Standard Operating Procedure for Clone Stability Assessment in Biosimilar Cell Line Development

1. Purpose

To define the procedure for evaluating the genetic and phenotypic stability of biosimilar-producing clones across extended cell passages under laboratory conditions.

2. Scope

This SOP applies to Cell Line Development (CLD) and Analytical Development teams conducting stability studies on selected biosimilar clones before Master Cell Bank creation.

3. Responsibilities

• CLD Scientist: Maintains cell cultures across passages and prepares samples.
• Analytical Scientist: Performs expression testing, gene copy number analysis, and glycosylation profiling.
• QA Representative: Reviews test logs and verifies data consistency and documentation.

4. Accountability

The Head of CLD is accountable for ensuring that only genetically and phenotypically stable clones are advanced to cell banking and further development stages.

5. Procedure

5.1 Clone Expansion for Stability Study

1. Thaw selected clone and culture in triplicate flasks.
2. Maintain continuous passaging every 3–4 days for a total of 60 population doublings (or 30 passages).
3. Document cell morphology, viability, and doubling time at each passage.

5.2 Sampling Strategy

1. Collect cell samples at initial passage (P0), mid-point (P15), and final passage (P30).
2. Preserve samples for:
   • mRNA expression (RT-PCR)
   • Protein expression (ELISA/Western blot)
   • Gene copy number (qPCR)
   • Glycan profiling (HPLC)

5.3 Expression Analysis

1. Quantify biosimilar protein at each sampling point using validated method (e.g., ELISA).
2. Compare final expression level with initial value. Acceptable drift: ±20%.

5.4 Gene Copy Number Analysis

1. Extract genomic DNA and perform qPCR using transgene-specific primers.
2. Use single-copy gene (e.g., GAPDH) for normalization.
3. Document results in Clone Stability Log (Annexure-1).

5.5 Glycosylation Profile Monitoring

1. Analyze glycan pattern via 2-AB labeled HPLC or lectin blotting.
2. Ensure no major shift in sialylation or fucosylation profile.

5.6 Data Review and Acceptance Criteria

1. Clone is considered stable if:
   • Expression deviation ≤ ±20%
   • Copy number deviation ≤ 1 copy
   • Glycan profile remains within ±10% for major glycans
2. Summarize findings in Stability Study Summary Report.

6. Abbreviations

• CLD: Cell Line Development
• qPCR: Quantitative Polymerase Chain Reaction
• HPLC: High Performance Liquid Chromatography
• 2-AB: 2-Aminobenzamide

7. Documents

1. Clone Stability Log (Annexure-1)
2. Expression Comparison Sheet (Annexure-2)

8. References

• ICH Q5B – Genetic Stability Testing Guidelines
• WHO TRS 999 – Biosimilar Clone Monitoring Standards
• CDSCO Guidance on Cell Line Characterization

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Clone Stability Log

Annexure-2: Expression Comparison Sheet

Revision History:

Standard Operating Procedure for Productivity Testing in Small-Scale Biosimilar Cultures

1. Purpose

To describe the procedure for assessing productivity of biosimilar-expressing cell clones using small-scale cultures to support early clone ranking and upstream development.

2. Scope

This SOP applies to the Cell Line Development and Upstream Process Development teams evaluating recombinant protein production in shake flasks or deep well plates (DWP).

3. Responsibilities

• Upstream Scientist: Plans and executes productivity runs and documents results.
• CLD Technician: Performs culture maintenance and sampling.
• QA Representative: Verifies result entries and cross-checks against production logs.

4. Accountability

The Head of Upstream Development is accountable for ensuring the reliability of productivity data generated in small-scale models for biosimilar clone progression.

5. Procedure

5.1 Culture Setup

1. Revive selected clones from WCB or cryovials and expand in T-flasks.
2. Inoculate shake flasks (e.g., 250 mL with 50 mL working volume) or DWP (2 mL per well) at 0.3–0.5 × 10⁶ cells/mL.
3. Use production medium or serum-free platform media.
4. Incubate at 36–37°C, 5% CO₂, shaking at 120–180 rpm.

5.2 Sampling Schedule

1. Collect 1–2 mL samples on Day 0, 3, 5, and 7 (or as per protocol).
2. Measure viable cell density and viability using Trypan Blue or automated cell counter.
3. Centrifuge and collect supernatants for protein analysis.

