SOP for Aseptic Culture, Expansion, and Maintenance of Mammalian Cells (HEK293, CHO)

1.0 Objective

To define the standardized procedure for aseptic handling, culture initiation, passaging, and expansion of mammalian cells (HEK293 and CHO) for use in gene therapy manufacturing processes, ensuring full compliance with current Good Manufacturing Practices (cGMP) per 21 CFR Parts 210/211 and EMA guidelines.

2.0 Scope

This SOP applies to all operations involving mammalian cell lines (adherent and suspension) within Grade B or C cleanroom environments of the Gene Therapy Manufacturing Unit.

Includes the following:

• Cryopreserved cell thawing

• Cell expansion

• Routine passaging

• Maintenance and harvest for downstream processing

3.0 Responsibility

4.0 References

• 21 CFR Part 210/211

• EMA Annex 1: Manufacture of Sterile Medicinal Products

• USP <1046> Cell and Gene Therapy Products

• JSA/SOP/GEN/EM/002 – Environmental Monitoring

• JSA/SOP/QA/DEV/003 – Deviation Management

5.0 Definitions

• HEK293: Human Embryonic Kidney cells commonly used in viral vector production.

• CHO: Chinese Hamster Ovary cells used for recombinant protein expression.

• SFM: Serum-Free Medium.

• Passage Number: Number of subculture cycles undergone by a cell population.

6.0 Materials and Equipment

6.1 Consumables:

• Cryovials with HEK293/CHO cell stocks

• FreeStyle™ F17 or equivalent serum-free media

• TrypLE™ Express (Gibco)

• USP-grade DMSO

• DPBS (Dulbecco's Phosphate Buffered Saline)

• Sterile 15 mL and 50 mL conical tubes

• T75/T175 flasks and sterile spinner flasks or shake flasks

6.2 Equipment:

• Class II Biological Safety Cabinet (NuAire NU-543 or equivalent)

• CO₂ Incubator (Thermo Scientific HERAcell™)

• Centrifuge (refrigerated, swing bucket rotor)

• Automated cell counter (Nexcelom Cellometer® or Vi-Cell XR)

• Orbital shaker (for suspension cultures)

• Water bath (37°C)

• Microscope (phase contrast)

1.0 Objective

To establish standardized procedures for the aseptic culture, expansion, and maintenance of mammalian cell lines (HEK293, CHO) used in gene therapy production, in compliance with cGMP guidelines (21 CFR Parts 210 and 211) and EMA regulations.

2.0 Scope

This SOP applies to all activities involving mammalian cell culture operations in ISO Class 7 (Grade C) and ISO Class 5 (Grade B) cleanrooms. It covers operations from thawing of cryopreserved cells to routine expansion and harvest, for both adherent and suspension-based cultures.

3.0 Responsibilities

4.0 Materials and Equipment

4.1 Consumables

• Serum-free media (e.g., FreeStyle™ F17)
• TrypLE™ Express Enzyme
• DMSO, USP Grade
• Dulbecco’s Phosphate Buffered Saline (DPBS)
• Cell culture flasks (T75/T175)
• Cryovials and conical tubes (15 mL and 50 mL)

4.2 Equipment

• Class II Biosafety Cabinet (e.g., NuAire NU-543)
• CO₂ Incubator (e.g., Thermo Scientific HERAcell™)
• Automated Cell Counter (e.g., Nexcelom Cellometer®)
• Refrigerated Centrifuge
• Inverted Phase-Contrast Microscope
• Liquid Nitrogen Freezer (Vapor Phase, -150°C)

5.0 Procedure

5.1 Thawing Cryopreserved Cells

1. Remove a vial of cryopreserved cells from the vapor-phase liquid nitrogen storage.
2. Immediately thaw in a 37°C water bath for no more than 90 seconds.
3. Wipe the vial with 70% IPA and transfer contents to a sterile 15 mL tube.
4. Add 9 mL of pre-warmed complete medium dropwise.
5. Centrifuge at 300 x g for 5 minutes with brake OFF.
6. Aspirate supernatant, resuspend the pellet in fresh complete medium to a concentration of 0.5 × 10⁶ cells/mL.
7. Transfer to T75 flask and incubate at 37°C in 5% CO₂.

