Standard Operating Procedure for Hold Time Validation of Intermediate Products in Biosimilar Manufacturing

Department	Biosimilars
SOP No.	SOP/BS/178/2025
Supersedes	SOP/BS/178/2022
Page No.	Page 1 of 10
Issue Date	04/05/2025
Effective Date	06/05/2025
Review Date	04/05/2026

1. Purpose

To define the procedure for validating the maximum allowable hold times for intermediate biosimilar products between downstream processing steps to ensure stability and product quality are maintained during temporary storage.

2. Scope

This SOP applies to all intermediate pools and bulk drug substances held at various stages of purification, including post-capture, post-viral filtration, and post-UF/DF in biosimilar manufacturing processes.

3. Responsibilities

• Production: Store intermediates under validated conditions and record holding times accurately.
• QC: Test stability-indicating parameters at designated intervals during the hold study.
• QA: Review validation protocol, approve final hold time, and ensure adherence in batch operations.

4. Accountability

The Head of Process Development is accountable for defining, executing, and updating hold time validation studies for all biosimilar intermediate stages.

5. Procedure

5.1 Study Design

1. Identify all hold points in the manufacturing process where intermediate product is stored before the next unit operation.
2. Define expected maximum hold duration based on operational planning (e.g., 24–96 hours).
3. Design validation protocol including:
   • Storage container type
   • Storage temperature (e.g., 2–8°C, 20–25°C)
   • Sampling time points (e.g., 0, 12, 24, 48, 72 hours)
   • Analytical test panel

5.2 Sample Collection and Testing

1. At each time point, collect representative samples under aseptic conditions.
2. Perform tests such as:
   • Appearance
   • pH and conductivity
   • Protein concentration
   • Purity (SEC-HPLC)
   • Microbial limit and endotoxin (for longer hold times)
3. Document results in Annexure-1.

5.3 Acceptance Criteria

1. Intermediates should maintain:
   • Monomer content ≥ 95%
   • Protein loss ≤ 5%
   • No increase in endotoxin/microbial load beyond specification
2. If criteria are met at all time points, approve the longest time as validated hold duration.

5.4 Implementation and Monitoring

1. Apply validated hold times in batch manufacturing records (BMR).
2. Monitor real-time hold durations for each lot and ensure compliance with validated limits.
3. Any deviation must be investigated and documented in Annexure-2.

6. Abbreviations

• UF/DF: Ultrafiltration/Diafiltration
• SEC-HPLC: Size Exclusion Chromatography–High Performance Liquid Chromatography
• BMR: Batch Manufacturing Record
• EU: Endotoxin Units

7. Documents

1. Hold Time Validation Data Sheet – Annexure-1
2. Deviation and Investigation Record – Annexure-2

8. References

• ICH Q8 – Pharmaceutical Development
• WHO TRS 1004 – GMP Guidelines for Biotherapeutic Products
• FDA Guidance – Process Validation: General Principles and Practices

9. SOP Version

Version: 2.0

10. Approval Section

	Prepared By	Checked By	Approved By
Signature
Date
Name
Designation
Department

11. Annexures

Annexure-1: Hold Time Validation Data Sheet

Time Point	Appearance	pH	Protein (mg/mL)	Purity (%)	Microbial Load (cfu/mL)	Endotoxin (EU/mL)	Remarks
0 hr	Clear	7.0	12.5	98.1	<1	0.04	Baseline
72 hr	Clear	7.1	12.4	97.8	<1	0.06	Pass

Annexure-2: Deviation and Investigation Record

Date	Intermediate Stage	Deviation Description	Impact	Corrective Action	Approved By
03/05/2025	Post-Polishing	Hold exceeded by 6 hours	Batch held under quarantine	Retested, no quality impact	QA Manager

Revision History:

Revision Date	Revision No.	Details	Reason	Approved By
04/05/2025	2.0	Included microbial and endotoxin testing for extended hold studies	GMP compliance enhancement