Standard Operating Procedure for Southern Blot Analysis in Biosimilar Cell Lines

Department	Biosimilars
SOP No.	SOP/BS/018/2025
Supersedes	SOP/BS/018/2022
Page No.	Page 1 of 14
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the method for performing Southern blot analysis for confirming transgene integration, determining gene copy number, and detecting rearrangements in genetically modified biosimilar-producing cell lines.

2. Scope

This SOP applies to the Molecular Biology and Cell Line Development teams responsible for genetic characterization and verification of recombinant clones used in biosimilar production.

3. Responsibilities

• Molecular Biologist: Carries out the complete Southern blot procedure and interprets results.
• Lab Assistant: Prepares reagents, assists with electrophoresis and blotting steps.
• QA Representative: Verifies documentation and ensures traceability of results.

4. Accountability

The Head of Molecular Biology is accountable for ensuring the accurate execution and compliance of Southern blot assays as part of clone characterization in biosimilar programs.

5. Procedure

5.1 Genomic DNA Preparation

1. Isolate high molecular weight DNA from 1 × 10⁷ cells using phenol-chloroform extraction.
2. Check DNA quality using agarose gel electrophoresis and spectrophotometry.

5.2 Restriction Enzyme Digestion

1. Digest 10 µg genomic DNA with restriction enzymes that cut outside the transgene insertion site.
2. Use at least 1 unit of enzyme per µg DNA and incubate overnight at 37°C.

5.3 Gel Electrophoresis and Denaturation

1. Run digested DNA on 0.8% agarose gel at 40V overnight.
2. Soak gel in 0.25M HCl for 10 minutes to depurinate.
3. Denature in 1.5M NaCl, 0.5M NaOH for 30 minutes, followed by neutralization in 1.5M NaCl, 0.5M Tris-HCl pH 7.5.

5.4 Capillary Blotting

1. Transfer DNA onto a nylon membrane overnight via capillary transfer in 20X SSC buffer.
2. Cross-link DNA to membrane using UV crosslinker or baking at 80°C for 2 hours.

5.5 Probe Preparation and Hybridization

1. Label the DNA probe (complementary to transgene) using radioactive or digoxigenin-based labeling.
2. Prehybridize membrane in hybridization buffer for 1 hour at 42°C.
3. Add labeled probe and hybridize overnight at 42–68°C depending on probe type.

5.6 Washing and Detection

1. Wash membrane with decreasing stringency buffers (e.g., 2X SSC, 0.1X SSC).
2. Detect signals using X-ray film (radioactive) or chemiluminescent substrate (DIG).
3. Interpret number and intensity of bands to determine gene copy number and integration pattern.
4. Document in Southern Blot Result Log (Annexure-1).

6. Abbreviations

• SSC: Saline-Sodium Citrate Buffer
• DIG: Digoxigenin
• UV: Ultraviolet
• X-ray: Radiographic Detection Method

7. Documents

1. Southern Blot Result Log (Annexure-1)
2. Probe Synthesis and Labeling Record (Annexure-2)

8. References

• WHO TRS 999 – Genetic Stability Testing of Cell Substrates
• ICH Q5B – Quality of Biotechnological Products: Analysis of Expression Constructs
• CDSCO Guidelines on Recombinant Cell Line Characterization

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Southern Blot Result Log

Date	Clone ID	Restriction Enzyme	Band Count	Band Size(s)	Remarks	Operator
03/05/2025	CL-BS-018	EcoRI	3	3.2kb, 2.8kb, 1.5kb	Multiple integrations	Rajesh Kumar

Annexure-2: Probe Synthesis and Labeling Record

Probe Name	Target Gene	Labeling Method	Lot No.	Prepared By	Date
pEPO-3	rhEPO	DIG	DG2025	Sunita Reddy	02/05/2025

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated hybridization buffer and added DIG detection method	Annual SOP update