Standard Operating Procedure for Cell Culture Media Optimization in Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/033/2025
Supersedes	SOP/BS/033/2022
Page No.	Page 1 of 13
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the procedure for optimizing cell culture media used for biosimilar production, aimed at improving cell growth, viability, product yield, and critical quality attributes (CQAs).

2. Scope

This SOP applies to the upstream process development team involved in screening, testing, and refining basal and feed media compositions used for CHO, HEK293, or other mammalian cells producing biosimilars.

3. Responsibilities

• Process Development Scientist: Designs and executes media optimization experiments.
• Analytical Support Team: Performs titer, metabolite, and quality assessments.
• QA Officer: Ensures all media development experiments are documented and controlled.

4. Accountability

The Head of Upstream Process Development is accountable for finalizing the media formulation used in GMP production batches.

5. Procedure

5.1 Media Selection and Preparation

1. Select initial media formulations based on literature, platform process, or commercially available basal media (e.g., CD-CHO, Ex-Cell).
2. Prepare basal media according to manufacturer’s instructions. Filter sterilize using 0.22 µm PES filters.
3. Formulate feed media supplements (glucose, amino acids, vitamins, lipids) as per pre-defined concentration ranges.

5.2 Experimental Design

1. Use Design of Experiments (DoE) approach:
   • Full factorial or fractional factorial designs
   • Plackett-Burman for screening
   • Central Composite Design (CCD) for optimization
2. Evaluate 3 to 5 media variables in combination (e.g., glutamine, trace metals, iron).

5.3 Small-Scale Testing

1. Seed cells into 50 mL shake flasks or deep-well plates at a density of 2–3 × 10⁵ cells/mL.
2. Incubate at 37°C, 5% CO₂, 80% humidity with shaking at 120 rpm.
3. Collect samples on Days 3, 5, 7 for:
   • Viable cell density and % viability (Trypan Blue, automated counters)
   • Glucose, lactate, ammonium
   • pH and osmolality
   • Product titer via HPLC or ELISA

5.4 Criteria for Optimization

1. Media formulation should support:
   • >90% viability till Day 7
   • High viable cell density (>10⁷ cells/mL)
   • Stable pH and osmolality (<340 mOsm/kg)
   • Titer improvement of ≥20% over baseline

5.5 Confirmation and Scale-up

1. Confirm selected media formulation in 1L spinner flasks or 2L benchtop bioreactors.
2. Monitor same parameters and compare with shake flask results.
3. If consistent, document final media composition and assign a unique formulation code.

5.6 Documentation

1. Record all batch-wise results in the Media Optimization Log (Annexure-1).
2. Prepare the Final Media Development Report (Annexure-2) for approval and QA archival.

6. Abbreviations

• DoE: Design of Experiments
• CQA: Critical Quality Attribute
• CHO: Chinese Hamster Ovary
• RPM: Revolutions Per Minute

7. Documents

1. Media Optimization Log (Annexure-1)
2. Final Media Development Report (Annexure-2)

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 999 – GMP for Biologicals
• USP <1046> – Cell Culture Media

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Media Optimization Log

Date	Media ID	Cell Line	Titer (g/L)	Viability (%)	Remarks	Scientist
02/05/2025	M033-V5	CHO-K1	3.5	94.2	Accepted	Sunita Reddy

Annexure-2: Final Media Development Report

Media Code	Composition Summary	Cell Line	Yield Improvement	Recommended For	Approved By
M033-V5	CD-CHO + Glutamine + Lipid Mix A	CHO-K1	+28%	MCB Expansion

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Updated with DoE and metabolite analysis section	Process optimization enhancement