Standard Operating Procedure for Transformation and Electroporation in Biosimilar Development

Department	Biosimilars
SOP No.	SOP/BS/005/2025
Supersedes	SOP/BS/005/2022
Page No.	Page 1 of 13
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To outline the standardized procedure for introducing recombinant plasmids into host cells using chemical transformation and electroporation methods in biosimilar development workflows.

2. Scope

This SOP is applicable to laboratory personnel involved in bacterial transformation processes using competent E. coli strains within the Biosimilars R&D facility.

3. Responsibilities

• Research Associate: Performs transformation and electroporation procedures.
• Technician: Prepares reagents and media, and assists in plate incubation and recovery steps.
• QA Officer: Reviews transformation efficiency and compliance documentation.

4. Accountability

The Head of Biosimilars R&D is accountable for ensuring that transformation protocols are conducted according to validated procedures and that resulting data is accurately recorded.

5. Procedure

5.1 Materials Required

• Chemically competent or electrocompetent E. coli cells
• Plasmid DNA (purified)
• SOC medium
• Agar plates with appropriate antibiotic
• Electroporator (for electroporation method)

5.2 Chemical Transformation Procedure

1. Thaw 100 µL of chemically competent cells on ice.
2. Add 1–5 ng of plasmid DNA gently and mix by tapping.
3. Incubate the mixture on ice for 30 minutes.
4. Heat shock at 42°C for 45 seconds.
5. Immediately transfer tubes to ice for 2 minutes.
6. Add 900 µL of SOC medium and incubate at 37°C for 1 hour with shaking (200 rpm).
7. Plate 100–200 µL on LB agar containing the appropriate antibiotic.
8. Incubate overnight at 37°C.

5.3 Electroporation Procedure

1. Thaw 50 µL of electrocompetent cells on ice.
2. Add 1–2 µL of plasmid DNA, mix gently.
3. Transfer to pre-chilled 0.1 cm electroporation cuvette.
4. Electroporate at 1.8 kV, 25 µF, 200 ohms (or follow equipment-specific settings).
5. Immediately add 950 µL of SOC medium and transfer to sterile tube.
6. Recover at 37°C for 1 hour, shaking at 200 rpm.
7. Plate appropriate volumes on selective agar and incubate overnight.

5.4 Post-Transformation Analysis

1. Count colonies and record transformation efficiency using formula:
   
   Efficiency (cfu/µg DNA) = (colonies × dilution factor) / amount of DNA (µg)
2. Log entries in Transformation Logbook (Annexure-1).

5.5 Storage and Record Keeping

1. Inoculate confirmed transformants into LB broth and prepare glycerol stocks for long-term storage.
2. Update transformation records and vector maps.

6. Abbreviations

• SOP: Standard Operating Procedure
• SOC: Super Optimal Broth with Catabolite Repression
• cfu: Colony Forming Units

7. Documents

1. Transformation Logbook (Annexure-1)
2. Glycerol Stock Record (Annexure-2)

8. References

• ICH Q5D: Cell Substrate Derivation and Characterization
• WHO TRS 978 – Annex on Recombinant DNA Technology
• CDSCO R&D Laboratory Guidelines

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Transformation Logbook

Date	Strain	Plasmid	Method	Colonies	Efficiency	Remarks
02/05/2025	DH5α	pUC19	Chemical	850	4.2 × 106	Accepted

Annexure-2: Glycerol Stock Record

Date	Clone ID	Plasmid	Volume	Storage Temp	Prepared By
03/05/2025	CL-DH5α-pUC19	pUC19	1 mL	-80°C	Rajesh Kumar

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated transformation efficiency logging and included electroporation steps	Annual revision