Biosimilars: SOP for Gene Synthesis and Sequence Verification – V 2.0

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Biosimilars: SOP for Gene Synthesis and Sequence Verification – V 2.0


Standard Operating Procedure for Gene Synthesis and Sequence Verification in Biosimilar Development

Department Biosimilars
SOP No. SOP/BS/002/2025
Supersedes SOP/BS/002/2022
Page No. Page 1 of 13
Issue Date 04/05/2025
Effective Date 06/05/2025
Review Date 04/05/2026

1. Purpose

To describe the standardized procedure for gene synthesis and verification of nucleotide sequences to ensure fidelity and regulatory compliance in biosimilar product development.

2. Scope

This SOP is applicable to the Biosimilars R&D and Molecular Biology teams responsible for designing, outsourcing, receiving, and verifying gene constructs used in biosimilar expression systems.

3. Responsibilities

  • Molecular Biologist: Designs gene sequences and validates synthetic constructs post-delivery.
  • R&D Scientist: Initiates synthesis requests and coordinates with vendors.
  • QA: Reviews sequence verification reports and documentation.

4. Accountability

The Head of Biosimilars R&D is accountable for ensuring that gene constructs are correctly synthesized and sequence-verified prior to downstream applications.

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5. Procedure

5.1 Gene Design and Optimization

  1. Design the coding sequence of the biosimilar target protein based on reference sequence from public databases (e.g., GenBank).
  2. Use codon optimization tools to adapt the sequence to the host expression system (CHO, E. coli, HEK293).
  3. Ensure inclusion of necessary elements such as restriction sites, Kozak sequence, or signal peptides as per application.

5.2 Sequence Validation and Approval

  1. Submit the final DNA construct design for internal review using the Gene Design Approval Form (Annexure-1).
  2. Obtain QA clearance for synthesis initiation.

5.3 Ordering and Vendor Management

  1. Select vendor based on approved supplier list for gene synthesis (e.g., GenScript, IDT).
  2. Place purchase order specifying:
    • Sequence length and cloning vector
    • Restriction sites and tags
    • Required delivery format (plasmid or lyophilized)
  3. Maintain vendor communication log (Annexure-2).

5.4 Receipt and Initial Inspection

  1. On receipt, verify labeling, packaging, and physical integrity.
  2. Cross-check delivered sequence with purchase order.
  3. Record in Gene Receipt Log (Annexure-3).
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5.5 Sequence Verification

  1. Extract plasmid DNA using miniprep kit.
  2. Perform Sanger sequencing using M13 or specific primers.
  3. Align sequencing chromatogram with reference design using tools such as SnapGene or BLAST.
  4. Document base accuracy and mutations (if any) in the Sequence Verification Report (Annexure-4).

5.6 Discrepancy Handling

  1. In case of mismatch:
    • Notify vendor immediately.
    • Reorder corrected construct after necessary approvals.

5.7 Archiving and Documentation

  1. Store physical DNA sample in -20°C plasmid archive with label and code.
  2. Archive all sequence files (.ab1, .seq), order forms, and approval documents.

6. Abbreviations

  • SOP: Standard Operating Procedure
  • QA: Quality Assurance
  • R&D: Research and Development
  • BLAST: Basic Local Alignment Search Tool

7. Documents

  1. Gene Design Approval Form (Annexure-1)
  2. Vendor Communication Log (Annexure-2)
  3. Gene Receipt Log (Annexure-3)
  4. Sequence Verification Report (Annexure-4)

8. References

  • ICH Q5B: Analysis of the Expression Construct in Cells Used for Production of r-DNA Derived Protein Products
  • WHO Guidelines on DNA Vaccines and Plasmid Vectors
  • CDSCO India Guidance on Genetic Engineering Techniques
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9. SOP Version

Version: 2.0

10. Approval Section

Prepared By Checked By Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Gene Design Approval Form

Gene Name rhEPO
Sequence Length 582 bp
Host System CHO-K1
Optimized Yes
Approved By QA

Annexure-2: Vendor Communication Log

Date Vendor Communication Mode Summary
18/04/2025 GenScript Email Sequence and vector confirmed

Annexure-3: Gene Receipt Log

Date Construct ID Received From Condition Checked By
26/04/2025 GENE-RH001 GenScript Intact Sunita Reddy

Annexure-4: Sequence Verification Report

Date Sample ID Sequencing Method Match with Reference (%) Status
28/04/2025 GENE-RH001 Sanger 100% Accepted

Revision History:

Revision Date Revision No. Revision Details Reason for Revision Approved By
04/05/2025 2.0 Updated to include digital verification tools Annual review and enhancement
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