5.3 Protein Quantification

1. Quantify expressed protein using:
   • ELISA for biosimilar mAb or recombinant protein
   • Protein A HPLC for monoclonal antibodies
2. Prepare standard curves using reference standards.
3. Calculate yield in mg/L and document in Productivity Log (Annexure-1).

5.4 Data Analysis

1. Determine specific productivity (qP) = Protein (pg/cell/day).
2. Rank clones by final titer and qP value.
3. Compare against productivity benchmarks from historical data or innovator values.

6. Abbreviations

• DWP: Deep Well Plate
• qP: Specific Productivity
• ELISA: Enzyme-Linked Immunosorbent Assay
• HPLC: High Performance Liquid Chromatography

7. Documents

1. Productivity Testing Log (Annexure-1)
2. Cell Growth and Viability Log (Annexure-2)

8. References

• ICH Q6B – Specifications: Test Procedures and Acceptance Criteria
• WHO Guidelines for Cell-Based Biosimilar Characterization
• CDSCO Biosimilar Guidelines (India)

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Productivity Testing Log

Annexure-2: Cell Growth and Viability Log

Revision History:

Standard Operating Procedure for Glycosylation Analysis in Biosimilar Clone Selection

1. Purpose

To define the procedure for analyzing glycosylation profiles of expressed biosimilar proteins in mammalian cell clones, facilitating clone selection based on critical quality attributes (CQAs).

2. Scope

This SOP applies to Analytical Development, Cell Line Development, and Quality Control teams involved in assessing glycan structures during biosimilar clone screening.

3. Responsibilities

• Analytical Scientist: Executes glycan release, labeling, and chromatography or mass spec analysis.
• CLD Scientist: Provides purified protein samples for analysis.
• QA Representative: Reviews and verifies analysis logs and compliance to regulatory expectations.

4. Accountability

The Head of Analytical Development is accountable for ensuring accurate glycan profiling and interpretation to guide clone selection in biosimilar programs.

5. Procedure

5.1 Sample Preparation

1. Collect 0.5–1.0 mg purified biosimilar protein from each clone.
2. Buffer-exchange to PBS if required using desalting columns.
3. Record sample details in Glycosylation Sample Log (Annexure-1).

5.2 N-linked Glycan Release

1. Denature protein at 95°C for 10 minutes in presence of SDS and DTT.
2. Add NP-40 and PNGase F enzyme; incubate at 37°C for 18 hours.

5.3 Glycan Labeling

1. Dry released glycans using vacuum concentrator.
2. Label with 2-AB (2-aminobenzamide) or procainamide dye in sodium cyanoborohydride solution.
3. Incubate at 65°C for 2 hours in the dark.

5.4 HPLC/UPLC Analysis

1. Inject labeled glycans onto amide column (e.g., BEH Glycan) connected to HPLC/UPLC system.
2. Run with gradient of acetonitrile and ammonium formate buffer.
3. Acquire fluorescence signal and calculate relative peak areas.

5.5 Optional Mass Spectrometry

1. Desalt labeled glycans using HILIC cartridges.
2. Analyze using LC-MS or MALDI-TOF to determine glycan composition.

5.6 Lectin Blotting (Optional)

1. Run protein sample on SDS-PAGE and transfer to membrane.
2. Block membrane and incubate with biotinylated lectins (e.g., SNA, ConA).
3. Detect with streptavidin-HRP and chemiluminescent substrate.

5.7 Data Interpretation and Reporting

1. Assign glycan peaks using reference ladder or glycan database.
2. Calculate % abundance of high mannose, complex, sialylated, and fucosylated glycans.
3. Compare profiles with innovator molecule specifications.
4. Record results in Glycan Profile Log (Annexure-2).

6. Abbreviations

• HPLC: High Performance Liquid Chromatography
• PNGase F: Peptide:N-Glycosidase F
• 2-AB: 2-Aminobenzamide
• SNA: Sambucus Nigra Agglutinin

7. Documents

1. Glycosylation Sample Log (Annexure-1)
2. Glycan Profile Log (Annexure-2)

8. References

• ICH Q6B – Test Procedures and Acceptance Criteria for Biotech Products
• WHO TRS 1004 – Glycosylation Evaluation Guidelines
• CDSCO Biosimilar Quality Guidelines (India)

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Glycosylation Sample Log

Annexure-2: Glycan Profile Log

Revision History:

Standard Operating Procedure for Protein Expression Profiling in Biosimilar-Producing Cell Lines

1. Purpose

To define the procedure for evaluating biosimilar protein expression levels in engineered cell clones using SDS-PAGE, Western blotting, and densitometry techniques for clone selection and development decisions.

