Standard Operating Procedure for Cell Density Monitoring (VCD, OD600) in Bioreactor Operations for Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/080/2025
Supersedes	SOP/BS/080/2022
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 standardized procedure for monitoring cell density in bioreactor operations using Viable Cell Density (VCD) via hemocytometer/automated counter and Optical Density (OD600) using a spectrophotometer during biosimilar production.

2. Scope

This SOP applies to upstream bioprocessing units involved in biosimilar production utilizing mammalian or microbial systems requiring real-time cell density monitoring.

3. Responsibilities

• Bioprocess Technician: Collect and measure cell density at defined intervals and maintain logs.
• QC Analyst: Calibrate instruments and validate measurement accuracy.
• QA Personnel: Review data logs and ensure SOP compliance.

4. Accountability

The Upstream Processing Manager is accountable for accurate implementation and compliance with cell density monitoring protocols.

5. Procedure

5.1 Sampling for Cell Density

1. Collect ~5 mL of culture fluid aseptically using SOP/BS/078/2025.
2. Transfer to labeled tubes and proceed with VCD or OD600 measurement immediately.

5.2 VCD Measurement Using Hemocytometer or Cell Counter

1. Mix 100 µL of sample with 100 µL of 0.4% trypan blue solution.
2. Load 10 µL into hemocytometer and observe under microscope (manual method) or use automatic cell counter.
3. Count viable (unstained) and non-viable (blue) cells in 4 corner squares.
4. Calculate VCD using:
   • VCD (cells/mL) = Average viable count × Dilution Factor × 10⁴
5. Log data in Annexure-1.

5.3 OD600 Measurement (Primarily for Microbial Culture)

1. Zero the spectrophotometer with fresh culture medium as blank at 600 nm.
2. Measure absorbance of culture sample diluted to fall within 0.1–1.0 OD range.
3. Use conversion factor for approximate CFU/mL if required.
4. Record OD600 readings and time in Annexure-2.

5.4 Instrument Calibration

1. Calibrate cell counter monthly using standard beads or reference slides.
2. Verify OD600 accuracy quarterly using traceable reference standards.

5.5 Documentation

1. Enter data in Bioreactor Monitoring Log and VCD/OD Log (Annexures 1 & 2).
2. Review trends to monitor growth rate and trigger feeding or harvest decisions.

6. Abbreviations

• VCD: Viable Cell Density
• OD600: Optical Density at 600 nm
• CFU: Colony Forming Units

7. Documents

1. VCD Measurement Log – Annexure-1
2. OD600 Monitoring Sheet – Annexure-2

8. References

• USP <1046> – Cell Viability Assays
• ICH Q9 – Quality Risk Management

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: VCD Measurement Log

Date	Time	Batch No.	VCD (cells/mL)	Viability (%)	Operator
04/05/2025	10:00	BS-080-B01	6.2 × 106	94%	Rajesh Kumar

Annexure-2: OD600 Monitoring Sheet

Date	Time	Batch No.	OD600	Dilution Factor	Operator
04/05/2025	10:15	BS-080-M01	0.78	10x	Sunita Reddy

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added automated counter method and OD600 calibration	Technology Upgrade