Fermentation in the biopharmaceutical industry

Fermentation in the biopharmaceutical industry

2020-08-31T08:42:18+00:00July 7th, 2020|

1. Background

Many biopharmaceutical and biological products are based on (different) cell-type fermentation. Biopharmaceutical products can be either intracellular or extracellular metabolites that have a pharmacological effect. Fermentation, as a whole, includes upstream and downstream good manufacturing practice (GMP) multi-stage processes whose purpose is to yield the required biopharmaceutical therapeutic material that is pure and  active, and which is released according to relevant quality standards and specifications.

Fermentation can begin using either genetically modified or unmodified cells such as bacteria, fungi, plant and mammalian material.

Usually, large scale fermentation processes will be performed in stainless steel or single use bioreactors (for more information about single use reactors, refer to single use equipment in the biopharmaceutical industry).

At the cell inoculation and fermentation stages, biological mass (products or metabolites) will be accumulated in the bioreactor as a result of the cell growth and proliferation processes.

The biological mass in the bioreactor should meet quality and microbial standards during and after the upstream fermentation stages to assure the presence of the “right” microorganisms, cell type populations and/or metabolites in the bioreactor.

As part of the downstream process stage, the biological therapeutic material will be purified through several extraction, separation and purification processes until the required identity, concentration, purity and quality of the therapeutic biological material/product is achieved.

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2. Inoculums preservation and storage

Cell culture preservation and storage can be performed using the following preservation and storage techniques:
2.1 Agar slant/dish
Usually being used in microbiology laboratories and not for animal cells culture.
Storage conditions: 2-8°C.
Maximal storage period: 12 month.

2.2 Ultra freezing
Fits all cell culture and is being used for industrial usage.
Storage conditions: (-70)°C for bacteria and fungi (freezer) and (-196)°C for plant and mammalian cell culture (liquid Nitrogen cryostat).
Maximal storage time: 5 years.

2.3 Lyophilization
Liquid drying technique based on gas sublimation principles under controlled (low) temperatures and pressure conditions. Lyophilization is suitable for bacteria and fungi cells storage and is used for industrial usage. Lyophilization is relatively costly but is considered as high quality, yield and validated preservation technique.
Storage conditions: Room temperature.
Maximal storage period: 10 years

2.4 Air drying
Low humidity air and “dry conditions” will cause “spore former” bacteria and fungi cells to create spores and as a result enable cell storage.
Storage conditions: Room temperature.
Maximal storage period: Years (depends on cell type)

3. Cell banks

3.1 Cell storage for industrial usage (immediate and long term) purposes should minimize potential risks such as cells stress, impurities and mix ups in order to assure preserved cells vitality, condition, purity and identification are being kept during short and prolonged storage periods.

3.2 Cell storage in cell banks is being performed in controlled conditions, using validated and calibrated main storage equipment (usually freezers) in addition to backup equipment, both usually will include redundancy between cooling systems, monitored storage conditions and other GMP (Good Manufacturing Practice) standards should be kept in order to avoid temperature fluctuations, damage and cell stress during the storage period.

3.3 Cell banks storage system should be stable, backed up (connected to backup generators) and validated prior to usage.

3.4 Cell bank content should be managed according to all GMP (Good Manufacturing Practice) and GSP (Good Storage Practice) standards. Moreover, cell banks storage system should be connected to monitoring and alarm system that were validated successfully as part of a computerized system/software validation program.
There are two levels of cell banks that are used for clinical trials and industrial usage:
3.4.1 Master cell bank- Used for working cell bank creation and long term storage. Usually stored cells culture will be diluted after thawing.
Master cell bank should be continuously monitored and tested periodically for cell identification, viability, purity, phenotype, genotype, stability and storage tubes identity and labeling.
3.4.2 Working cell bank- Used for short and medium term cell storage for cell culture trials/research and fermentation purposes on a day-to-day basis. Working cell bank should be periodically tested for cells viability and identification and should be maintained in a validated state.
3.5 Using a biological laminar air flow hood, the frozen cell tube (after graduate thawing), colony or lyophilisate are transferred into Erlenmeyer containing the appropriate sterile medium in the required volume. The Erlenmeyer is being incubated over night in shaker incubator at the required incubation temperature (and shaking speed) defined in table No.1, till the required turbidity level is achieved.
Using aseptic techniques in biological laminar air flow hood, the required inoculums volume will be transferred into the sterile bio-reactor for the upstream fermentation stage.

