Standard Operating Procedure for Small-Scale Expression Screening in Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/034/2025
Supersedes	SOP/BS/034/2022
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 procedure for evaluating the expression of biosimilar-producing clones in small-scale formats to determine protein titer, cell growth characteristics, and product quality prior to scale-up.

2. Scope

This SOP applies to upstream development personnel responsible for selecting high-expressing recombinant clones using small-volume cultures such as deep-well plates or shake flasks.

3. Responsibilities

• Research Associate: Prepares culture media, inoculates clones, monitors growth, and performs titer assays.
• Scientist: Designs experiments and interprets data for clone selection.
• QA Officer: Reviews documentation and ensures traceability of clone tracking and data capture.

4. Accountability

The Head of Upstream Process Development is accountable for the implementation and validation of clone screening procedures.

5. Procedure

5.1 Inoculum Preparation

1. Revive individual clones from cryopreserved stocks using selective media containing appropriate antibiotics or selection pressure (e.g., MTX or puromycin).
2. Expand cultures in 24-well deep-well plates or 50 mL shake flasks in a 37°C, 5% CO₂ incubator with shaking at 120 rpm.

5.2 Culture Conditions

1. Seed cells at 2 × 10⁵ cells/mL in 5–10 mL working volume per flask/well.
2. Incubate for 7 days, sampling on Day 3, 5, and 7.
3. Monitor viable cell density, % viability, and glucose/lactate levels.

5.3 Protein Expression Analysis

1. Harvest 1 mL culture supernatant from each sample timepoint.
2. Quantify protein titer using:
   • ELISA (sandwich or indirect)
   • SDS-PAGE densitometry (for relative comparison)
3. Record all values in Expression Screening Log (Annexure-1).

5.4 Criteria for Clone Advancement

1. Clone must meet:
   • Viability > 85% on Day 7
   • Titer ≥ 0.8 g/L
   • No abnormal cell morphology or excessive foaming

5.5 Selection and Documentation

1. Rank clones based on productivity and growth performance.
2. Document final clone selection in the Clone Selection Summary (Annexure-2).

6. Abbreviations

• ELISA: Enzyme-Linked Immunosorbent Assay
• SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
• CO₂: Carbon Dioxide
• MTX: Methotrexate

7. Documents

1. Expression Screening Log (Annexure-1)
2. Clone Selection Summary (Annexure-2)

8. References

• ICH Q5B – Expression Characterization of Recombinant Proteins
• WHO TRS 999 – GMP for Biotherapeutic Products

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Expression Screening Log

Clone ID	Viability (%)	Titer (g/L)	Day 7 VCD	Analyst	Remarks
C-034-A1	91.5	1.12	1.8×10⁷	Rajesh Kumar	High producer

Annexure-2: Clone Selection Summary

Selected Clone	Media Used	Final Titer	Rank	Selected For	Approved By
C-034-A1	CD-CHO	1.12 g/L	1	MCB Development

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added SDS-PAGE as optional screening method	Process optimization