2. Scope

This SOP is applicable to all personnel in the Biosimilars department responsible for assessing recombinant protein expression levels in stably transfected or transiently expressing mammalian cell lines.

3. Responsibilities

• Research Scientist: Designs experimental layout and reviews results.
• Lab Technician: Carries out protein extraction, electrophoresis, blotting, and quantification.
• QA Officer: Ensures records are maintained and verifies consistency with GMP documentation practices.

4. Accountability

The Head of Analytical Development is accountable for the accuracy and regulatory compliance of protein expression data used for biosimilar development and clone selection.

5. Procedure

5.1 Sample Collection and Protein Extraction

1. Harvest 1–2 × 10⁶ cells by centrifugation at 300 × g for 5 min.
2. Wash cell pellet with ice-cold PBS.
3. Lyse cells in RIPA buffer containing protease inhibitors.
4. Incubate on ice for 30 minutes and centrifuge at 12,000 × g for 15 minutes.
5. Collect supernatant and measure protein concentration using BCA assay.

5.2 SDS-PAGE

1. Load 20–40 µg of total protein per lane on a 10–12% SDS-PAGE gel.
2. Run at 120V until dye front reaches bottom of gel.

5.3 Western Blotting

1. Transfer proteins to PVDF membrane at 100V for 90 minutes in transfer buffer.
2. Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature.
3. Incubate with primary antibody (e.g., anti-rhEPO) overnight at 4°C.
4. Wash and incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.
5. Detect signals using chemiluminescent substrate and capture on X-ray film or imaging system.

5.4 Densitometry and Data Analysis

1. Scan membrane and quantify band intensity using image analysis software (e.g., ImageJ).
2. Normalize expression to housekeeping protein (e.g., β-actin).
3. Document results in Expression Profiling Log (Annexure-1).

6. Abbreviations

• SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
• HRP: Horseradish Peroxidase
• BCA: Bicinchoninic Acid
• PVDF: Polyvinylidene Fluoride

7. Documents

1. Expression Profiling Log (Annexure-1)
2. Protein Quantification Log (Annexure-2)

8. References

• ICH Q6B – Specifications: Test Procedures and Acceptance Criteria
• WHO Guidelines for Biosimilar Characterization
• MIQE Guidelines – Western Blotting Best Practices

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Expression Profiling Log

Annexure-2: Protein Quantification Log

Revision History:

Standard Operating Procedure for mRNA Transcript Analysis using RT-PCR in Biosimilar Development

1. Purpose

To define the standard procedure for analyzing mRNA transcripts of biosimilar genes in transfected cell clones using reverse transcription polymerase chain reaction (RT-PCR).

2. Scope

This SOP applies to molecular biology and analytical teams responsible for evaluating transcriptional expression of biosimilar genes in cell line development processes.

3. Responsibilities

• Molecular Biologist: Performs total RNA isolation, cDNA synthesis, and RT-PCR.
• Research Assistant: Maintains reagents, prepares reaction setups, and performs gel analysis.
• QA Officer: Verifies experimental records and ensures compliance with documentation standards.

4. Accountability

The Head of Molecular Biology is accountable for ensuring transcript analysis is accurate, validated, and compliant with biosimilar development protocols.

5. Procedure

5.1 Total RNA Isolation

1. Harvest cells (≥1 × 10⁶) and wash with ice-cold PBS.
2. Lyse cells using TRIzol reagent or column-based RNA extraction kits.
3. Quantify RNA using a spectrophotometer (A260/A280 ratio ~2.0).
4. Check RNA integrity on 1% agarose gel (28S/18S rRNA bands).

5.2 DNase Treatment

1. Treat 1 µg of RNA with DNase I to remove genomic DNA contamination.
2. Incubate at 37°C for 30 minutes and inactivate DNase as per reagent protocol.