4. Growth substances

4.1 Water
Water purity grade for media preparation can begin as Purified Water (PW) for bacteria and yeast, PW and HPW (Highly Purified Water) for plant cells and Highly Purified Water (HPW) and Water For Injection (WFI) for mammalian cells fermentation.
Water system should be of sanitary design, pharmaceutical grade and should be validated before usage and continuously monitored.

4.2 Air
Air is an Oxygen source for cell growth as it contains ~21% O2 and therefore should be supplied and dissolved in the media during fermentation. Moreover, instrument air is supplied for fermentation process equipment components pressurization and pneumatic operation purposes.
Process air that is in contact with vessel/equipment inner surface, media or product should be dry, particle, microorganisms and oil free air.
Oil Free Compressed Air system should be pharmaceutical grade and should be validated before usage and continuously monitored.

4.3 Carbon
Carbohydrates, Glucose, Glycerol, Lactose etc. are essential basic substance for cell growth, proliferation.
Carbohydrates cannot be used for animal cells media.

4.4 Nitrogen
Amino acids, Ammonia, yeast extract etc. are essential basic substance for cell growth and proliferation.

4.5 Macro elements
Salts that contain elements such as Na, Cl, Mg, S, K

4.6 Micro elements
Salts that are required in low quantities for fermentation

4.7 Serum
Serum usage as a growth media is usually forbidden for usage in large quantities. Serum free media can be purchased or developed for specific fermentation process needs and is used as a substitute serum. Serum is very rich in essential substances and is suitable for most mammalian cell lines growth. Fetal calf/calf serums usually characterized in ‘lot to lot’ variation and may contain Endotoxins, viruses, hormones etc.
Before usage, serum will be analyzed at least for sterility (including mycoplasma detection), growth curves, cloning efficiency and visually (microscope).

4.8 Osmotic pressure maintaining agents
4.9 Vitamins
4.10 Buffer
Buffers are added to media, in order to avoid pH fluctuations during fermentation.
4.11 Anti-foam agents
Usually, for large scale fermentation processes and as result of high sheer mixing forces, form may be created. Silicone/vegetable oils and other anti-foam agents should be added in addition to anti-foam mechanical components that can be installed in the bio-reactor.
(For more information refer to Hebrew article on fluid mixing technology in the biotechnology, pharmaceutical, cosmetics and food industry)

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5. Inoculum growth

There are many challenges in inoculum growth, such as keeping cells viable after recovery, genotypic identity after storage, high levels of biological mass, etc.

Table 1.  Cell Growth Conditions

Fermentation parameter

Bacterial cells

Yeast
cells

Mold
cells

Plant
cells

Mammalian cells

pH

6.0-7.5

4.5-6.0

4.5-7.0

6.8-7.6

6.9-7.6

Temperature [°C]

30-37

30-37

30-37

24-28

36-38

Aeration

Aerobic & Anaerobic

Aerobic & Anaerobic

Aerobic

Aerobic

Aerobic

Inoculum volume/total

1-5%

1-10%

1-10%

10-20%

105/ml

6. Fermentation

Generally, there are two different cell fermentation techniques:

6.1 Solid state fermentation- Cell growth on a solid substrate.

6.2 Submerged fermentation- Cells are submerged in the media and separated to batch, fed batch, continuous and perfusion fermentation.

6.2.1 Batch fermentation- Non-continuous process. No substrate and/or cells are being added into the bio-reactor during the entire fermentation process (except for air, anti foam and pH adjustment solutions).

6.2.2 Fed batch fermentation- Non-continuous process. Part of media substrates are being fed into the bio-reactor during fermentation process (Carbon, Nitrogen sources etc.) in addition to air, anti foam and pH adjustment solutions addition.

Figure No. 1- Batch Vs. fed batch fermentation

6.2.3 Continuous fermentation

Fresh media is added into the bio-reactor in the same rate spent media is being taken out of the bio-reactor. Cells are kept in the logarithmic phase longer in comparison to other fermentation techniques (ideal for biological materials/products that are being produced by the cells in the logarithmic phase of the cells).

6.2.4 Perfusion fermentation

Cells are immobilized on beads/discs or being filtered and retained in the bio-reactor through the fermentation process and are not being taken out of the bio-reactor during spent media take out as in continuous fermentation (higher biological mass yield).

7. In-process controls

Critical parameters through the fermentation process should be controlled, monitored and tested as part of the validation stage (OQ, PQ and PPQ). Critical parameters should be kept within defined validated limits during fermentation, separation and purification and filling processes.

