name/SRRDU Code: Manufacturer: Batch No: Mfg Date:

S.No StreptokinaseIU/ml

(Final concentration)

Streptokinase*

10 IU/ml

Buffer-C

(µl)

Chromogenic Substrate(1X)

(µl)

Incubate

at 370

C for

20m

in u

p t

o a

ppear

ance o

f pale

yello

w c

olo

ur,

by k

eepin

g s

topw

atch a

dd 5

0%

GA

A w

ith

10 s

ec in

terv

al t

o e

ach tube

50% Glacial Acetate(µl)

Load into Microplate

O.D at 405nm

1 Blank 0 60 40 50

2 Ref. Std 0.5 3 57 40 50

3 Ref. Std 0.5 3 57 40 50

4 Ref. Std 1.0 6 54 40 50

5 Ref. Std 1.0 6 54 40 50

6 Ref. Std 2.0 12 48 40 50

7 Ref. Std 2.0 12 48 40 50

8 Ref. Std 4.0 24 36 40 50

9 Ref. Std 4.0 24 36 40 50

10 Sample 0.5 3 57 40 50

11 Sample 0.5 3 57 40 50

12 Sample 1.0 6 54 40 50

13 Sample 1.0 6 54 40 50

14 Sample 2.0 12 48 40 50

15 Sample 2.0 12 48 40 50

16 Sample 4.0 24 36 40 50

17 Sample 4.0 24 36 40 50

* Ref. 77Std and Sample concentration stock 10 IU/ml used to set the reaction in duplicates Details of vials used in the test:


NIB/EHL/GM/01

1.12. References:

i. Indian Pharmacopoeia 2010

ii. Concern SOPs of test parameters, equipment, QA/QC SOPs.

iii. Quality policy

iv. ELSEVIER Journal-Current opinion in Biotechnology 2003, 14:1-7.