5.3 cDNA Synthesis

1. Use 1 µg of DNase-treated RNA in a 20 µL reverse transcription reaction.
2. Include oligo(dT) or random hexamer primers, dNTPs, and reverse transcriptase enzyme.
3. Incubate as follows:
   • 25°C for 10 minutes
   • 42°C for 60 minutes
   • 70°C for 15 minutes (enzyme inactivation)

5.4 PCR Amplification of Target Transcript

1. Set up PCR using gene-specific primers (e.g., for rhEPO or mAb light chain).
2. Prepare 25 µL reaction mixtures using cDNA, Taq polymerase, and buffer system.
3. Thermal cycling conditions:
   • Initial denaturation: 95°C for 3 min
   • 35 cycles: 95°C for 30 sec, 58°C for 30 sec, 72°C for 1 min
   • Final extension: 72°C for 5 min

5.5 Gel Electrophoresis and Documentation

1. Run PCR product on 1.5% agarose gel with ethidium bromide or GelRed.
2. Capture gel image and note band size and intensity.
3. Record results in the RT-PCR Expression Log (Annexure-1).

6. Abbreviations

• RT-PCR: Reverse Transcription Polymerase Chain Reaction
• RNA: Ribonucleic Acid
• cDNA: Complementary DNA
• PBS: Phosphate Buffered Saline

7. Documents

1. RT-PCR Expression Log (Annexure-1)
2. RNA Quality Assessment Sheet (Annexure-2)

8. References

• WHO TRS 999 – Molecular Characterization of Cell Lines
• MIQE Guidelines for RT-PCR Reporting
• ICH Q5B – Analysis of Expression Constructs

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: RT-PCR Expression Log

Annexure-2: RNA Quality Assessment Sheet

Revision History:

Standard Operating Procedure for Southern Blot Analysis in Biosimilar Cell Lines

1. Purpose

To define the method for performing Southern blot analysis for confirming transgene integration, determining gene copy number, and detecting rearrangements in genetically modified biosimilar-producing cell lines.

2. Scope

This SOP applies to the Molecular Biology and Cell Line Development teams responsible for genetic characterization and verification of recombinant clones used in biosimilar production.

3. Responsibilities

• Molecular Biologist: Carries out the complete Southern blot procedure and interprets results.
• Lab Assistant: Prepares reagents, assists with electrophoresis and blotting steps.
• QA Representative: Verifies documentation and ensures traceability of results.

4. Accountability

The Head of Molecular Biology is accountable for ensuring the accurate execution and compliance of Southern blot assays as part of clone characterization in biosimilar programs.

5. Procedure

5.1 Genomic DNA Preparation

1. Isolate high molecular weight DNA from 1 × 10⁷ cells using phenol-chloroform extraction.
2. Check DNA quality using agarose gel electrophoresis and spectrophotometry.

5.2 Restriction Enzyme Digestion

1. Digest 10 µg genomic DNA with restriction enzymes that cut outside the transgene insertion site.
2. Use at least 1 unit of enzyme per µg DNA and incubate overnight at 37°C.

5.3 Gel Electrophoresis and Denaturation

1. Run digested DNA on 0.8% agarose gel at 40V overnight.
2. Soak gel in 0.25M HCl for 10 minutes to depurinate.
3. Denature in 1.5M NaCl, 0.5M NaOH for 30 minutes, followed by neutralization in 1.5M NaCl, 0.5M Tris-HCl pH 7.5.

5.4 Capillary Blotting

1. Transfer DNA onto a nylon membrane overnight via capillary transfer in 20X SSC buffer.
2. Cross-link DNA to membrane using UV crosslinker or baking at 80°C for 2 hours.

5.5 Probe Preparation and Hybridization

1. Label the DNA probe (complementary to transgene) using radioactive or digoxigenin-based labeling.
2. Prehybridize membrane in hybridization buffer for 1 hour at 42°C.
3. Add labeled probe and hybridize overnight at 42–68°C depending on probe type.

5.6 Washing and Detection

1. Wash membrane with decreasing stringency buffers (e.g., 2X SSC, 0.1X SSC).
2. Detect signals using X-ray film (radioactive) or chemiluminescent substrate (DIG).
3. Interpret number and intensity of bands to determine gene copy number and integration pattern.
4. Document in Southern Blot Result Log (Annexure-1).

