Standard Operating Procedure for Cell Viability Testing (Trypan Blue, Flow Cytometry) in Bioreactor Operations for Biosimilars

Department	Biosimilars
SOP No.	SOP/BS/085/2025
Supersedes	SOP/BS/085/2022
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 assessing cell viability in bioreactor cultures using the Trypan Blue exclusion method and flow cytometry, ensuring accurate evaluation of culture health during biosimilar production.

2. Scope

This SOP applies to upstream bioprocesses where routine monitoring of cell viability is required to evaluate culture performance and trigger critical process decisions.

3. Responsibilities

• Bioprocess Technician: Collect and process samples for viability testing.
• QC Analyst: Conduct viability assays and record results.
• QA Officer: Review viability data and investigate deviations if viability falls below threshold limits.

4. Accountability

The Upstream Production Manager is accountable for ensuring implementation of viability testing protocols and compliance with specifications.

5. Procedure

5.1 Sample Collection

1. Withdraw 5 mL of culture fluid aseptically from the bioreactor using in-process sampling SOP/BS/078/2025.
2. Transfer sample to a sterile, labeled tube and process immediately.

5.2 Trypan Blue Exclusion Method

1. Mix 100 µL of cell suspension with 100 µL of 0.4% Trypan Blue solution.
2. Load 10 µL onto a hemocytometer and observe under 100x magnification.
3. Count viable (unstained) and non-viable (blue) cells in four corner squares.
4. Calculate viability percentage:
   • Viability (%) = (Number of viable cells / Total cells) × 100
5. Log results in Annexure-1.

5.3 Flow Cytometry Method

1. Stain cells with 7-AAD or PI dye according to manufacturer’s instructions.
2. Run sample through flow cytometer and gate viable vs. non-viable populations.
3. Ensure at least 10,000 events are collected per sample.
4. Record fluorescence-based viability percentage in Annexure-2.

5.4 Frequency

1. Perform cell viability testing:
   • Before feed addition
   • At mid-log phase
   • Prior to harvest

5.5 Acceptance Criteria

1. Viability must be ≥85% to proceed with feeding or product harvest.
2. Values below 80% require investigation and deviation reporting.

6. Abbreviations

• PI: Propidium Iodide
• QC: Quality Control
• 7-AAD: 7-Aminoactinomycin D

7. Documents

1. Trypan Blue Viability Log – Annexure-1
2. Flow Cytometry Viability Report – Annexure-2

8. References

• USP <1046> – Cell Viability Assays
• ICH Q6B – Specifications for Biotechnological 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: Trypan Blue Viability Log

Date	Time	Batch No.	Total Cells	Viable Cells	Viability (%)	Operator
04/05/2025	10:30	BS-085-B01	2.5 × 10⁶	2.2 × 10⁶	88%	Rajesh Kumar

Annexure-2: Flow Cytometry Viability Report

Date	Time	Batch No.	Live Population (%)	Dead Population (%)	Dye Used	Operator
04/05/2025	14:45	BS-085-B01	91.2	8.8	7-AAD	Sunita Reddy

Revision History:

Revision Date	Revision No.	Revision Details	Reason for Revision	Approved By
04/05/2025	2.0	Added flow cytometry method and 7-AAD usage for high-throughput viability assessment	Process Expansion