7.1   Biological parameters

Culture purity (in every stage, starting from the inoculums stage) will be verified using microscope, agar rich medium and selective medium (Petri dish incubation) through the entire fermentation process in addition to cells and product concentrations, microbial contamination testing etc.

7.2   Physical parameters

Temperature, pressure, flow rates (gases/liquid), agitation speed, foam detection, viscosity, biological mass concentration weight/volume, turbidity etc.

7.3   Chemical parameters

pH, dissolved gases concentration (O2, CO2), redox potential, gases concentration in outlet etc.

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8. Sterilization

8.1   Most media types will be sterilized using heat, except for animal cell media that is usually sensitive for high temperatures, therefore will be sterilized usually by filtration.

8.2   All equipment components that are in direct or indirect contact with substances or products should be constructed of stainless steel 316 with a low roughness level.

8.3   Instrumentation elements that are in direct or indirect contact with substances or products should be constructed of stainless steel 316 with low roughness level and sterile.

8.4   Sterilization and sterile storage hold times parameters and procedures should be validated in addition to Cleaning Validation and clean hold time validations.

8.5   Sterilization is more critical at downstream stages than upstream stages due to risk based approach.

9. Qualification and validation

9.1   Utilities Validation

Critical utilities will be validated for Installation and Operational Qualification (IQ and OQ). Non critical utilities (have no impact on product quality and/or GMP) can be qualified by commissioning only.

9.2   Fermentation equipment Validation

Fermentation equipment such as bio-reactors, pumps, fermentors, vessels should be validated for Installation, Operational and Performance Qualification (IQ, OQ and PQ)

9.3   Fermentation, separation and purification system Validation

Equipment should be validated for Cleaning Validation and clean hold time.

9.4   Analytical equipment and system Validation

Should be validated for Installation and Operational Qualification (IQ, OQ) as well as for Computerized System Validation (CSV) in case system includes software according to Annex 11, 21CFR part 11 and GAMP 5 requirements.

(For more information about computerized system validation refer to CE/FDA compliant Computerized System and Software Validation).

9.5   Analytical methods Validation

Analytical methods in usage for product detection, impurities, cleaning agents and microbial detection should be validated.

9.6   CIP (Clean In Place) skids Validation

Should be validated for Installation, Operational and Performance Qualification including spray device qualification (spray devices installed on equipment).

9.7   Control panel and HMI Validation

Equipment and skids control panels and HMIs will be validated for Computerized System Validation (CSV) according to Annex 11, 21CFR part 11 and GAMP 5 requirements.

(For more information about software and Computerized System Validation refer to CE/FDA compliant Computerized System and Software Validation)

9.8   SIP (Steam In Place) Validation

Sterilization and sanitization processes should be validated for Installation, Operational and Performance Qualification (IQ, OQ and PQ).

9.9   Sterile hold time Validation

Sterile state of equipment during storage should be validated in case the equipment is not being sterilized prior batch production.

9.10  Cleaning Validation

Equipment cleaning processes such as bio-reactors, vessels, chromatography columns, ultra filtration, small equipment and others should be validated (For more information refer to Cleaning Validation article).

9.11  Clean hold time Validation

Cleaned equipment storage period and conditions after cleaning should be validated.

(For more information refer to Cleaning Validation article).

9.12  Growth promotion Validation

Growth promotion factors in media should be validated using cell vitality and growth rates calculations.

9.13  Media shelf life storage Validation

Media shelf life growth material concentration and sterilization state should be validated for defined storage conditions and duration.

9.14  Product storage Validation

Batch storage conditions, duration, microbial contamination and shelf life should be validated.

9.15  Cell bank storage systems

Cryostates, freezers, refrigerators and freezing rooms should be validated for Installation, Operational and Performance Qualification (IQ, OQ and PQ).

(For more information about computerized system validation refer to CE/FDA compliant Computerized System and Software Validation).

9.16   Cell bank alarm and monitoring computerized system should be validated for Computerized System Validation (CSV) according to Annex 11, 21CFR part 11 and GAMP 5.

9.17  Product manufacturing procedure Validation

The entire manufacturing process should be validated for Process Performance Qualification (PPQ) for the required number of batches.

9.18  Shipment Validation

Final product shipment and supply chain equipment should be validated for Installation, Operational and Performance Qualification (IQ, OQ and PQ) for transportation system as well as for transportation routs (for more information refer to article Good Storage and Distribution Practice, cold chain safety and validation)

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