SOP for Cell Culture Scale-Up and Bioreactor Inoculation for Gene Therapy Production

1.0 Objective

To establish a standardized procedure for the aseptic scale-up of mammalian cells (e.g., HEK293, CHO) from shake flasks to production-scale bioreactors (5L–200L), ensuring cell viability and performance consistency for gene therapy vector manufacturing under GMP-compliant conditions.

2.0 Scope

This SOP applies to all upstream processing personnel involved in the scale-up of suspension/adherent mammalian cells and bioreactor inoculation in Grade B/C cleanroom environments of gene therapy manufacturing facilities.

3.0 Responsibilities

4.0 References

• 21 CFR 210/211 – cGMP for Finished Pharmaceuticals

• EMA Guideline on Manufacture of ATMPs (EMA/CAT/GTWP/671639/2008)

• ICH Q7 – Good Manufacturing Practice for Active Pharmaceutical Ingredients

• Manufacturer instructions: Sartorius® BIOSTAT STR, Thermo™ HyPerforma™ Bioreactor Systems

5.0 Definitions

• Seed Train: Stepwise expansion of cells from thaw to production scale.

• Viable Cell Density (VCD): Number of living cells per mL.

• DO: Dissolved Oxygen, typically maintained at 40–50%.

• pCO₂: Partial pressure of carbon dioxide, regulated during scale-up.

6.0 Materials and Equipment

6.1 Materials

• Serum-free suspension media (e.g., FreeStyle™ F17, BalanCD™ HEK293)

• Glucose solution (200 g/L, sterile)

• Antifoam C (USP grade)

• DPBS (Dulbecco’s Phosphate Buffered Saline)

• Mycoplasma and sterility test kits

6.2 Equipment

• Class II BSC

• CO₂ Incubator (Thermo HERAcell™ 150i)

• Orbital shakers (Infors HT Multitron®)

• 125 mL–3L shake flasks (Corning® or equivalent)

• Bioreactor (5L, 20L, 50L, 200L Sartorius® BIOSTAT STR or equivalent)

• Cell Counter (Vi-CELL™, Cellometer®, or NucleoCounter®)

• Peristaltic pump (Watson Marlow)

• pH and DO sensors (calibrated)

SOP for Bioreactor Operation and Control Strategy for Gene Therapy Production

1.0 Objective

To define the procedures and control strategies for operating single-use or stainless-steel bioreactors used in the upstream production of viral vectors and gene therapy products, ensuring consistency, sterility, and optimal process parameters under GMP-compliant conditions.

2.0 Scope

This SOP applies to all production-scale bioreactor operations (5L to 200L and above) for HEK293, CHO, or other mammalian cell lines used in gene therapy manufacturing within Grade C cleanrooms.

3.0 Responsibilities

4.0 References

• 21 CFR Part 210/211

• EudraLex Volume 4, Annex 2 – Manufacture of Biological Products

• ICH Q8 – Pharmaceutical Development

• Manufacturer Manuals: Sartorius® BIOSTAT STR, Thermo Scientific™ HyPerforma™

• ASTM E2500 – Standard Guide for Specification, Design, and Verification of Pharmaceutical Systems

5.0 Definitions

• PID: Proportional–Integral–Derivative controller used for process control loops.

• Cascade Control: Multi-loop control system typically used for pH and DO.

• Control Set Point: Target value for a controlled parameter (e.g., pH, DO, Temp).

• SIP: Steam-in-place sterilization (for stainless-steel systems).

• SCADA: Supervisory Control and Data Acquisition system.

6.0 Materials and Equipment

6.1 Materials

• Calibrated pH and DO probes

• Antifoam C

• Filtered glucose and base solutions

• Disposable bioreactor bags (for SU systems)

6.2 Equipment

• Single-use or stainless-steel bioreactors (5L–200L)

• Control system with HMI/SCADA

• Gas supply lines and MFCs for Air, O₂, CO₂, N₂

• Inline pH and DO sensors

• Peristaltic pumps for feed/base addition

• Load cells or balances for weight-based control

• Alarm and data logging systems