Standard Operating Procedure for ELISA-Based Screening of Clones in Biosimilar R&D
| Department | Biosimilars |
|---|---|
| SOP No. | SOP/BS/013/2025 |
| Supersedes | SOP/BS/013/2022 |
| Page No. | Page 1 of 12 |
| Issue Date | 04/05/2025 |
| Effective Date | 06/05/2025 |
| Review Date | 04/05/2026 |
1. Purpose
To establish a standardized method for using Enzyme-Linked Immunosorbent Assay (ELISA) to screen biosimilar-producing cell clones based on protein expression levels in the culture supernatant.
2. Scope
This SOP applies to biosimilar research and development laboratories using ELISA-based assays to select high-expressing clones during early-phase clone screening.
3. Responsibilities
- Research Associate: Prepares samples, standards, and performs ELISA procedures.
- Lab Technician: Assists in plate preparation, reagent handling, and recordkeeping.
- QA Representative: Ensures compliance of assay data and documentation standards.
4. Accountability
The Head of Analytical Development is accountable for the implementation and validation of the ELISA procedure for clone screening purposes.
5. Procedure
5.1 Materials and Reagents
- Pre-coated ELISA 96-well plates or uncoated plates with capture antibody
- Standards and controls (known concentration of biosimilar protein)
- Culture supernatants from clones
- Detection antibody (biotinylated or HRP-conjugated)
- Substrate solution (TMB)
- Stop solution (e.g., 2N H2SO4)
- Wash buffer (PBS-Tween 20)
- Plate washer or multichannel pipette
- Microplate reader
5.2 Sample Preparation
- Collect culture supernatants after 48–72 hours of clone growth.
- Centrifuge at 3000 rpm for 10 minutes to remove cell debris.
- Store aliquots at -20°C if not used immediately.
5.3 Assay Setup
- Add 100 µL of standards, controls, and samples in duplicates to the plate.
- Incubate for 1–2 hours at room temperature or as per kit instructions.
- Wash plate 3–5 times with PBS-Tween using plate washer or manually.
- Add 100 µL of detection antibody to each well and incubate again as directed.
- Repeat washing step, then add 100 µL TMB substrate and incubate in dark.
- Stop reaction after color development using 100 µL stop solution.
- Read absorbance at 450 nm using a microplate reader.
5.4 Data Analysis
- Generate standard curve from absorbance readings of known standards.
- Calculate protein concentrations in each clone sample using curve-fit equations.
- Record expression levels in the ELISA Clone Screening Log (Annexure-1).
5.5 Result Interpretation and Documentation
- Mark clones with expression ≥80% of control as “Positive.”
- Verify plate layout, sample positions, and replicate consistency.
- Review assay with QA before shortlisting clones.
6. Abbreviations
- ELISA: Enzyme-Linked Immunosorbent Assay
- HRP: Horseradish Peroxidase
- TMB: Tetramethylbenzidine
- PBS: Phosphate Buffered Saline
7. Documents
- ELISA Clone Screening Log (Annexure-1)
- Standard Curve Record Sheet (Annexure-2)
8. References
- ICH Q6B – Specifications: Test Procedures and Acceptance Criteria for Biotech Products
- WHO Guidelines on Quality Control of Biotherapeutics
- CDSCO Biologics Assay Guidelines
9. SOP Version
Version: 2.0
10. Approval Section
| Prepared By | Checked By | Approved By | |
|---|---|---|---|
| Signature | |||
| Date | |||
| Name | |||
| Designation | |||
| Department |
11. Annexures
Annexure-1: ELISA Clone Screening Log
| Date | Clone ID | Sample Type | Absorbance (450 nm) | Expression Level (ng/mL) | Result | Operator |
|---|---|---|---|---|---|---|
| 03/05/2025 | CL-013 | Supernatant | 1.238 | 96.5 | Positive | Rajesh Kumar |
Annexure-2: Standard Curve Record Sheet
| Standard (ng/mL) | Absorbance (450 nm) |
|---|---|
| 0 | 0.032 |
| 10 | 0.210 |
| 25 | 0.456 |
| 50 | 0.782 |
| 100 | 1.295 |
| 200 | 1.890 |
Revision History:
| Revision Date | Revision No. | Revision Details | Reason for Revision | Approved By |
|---|---|---|---|---|
| 04/05/2025 | 2.0 | Added curve log annexure and standard interpretation | Annual SOP update |