Standard Operating Procedure for Vector Design and Cloning in Biosimilar Research

Department	Biosimilars
SOP No.	SOP/BS/003/2025
Supersedes	SOP/BS/003/2022
Page No.	Page 1 of 14
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To describe the procedure for designing plasmid expression vectors and performing gene cloning required for biosimilar development, ensuring proper integration, regulatory compliance, and traceability of constructs.

2. Scope

This SOP is applicable to R&D personnel in the Biosimilars department who are involved in cloning biosimilar gene constructs into vectors for expression in host cell lines such as CHO, HEK293, or E. coli.

3. Responsibilities

• Molecular Biology Scientist: Designs vector maps, selects restriction sites, and performs ligation.
• Research Assistant: Executes the cloning process and confirms insert integration.
• QA Representative: Verifies and approves cloning results and sequence confirmations.

4. Accountability

The Head of Molecular Biology is accountable for ensuring cloning procedures are conducted as per defined protocols and all documentation is complete and accurate.

5. Procedure

5.1 Vector Backbone Selection

1. Select suitable plasmid backbone (e.g., pcDNA3.1, pET28a, pCMV6) based on host system and expression requirements.
2. Confirm:
   • Selectable marker (Ampicillin, Kanamycin, Hygromycin)
   • Promoter compatibility (CMV, T7, EF-1α)
   • Multiple Cloning Site (MCS) availability

5.2 Insert Preparation

1. Obtain synthesized gene insert or PCR-amplify target gene with designed primers.
2. Incorporate restriction enzyme recognition sites during primer design (e.g., EcoRI, HindIII).
3. Purify PCR product using a column purification kit and verify via agarose gel electrophoresis.

5.3 Vector Linearization

1. Digest the plasmid backbone with appropriate restriction enzymes.
2. Perform double digestion, if required, to prevent vector re-ligation.
3. Purify digested vector using gel extraction method.

5.4 Ligation of Insert into Vector

1. Set up ligation reaction using T4 DNA ligase at the recommended molar ratio (insert:vector = 3:1).
2. Incubate ligation reaction at 16°C overnight or at room temperature for 2 hours.

5.5 Transformation and Colony Screening

1. Transform ligation mix into competent E. coli DH5α cells.
2. Plate on antibiotic selection agar plates and incubate overnight at 37°C.
3. Pick colonies and inoculate into LB broth with appropriate antibiotic.

5.6 Confirmation of Cloning

1. Extract plasmid DNA using miniprep kit.
2. Verify insert via:
   • Restriction digestion pattern
   • PCR using gene-specific primers
   • Sanger sequencing for sequence confirmation
3. Record results in Cloning Verification Report (Annexure-1).

5.7 Storage and Documentation

1. Store confirmed clones in glycerol stocks at -80°C (Annexure-2: Clone Stock Log).
2. Archive sequencing results and annotated vector maps in shared database.

6. Abbreviations

• SOP: Standard Operating Procedure
• MCS: Multiple Cloning Site
• CMV: Cytomegalovirus Promoter
• PCR: Polymerase Chain Reaction
• QA: Quality Assurance

7. Documents

1. Cloning Verification Report (Annexure-1)
2. Clone Stock Log (Annexure-2)
3. Vector Design Checklist (Annexure-3)

8. References

• ICH Q5B: Expression Construct Analysis Guidelines
• WHO TRS 978 Annex on Recombinant Technology
• CDSCO Guidelines on Recombinant Product Development

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Cloning Verification Report

Date	Plasmid ID	Insert Verified	Sequencing Match (%)	Status
25/04/2025	pCMV-rhEPO	Yes	99.8%	Accepted

Annexure-2: Clone Stock Log

Date	Clone Name	Plasmid ID	Storage Temp	Stored By
26/04/2025	DH5α-pCMV-rhEPO	pCMV-rhEPO	-80°C	Rajesh Kumar

Annexure-3: Vector Design Checklist

Item	Details	Checked
Promoter	CMV	Yes
Selectable Marker	Ampicillin	Yes
Insert Direction	Correct	Yes
Sequencing Confirmed	Yes	Yes

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated SOP with sequence verification step and digital record archiving	Annual SOP revision