Standard Operating Procedure for Limiting Dilution Cloning in Biosimilar Development

Department	Biosimilars
SOP No.	SOP/BS/011/2025
Supersedes	SOP/BS/011/2022
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 a standard procedure for isolating single-cell-derived clones by limiting dilution cloning in order to establish monoclonal cell lines used in biosimilar product development.

2. Scope

This SOP applies to all R&D and cell line development personnel performing single-cell isolation for generating stable monoclonal cell populations expressing biosimilar proteins.

3. Responsibilities

• Research Scientist: Designs and executes cloning strategy, monitors clone growth.
• Lab Technician: Prepares media, dilutions, and culture plates.
• QA Personnel: Verifies clone isolation records and ensures traceability.

4. Accountability

The Head of Cell Line Development is accountable for ensuring proper implementation and documentation of limiting dilution cloning procedures.

5. Procedure

5.1 Cell Preparation

1. Harvest the transfected cells from log phase culture with viability ≥ 90%.
2. Determine cell concentration using hemocytometer or automated cell counter.

5.2 Dilution Series Preparation

1. Prepare serial dilutions in complete growth medium to achieve 1 cell/100 µL.
2. Dispense 100 µL per well into a 96-well flat-bottom tissue culture plate.
3. Use at least 3 plates to increase monoclonality chances.

5.3 Incubation and Observation

1. Incubate the plates at 37°C with 5% CO₂.
2. Check wells daily under inverted microscope for single-cell outgrowth.
3. Document wells showing growth from a single cell in Clone Seeding Log (Annexure-1).

5.4 Expansion of Positive Clones

1. When cell number increases to 50–100 cells (typically 7–14 days), transfer to a 24-well plate.
2. Gradually expand to 6-well, then T-25 flask with proper labeling and tracking.
3. Confirm monoclonality via observation logs or automated imaging (if available).

5.5 Record Keeping

1. Assign Clone IDs to successfully expanded clones.
2. Record growth parameters, morphology, and well ID in Clone Tracking Record (Annexure-2).

5.6 Troubleshooting

1. If multiple colonies are observed in a well, discard for cloning purposes.
2. If no growth is observed after 14 days, mark as non-viable.

6. Abbreviations

• SOP: Standard Operating Procedure
• QA: Quality Assurance
• CO₂: Carbon Dioxide

7. Documents

1. Clone Seeding Log (Annexure-1)
2. Clone Tracking Record (Annexure-2)

8. References

• ICH Q5D – Derivation and Characterization of Cell Substrates
• WHO Guidelines for Cell Line Characterization
• CDSCO Guidelines for Monoclonality Assurance

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Clone Seeding Log

Date	Plate ID	Well No.	Cell Count	Observation	Recorded By
03/05/2025	LD-01	B6	1	Single Cell Observed	Sunita Reddy

Annexure-2: Clone Tracking Record

Clone ID	Source Plate	Expansion Date	Media Used	Passage No.	Status
CL-CHO-11	LD-01:B6	10/05/2025	CHO DMEM + 10% FBS	1	Active

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added expanded annexures and clarified single-cell criteria	Annual review