Standard Operating Procedure for Preparation of Competent Cells in Biosimilar R&D

Department	Biosimilars
SOP No.	SOP/BS/004/2025
Supersedes	SOP/BS/004/2022
Page No.	Page 1 of 11
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the process for preparing chemically and electrocompetent E. coli cells for use in transformation procedures during biosimilar molecular biology workflows.

2. Scope

This SOP applies to laboratory personnel involved in preparing and storing competent bacterial cells within the Biosimilars R&D facility.

3. Responsibilities

• Research Associate: Carries out the competent cell preparation procedure and maintains documentation.
• Laboratory Technician: Prepares media and buffers, ensures sterile conditions, and labels vials.
• QA Officer: Verifies batch records and checks viability logs.

4. Accountability

The Head of Molecular Biology is accountable for ensuring that all competent cell preparations meet quality and transformation efficiency requirements.

5. Procedure

5.1 Reagents and Materials Required

• SOB medium or LB broth
• Calcium chloride (CaCl₂) solution (100 mM)
• Glycerol (10%) for storage
• Electroporation buffer (if preparing electrocompetent cells)
• Ice, sterile tubes, centrifuge

5.2 Culture Growth

1. Inoculate a single colony of E. coli DH5α into 5 mL LB broth and incubate overnight at 37°C with shaking (200 rpm).
2. Dilute 1:100 into 100 mL fresh LB medium and grow until OD₆₀₀ reaches 0.4–0.6.

5.3 Preparation of Chemically Competent Cells

1. Chill the culture on ice for 10–15 minutes.
2. Centrifuge at 4,000 rpm for 10 minutes at 4°C.
3. Discard supernatant and gently resuspend pellet in 50 mL of ice-cold 100 mM CaCl₂.
4. Incubate on ice for 30 minutes.
5. Repeat centrifugation and resuspend in 10 mL of ice-cold 100 mM CaCl₂ + 15% glycerol.
6. Aliquot 100 µL per sterile microcentrifuge tube and flash freeze in liquid nitrogen.
7. Store at -80°C.

5.4 Preparation of Electrocompetent Cells

1. Harvest cells as above once OD₆₀₀ reaches 0.5.
2. Wash cells three times with ice-cold sterile 10% glycerol.
3. Resuspend final pellet in 1 mL ice-cold 10% glycerol.
4. Aliquot into pre-chilled sterile tubes (50 µL each) and store at -80°C.

5.5 Documentation and Quality Check

1. Label all tubes with strain name, date, and preparation type (chemically/electrocompetent).
2. Log entries in Competent Cell Preparation Log (Annexure-1).
3. Test transformation efficiency using a standard plasmid (e.g., pUC19) and record in Transformation Efficiency Report (Annexure-2).

6. Abbreviations

• SOP: Standard Operating Procedure
• OD₆₀₀: Optical Density at 600 nm
• QA: Quality Assurance

7. Documents

1. Competent Cell Preparation Log (Annexure-1)
2. Transformation Efficiency Report (Annexure-2)

8. References

• ICH Q5A: Viral Safety Evaluation of Biotechnology Products
• WHO Guidelines for Recombinant DNA Products
• CDSCO Laboratory Practice Manual

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Competent Cell Preparation Log

Date	Strain	Type	OD600	Stored At	Prepared By
01/05/2025	E. coli DH5α	Chemically Competent	0.45	-80°C	Sunita Reddy

Annexure-2: Transformation Efficiency Report

Date	Strain	Plasmid	Colonies Obtained	Efficiency (cfu/µg DNA)	Status
02/05/2025	E. coli DH5α	pUC19	1200	6 × 106	Accepted

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Separated electrocompetent and chemical protocols	Version update