Standard Operating Procedure for Plasmid Amplification and Purification in Biosimilar Development

Department	Biosimilars
SOP No.	SOP/BS/006/2025
Supersedes	SOP/BS/006/2022
Page No.	Page 1 of 12
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the process for amplifying and purifying plasmid DNA from transformed bacterial cells to ensure high-quality DNA yield suitable for downstream biosimilar applications.

2. Scope

This SOP applies to research personnel in the Biosimilars department involved in plasmid preparation for gene expression, cloning, and sequence verification activities.

3. Responsibilities

• Research Associate: Executes bacterial growth and plasmid extraction.
• Laboratory Technician: Prepares media, stocks, and handles centrifugation and filtration steps.
• QA Officer: Reviews purity and concentration documentation.

4. Accountability

The Head of Biosimilars Molecular Biology is accountable for ensuring plasmid preparation is carried out per defined standards and the DNA meets required specifications.

5. Procedure

5.1 Inoculation and Culture

1. Inoculate a single colony of transformed E. coli into 5 mL of LB broth with appropriate antibiotic.
2. Incubate at 37°C with shaking (200 rpm) overnight.

5.2 Plasmid Amplification

1. Transfer overnight culture into 100 mL LB broth with antibiotic.
2. Incubate at 37°C with shaking for 8–10 hours until OD₆₀₀ reaches 2.0–2.5.

5.3 Harvesting Bacterial Cells

1. Transfer culture to centrifuge bottles and spin at 6,000 rpm for 10 minutes at 4°C.
2. Discard supernatant and proceed with plasmid extraction immediately or store pellet at -20°C.

5.4 Plasmid Purification

1. Use a commercial plasmid purification kit (e.g., Qiagen, Thermo Fisher).
2. Follow standard alkaline lysis procedure:
   • Resuspend in Buffer P1
   • Lyse with Buffer P2
   • Neutralize with Buffer N3
3. Centrifuge at 13,000 rpm for 10 minutes to pellet debris.
4. Apply supernatant to spin column and wash with ethanol-based buffer.
5. Elute plasmid in nuclease-free water or TE buffer (50–100 µL).

5.5 DNA Quantification and Quality Check

1. Measure DNA concentration using NanoDrop or equivalent spectrophotometer.
2. Check purity using A₂₆₀/A₂₈₀ ratio (acceptable: 1.8–2.0).
3. Record all data in Plasmid Quality Log (Annexure-1).

5.6 Agarose Gel Verification

1. Run 5 µL of plasmid DNA on 1% agarose gel with loading dye.
2. Visualize bands under UV transilluminator.
3. Confirm correct plasmid size and supercoiled form.

5.7 Documentation and Storage

1. Label purified plasmid tubes with ID, date, and concentration.
2. Store at -20°C or -80°C depending on long-term use.
3. Update Plasmid Storage Inventory (Annexure-2).

6. Abbreviations

• SOP: Standard Operating Procedure
• OD₆₀₀: Optical Density at 600 nm
• TE: Tris-EDTA Buffer
• QA: Quality Assurance

7. Documents

1. Plasmid Quality Log (Annexure-1)
2. Plasmid Storage Inventory (Annexure-2)

8. References

• ICH Q5B – Analysis of Expression Constructs
• WHO Technical Report Series 978 – Annex on DNA Products
• CDSCO Guidelines for Molecular Biology Procedures

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Plasmid Quality Log

Date	Plasmid ID	Concentration (ng/µL)	A260/A280	Purity Status	Tested By
02/05/2025	pCMV-rhEPO	205	1.92	Accepted	Sunita Reddy

Annexure-2: Plasmid Storage Inventory

Plasmid ID	Date	Volume (µL)	Location	Stored By
pCMV-rhEPO	02/05/2025	80	Freezer R-2 (-20°C)	Rajesh Kumar

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Expanded section on agarose gel confirmation and added storage tracking	Annual review