6. Abbreviations

• SSC: Saline-Sodium Citrate Buffer
• DIG: Digoxigenin
• UV: Ultraviolet
• X-ray: Radiographic Detection Method

7. Documents

1. Southern Blot Result Log (Annexure-1)
2. Probe Synthesis and Labeling Record (Annexure-2)

8. References

• WHO TRS 999 – Genetic Stability Testing of Cell Substrates
• ICH Q5B – Quality of Biotechnological Products: Analysis of Expression Constructs
• CDSCO Guidelines on Recombinant Cell Line Characterization

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Southern Blot Result Log

Annexure-2: Probe Synthesis and Labeling Record

Revision History:

Standard Operating Procedure for Gene Copy Number Determination in Biosimilar Cell Lines

1. Purpose

To establish a standardized method for determining the gene copy number of integrated biosimilar transgenes in cell lines using quantitative PCR (qPCR), aiding in clone selection and stability assessment.

2. Scope

This SOP applies to molecular biology and analytical development teams responsible for evaluating and validating biosimilar-producing clones through gene copy number quantification.

3. Responsibilities

• Molecular Biologist: Conducts DNA extraction, qPCR setup, and data analysis.
• Research Associate: Assists in sample preparation and maintains documentation.
• QA Officer: Verifies results and checks data integrity for regulatory compliance.

4. Accountability

The Head of Molecular Biology is accountable for ensuring accuracy and reproducibility of gene copy number data in biosimilar cell line evaluations.

5. Procedure

5.1 Genomic DNA Extraction

1. Harvest cells (≥1 × 10⁶) and wash with PBS.
2. Extract DNA using silica column-based kits or phenol-chloroform method.
3. Quantify and assess DNA purity using spectrophotometer (A260/A280 ratio 1.8–2.0).

5.2 Primer and Probe Design

1. Design primers targeting a region of the biosimilar transgene (e.g., Fc region for mAb genes).
2. Select a single-copy reference gene (e.g., β-actin or GAPDH).
3. Validate primer efficiency (90–110%) and specificity prior to use.

5.3 Real-Time PCR Setup

1. Prepare 20 µL reactions containing:
   • 10 µL SYBR Green or probe-based master mix
   • 1 µL each of forward and reverse primers (10 µM)
   • 1 µL genomic DNA (10–50 ng)
   • 7 µL nuclease-free water
2. Run samples in triplicates on a real-time PCR machine (e.g., ABI 7500).
3. Thermal cycling conditions:
   • Initial denaturation: 95°C for 10 min
   • 40 cycles: 95°C for 15 sec, 60°C for 60 sec

5.4 Data Analysis

1. Calculate ΔCt = Ct_transgene – Ct_reference.
2. Relative copy number = 2^-ΔΔCt compared to control clone or calibrator.
3. Include positive control (known copy number) and no template control (NTC).
4. Document results in Gene Copy Number Log (Annexure-1).

5.5 Acceptance Criteria

1. Coefficient of variation (CV) among replicates should be ≤5%.
2. Reference gene Ct range: 18–25.
3. Transgene Ct range: 18–30 depending on copy number.

6. Abbreviations

• qPCR: Quantitative Polymerase Chain Reaction
• DNA: Deoxyribonucleic Acid
• Ct: Cycle Threshold
• ΔCt: Difference in Ct values between target and reference genes

7. Documents

1. Gene Copy Number Log (Annexure-1)
2. qPCR Plate Map (Annexure-2)

8. References

• ICH Q6B – Test Procedures and Acceptance Criteria
• WHO TRS 1004 – Guidelines on Molecular Characterization
• MIQE Guidelines – Minimum Information for Publication of qPCR Experiments

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Gene Copy Number Log

Annexure-2: qPCR Plate Map

Revision History:

Standard Operating Procedure for Adaptation of Cells to Serum-Free Media in Biosimilar Development

1. Purpose

To describe the step-by-step procedure for adapting mammalian cell lines used in biosimilar production from serum-containing media to serum-free media to support regulatory compliance, scalability, and safety.

2. Scope

This SOP applies to all R&D and process development personnel involved in upstream cell culture operations for biosimilars where serum-free media is required for clone expansion or bioreactor inoculation.

