SOP for Testing for Bioburden of Fractional Solutions

1.0 OBJECTIVE

To check the bio-burden of fraction solution before sterile filtration.

2.0 SCOPE

This procedure is applicable to fraction solution of insulin product.

3.0 RESPONSIBILITY

3.1 Doing: Technical Assistant (Microbiologist)/Executive
3.2 Checking: Executive/ Manager

4.0 ACCOUNTABILITY

Head of the Department

5.0 PROCEDURE

5.1 Frequency
5.1.1 Each fraction of each product of insulin is to be taken for testing per month.
5.2 Sampling
5.2.1 Samples for testing are been send by Parenteral Dept. which should not be less than 100ml.
5.3 Preservation of sample
After sampling, the samples is kept at 2 to 8°C in the refrigerator if analysis cannot be performed within 8 hrs. The testing must be completed within 24 hours of sampling.
5.4 PROCEDURE:
5.4.1 The testing procedure is carried out under LAF aseptically.
5.4.2 The testing of sample carried out as specified method for specified product as given below

Name of product

Name of solution	Method used
Mixtard	Preservative, Buffer	B
	Preservative, Protamine & Insulin solution	D
	Protamine solution, Preservative Buffer& Insulin Solution	D
Human Mixtard	Preservative Buffer solution	B
	Preservative, Protamine & Insulin solution Buffer& Insulin solution	D
Human Monotard	MPO solution	E
	Insulin solution	C
	NaOH solution	B
	Insulin , MPO & Buffer solution	C
Human  Insulatard	Preservative  Buffer solution	B
	Preservative Protamine & Insulin solution	D
Lentard	Insulin solution	C
	Buffer solution	B
	Zn MPO solution	A
	Buffer solution	B
Actrapid	Preservative solution	B
	Insulin solution	C
	NaOH solution	B
Human Actrapid	NaOH solution	B

5.5 Preparation of Plates
5.5.1 Prepare the plates as per SOP.

5.6 METHOD - A

5.6.1 Membrane filter: -Nitrocellulose
5.6.2 Transfer 100ml sample to membrane filter & filter immediately.
5.6.3 Wash the membrane filter by passing through the filter 3 X 100ml Buffer
5.6.4 Transfer the membrane filter to the surface of a plate of TSA (SCD agar).

5.7 METHOD - B

5.7.1 Membrane filter:- Durapore
5.7.2 Transfer 100ml sample to a membrane filter & filter immediately.
5.7.3 Wash the membrane filter by passing through the filter 3 X 100ml Buffer
5.7.4 Transfer the membrane filter to the surface of TSA (SCD agar).

5.8 METHOD - C

5.8.1 Membrane filter:- Nitrocellulose
5.8.2 Transfer 100ml sample to a membrane filter & filter immediately.
5.8.3 Wash the membrane filter by first passing through the filter 10ml of 0.01 % w/v ascorbic acid solution in 0.9 % w/v NaCl.
5.8.4 Then pass through the filter 2 x 100ml Buffer
5.8.5 Transfer the membrane filter to the surface of a plate of TSA (SCD agar)

5.9 METHOD - D

5.9.1 Membrane filter:- nitrocellulose
5.9.2 Transfer 100ml sample to a membrane filter and filter immediately.
5.9.3 Wash the membrane filter by first passing through the 10ml of 0.01 1 w/v ascorbic acid solution in 0.9 % w/v NaCl.
5.9.4 Then pass through the filter 2 x 100ml Buffer
5.9.5 Transfer the membrane filter to the surface of a plate of TSA (SCD agar).

5.10 METHOD - E

5.10.1 Membrane filter:- Nitrocellulose
5.10.2 Transfer 100ml sample to a membrane filter and filter immediately.
5.10.3 Wash the membrane filter by passing through the filter 3 x 200ml Buffer
5.10.4 Transfer the membrane filter to the surface of TSA.

5.11 INCUBATION

5.11.1 Incubate at 30-35° for 5 days.
5.11.2 Count the number of colonies bacterial/fungal and report as cfu/100ml in Annexure-I
Limit : Not more than 10 cfu/ 100 ml
The fungal colony should be absent.

6.0 ABBREVIATIONS

6.1 SOP - Standard Operating Procedure

ANNEXURE-I

QUALITY CONTROL DEPARTMENT

MICROBIOLOGY LAB

BIOBURDEN OF FRACTIONAL SOLUTION

Name of Material:
Batch No:	Date of test:
Sampling Quantity:	AR.  No:
Method:  Membrane filtration	Slip No:
Medium:  Soyabean casein Digest agar	Medium Lot No.:
Incubation Temp: 30°C  - 35°C Incubation period: 5 days	Method No.:

Procedure:                      
(1)  Take 100 ml sample and filter aseptically through the filter specified in method.                                           

(2)  Wash the filter by passing the rinsing fluid specified in method.

Result:

Limit:  Not more than 10 cfu / 100ml.

            Fungal colony should be absent.

Remark:

Microbiologist	Date Of Completion	Checked By
Sign & Date		Sign & Date

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