Standard Operating Procedure for Restriction Enzyme Digestion and Mapping in Biosimilar Research

Department	Biosimilars
SOP No.	SOP/BS/007/2025
Supersedes	SOP/BS/007/2022
Page No.	Page 1 of 11
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To establish a standard method for performing restriction enzyme digestion and mapping of plasmid DNA for verification of insert orientation, integration, and overall vector integrity during biosimilar development.

2. Scope

This SOP applies to the Biosimilars R&D team engaged in molecular biology operations involving plasmid validation post-cloning and prior to expression studies.

3. Responsibilities

• Molecular Biologist: Designs digestion strategy, selects appropriate enzymes, and interprets results.
• Research Assistant: Prepares reaction mixes and performs electrophoresis.
• QA Personnel: Verifies documentation of mapping results and digestion patterns.

4. Accountability

The Head of Molecular Biology is accountable for ensuring all plasmid digestion and mapping procedures are compliant with molecular verification protocols and documented accordingly.

5. Procedure

5.1 Selection of Restriction Enzymes

1. Choose enzymes based on:
   • Vector backbone map
   • Insert flanking regions
   • Expected fragment sizes
2. Use double or single digests as needed to differentiate between orientations or configurations.

5.2 Preparation of Digestion Reaction

1. Prepare the reaction mix in a sterile microcentrifuge tube:
   • Plasmid DNA: 500–1000 ng
   • 10X Buffer: 2 µL
   • Restriction Enzyme(s): 1 µL each
   • Nuclease-Free Water: To make up to 20 µL
2. Gently mix by tapping and spin down briefly.
3. Incubate at 37°C for 1–2 hours (as per enzyme manufacturer guidelines).

5.3 Agarose Gel Electrophoresis

1. Prepare 1% or 1.5% agarose gel with safe DNA stain (e.g., ethidium bromide or SYBR Safe).
2. Load 5 µL of digested sample mixed with 1 µL of 6X loading dye.
3. Include 1 kb or 100 bp DNA ladder as reference.
4. Run gel at 80–100V for 45–60 minutes in 1X TAE buffer.
5. Visualize bands using UV transilluminator and photograph gel for records.

5.4 Mapping and Result Interpretation

1. Compare observed bands with predicted digestion pattern from in-silico map (e.g., SnapGene or NEBcutter).
2. Record:
   • Fragment sizes
   • Band intensity
   • Lane and ladder references
3. Document findings in Plasmid Mapping Record (Annexure-1).

5.5 Troubleshooting and Repeat Analysis

1. If unexpected bands appear:
   • Check for star activity or incomplete digestion.
   • Repeat with fresh enzyme or alternative buffer.
   • Use additional enzymes to confirm map.

5.6 Documentation and Archiving

1. Label gel image with date, sample ID, and enzyme names.
2. Attach to printed mapping report and file in project record folder.
3. Store digital copy in central repository with access control.

6. Abbreviations

• SOP: Standard Operating Procedure
• TAE: Tris Acetate EDTA Buffer
• QA: Quality Assurance

7. Documents

1. Plasmid Mapping Record (Annexure-1)
2. Gel Image Archive Log (Annexure-2)

8. References

• ICH Q5B – Expression Construct Analysis
• WHO Guidelines on DNA-Based Biologics
• CDSCO Recombinant Product Evaluation 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: Plasmid Mapping Record

Date	Plasmid ID	Enzymes Used	Expected Bands (bp)	Observed Bands (bp)	Status
03/05/2025	pCMV-rhEPO	EcoRI, HindIII	3000, 1800	3000, 1800	Verified

Annexure-2: Gel Image Archive Log

Date	Plasmid ID	Image Filename	Operator	Location
03/05/2025	pCMV-rhEPO	gel_rhEPO_03052025.jpg	Sunita Reddy	Lab Server/Gels/May25

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Clarified mapping interpretation and added Gel Image Log	Annual SOP update