3. Responsibilities

• Cell Culture Scientist: Designs and monitors the adaptation process.
• Technician: Performs daily cell culture maintenance and media exchanges.
• QA Officer: Reviews adaptation logs and confirms compliance with procedural requirements.

4. Accountability

The Head of Upstream Process Development is accountable for ensuring successful adaptation of production clones to serum-free conditions and maintaining traceable documentation.

5. Procedure

5.1 Preparation and Planning

1. Select serum-free medium compatible with the specific cell line (e.g., CD-CHO, ProCHO5).
2. Prepare complete serum-containing medium and serum-free medium (SFM) in advance.
3. Seed cells at 3–5 × 10⁵ cells/mL in serum-containing medium in T-75 flasks.

5.2 Stepwise Adaptation Schedule

1. Begin with a 75:25 mixture of serum-containing medium to SFM.
2. Allow cells to reach ~80% confluency or 1 × 10⁶ cells/mL.
3. Subculture using the next ratio: 50:50, then 25:75, and finally 100% SFM in successive passages.
4. Monitor cell viability, morphology, and doubling time after each adaptation step.
5. If viability drops below 85%, repeat the previous step before advancing.

5.3 Maintenance in Serum-Free Medium

1. Once cells grow stably in 100% SFM for at least 3 passages, consider adaptation complete.
2. Expand adapted cells for productivity testing and bioreactor inoculum.
3. Document final adaptation status in the Adaptation Completion Log (Annexure-2).

5.4 Troubleshooting

1. For poor growth or clumping, supplement with minimal FBS (≤1%) for one cycle.
2. Increase passage interval or seeding density if doubling time is extended.
3. Ensure media components (e.g., L-glutamine) are not degraded during storage.

6. Abbreviations

• SFM: Serum-Free Medium
• FBS: Fetal Bovine Serum
• QA: Quality Assurance
• CHO: Chinese Hamster Ovary cells

7. Documents

1. Adaptation Tracking Log (Annexure-1)
2. Adaptation Completion Log (Annexure-2)

8. References

• ICH Q5D – Cell Substrate Guidelines
• WHO TRS 978 – Guidelines for Serum-Free Media
• CDSCO Guidelines on Animal-Origin-Free Cell Culture Systems

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Adaptation Tracking Log

Annexure-2: Adaptation Completion Log

Revision History:

Standard Operating Procedure for Stable Cell Line Development in Biosimilar R&D

1. Purpose

To establish a standardized procedure for the development of stable mammalian cell lines expressing biosimilar therapeutic proteins. This includes transfection, clone selection, expansion, and characterization steps.

2. Scope

This SOP applies to biosimilar R&D personnel involved in generating and validating stable expression cell lines for preclinical and commercial biosimilar development.

3. Responsibilities

• Research Scientist: Designs and supervises the stable cell line generation workflow.
• Cell Culture Technician: Executes cell handling, selection, and clone expansion.
• QA Representative: Ensures adherence to procedural documentation and review of clone selection records.

4. Accountability

The Head of Cell Line Development is accountable for ensuring regulatory-compliant execution and traceability of stable cell line generation activities.

5. Procedure

5.1 Preparation for Transfection

1. Maintain parental cell lines (e.g., CHO, HEK293) in exponential growth phase.
2. Confirm cell viability (>90%) and absence of contamination.
3. Prepare transfection-grade plasmid DNA encoding the biosimilar gene of interest and selection marker.

5.2 Transfection

1. Transfect cells using electroporation or lipid-based reagent per protocol.
2. Plate transfected cells in T-75 or 6-well plates in antibiotic-free medium for 24–48 hours.

5.3 Antibiotic Selection

1. Determine kill curve for antibiotic (e.g., G418, Hygromycin) prior to application.
2. Add selection medium at predetermined lethal concentration and monitor for 10–14 days.
3. Change medium every 3–4 days.

5.4 Clone Isolation

1. Isolate single colonies using cloning rings or limiting dilution cloning.
2. Expand clones to 24-well plates, then to 6-well plates and T-25 flasks.
3. Maintain Clone Tracking Log (Annexure-1) throughout.

5.5 Expression Screening

1. Screen for expression using ELISA or Western blot.
2. Select top-performing clones with high and stable protein expression.
3. Document results in Expression Analysis Log (Annexure-2).

5.6 Clone Expansion and Characterization

1. Expand selected clones to generate enough biomass for further testing.
2. Confirm identity, gene copy number, productivity, and glycosylation profile.
3. Document characterization in Stable Clone Profile Sheet (Annexure-3).

5.7 Master Cell Bank (MCB) Generation

1. Freeze vials of confirmed stable clones under GMP-like conditions.
2. Label each vial with Clone ID, batch no., and date.
3. Store at -80°C and then transfer to liquid nitrogen storage.

6. Abbreviations

• CHO: Chinese Hamster Ovary
• MCB: Master Cell Bank
• ELISA: Enzyme-Linked Immunosorbent Assay
• QA: Quality Assurance

7. Documents

1. Clone Tracking Log (Annexure-1)
2. Expression Analysis Log (Annexure-2)
3. Stable Clone Profile Sheet (Annexure-3)

8. References

• ICH Q5D – Derivation and Characterization of Cell Substrates
• WHO Guidelines for Cell Line Development
• CDSCO Technical Guidelines on Cell Banks

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Clone Tracking Log

Annexure-2: Expression Analysis Log

Annexure-3: Stable Clone Profile Sheet

Revision History:

Standard Operating Procedure for Western Blotting for Protein Expression in Biosimilar Cell Clones

1. Purpose

To define the standardized procedure for detecting and analyzing biosimilar protein expression in cell lysates using SDS-PAGE followed by Western blotting during clone evaluation and selection.

2. Scope

This SOP is applicable to research and analytical staff involved in screening and confirming protein expression in cell-based biosimilar production systems using immunoblotting techniques.

3. Responsibilities

• Analytical Scientist: Performs protein extraction, SDS-PAGE, blotting, and detection.
• Lab Technician: Assists in reagent preparation and operation of blotting apparatus.
• QA Officer: Verifies blotting data and ensures procedural documentation is complete.

4. Accountability

The Head of Analytical Development is accountable for ensuring the Western blotting process is performed accurately and results are validated and recorded per regulatory guidelines.

5. Procedure

5.1 Sample Preparation

1. Harvest cells and wash with PBS.
2. Lyse cells using RIPA buffer supplemented with protease inhibitors.
3. Centrifuge at 13,000 rpm for 15 minutes at 4°C and collect supernatant.
4. Quantify protein concentration using BCA or Bradford assay.

5.2 SDS-PAGE Electrophoresis

1. Prepare 10–12% polyacrylamide gels depending on target protein size.
2. Mix equal volumes of sample (20–40 µg protein) with 2X Laemmli buffer and heat at 95°C for 5 minutes.
3. Load molecular weight marker and samples into gel wells.
4. Run gel at 100–120V until dye front reaches the bottom.

5.3 Transfer to Membrane

1. Assemble gel and PVDF/nitrocellulose membrane in blotting cassette.
2. Transfer proteins at 100V for 1 hour or 30V overnight at 4°C.
3. Confirm transfer by Ponceau S staining if required.

5.4 Blocking and Antibody Incubation

1. Block membrane with 5% skim milk or BSA in TBST for 1 hour at room temperature.
2. Incubate with primary antibody (e.g., anti-biosimilar protein) in blocking buffer for 2 hours or overnight at 4°C.
3. Wash membrane 3× with TBST, 5 minutes each wash.
4. Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature.
5. Repeat washing step post-secondary antibody incubation.

5.5 Detection and Documentation

1. Develop blot using chemiluminescent substrate.
2. Capture signal using gel documentation system or X-ray film.
3. Record exposure time, band intensity, and molecular weight in Western Blot Analysis Log (Annexure-1).

6. Abbreviations

• SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
• HRP: Horseradish Peroxidase
• TBST: Tris Buffered Saline with Tween 20
• BSA: Bovine Serum Albumin

7. Documents

1. Western Blot Analysis Log (Annexure-1)
2. Antibody and Reagent Inventory Sheet (Annexure-2)

8. References

• ICH Q6B – Specifications: Test Procedures for Biotechnological Products
• WHO Guidelines on Biosimilar Characterization
• CDSCO Analytical Method Validation Guidance

9. SOP Version

Version: 2.0

10. Approval Section

11. Annexures

Annexure-1: Western Blot Analysis Log

Annexure-2: Antibody and Reagent Inventory Sheet

Revision History: