Fermentation in the biopharmaceutical industry

Fermentation in the biopharmaceutical industry

2020-08-05T15:27:49+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.

2.  Inoculum preservation and storage

Cell culture preservation and storage may be performed using the following preservation and storage techniques:

2.1  Agar slant or plate

Usually used in microbiology laboratories and not for animal cell culture.

Storage conditions:  2-8°C.

Maximal storage period: 12 months.

2.2  Ultra freezing

Appropriate for all cell cultures and commonly used in industry.

Storage conditions: Freezing (-70)°C for bacteria and fungi, and cyrostat freezing with liquid nitrogen (-196)°C for plant and mammalian cell cultures.

Maximal storage period: 5 years.

2.3  Lyophilization

Lyophilization is a technique for drying liquids which is based on gas sublimation principles under controlled (low) temperature and pressure conditions. Lyophilization is suitable for bacteria and fungi cell storage and is in widespread use by industry. Lyophilization is relatively costly but is considered to be a high quality, high 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 immediate and long term industrial use should minimize potential risks such as cell stress, impurities and mix-ups to assure that preserved cell vitality, condition, purity and identification are maintained over short and prolonged periods of storage.

3.2  Cell storage in cell banks is performed in controlled conditions, using validated and calibrated main storage equipment (usually freezers), in addition to backup equipment which usually has the same types of cooling systems and monitored storage conditions. GMP (good manufacturing practice) principles should be followed to avoid temperature fluctuations, damage and cell stress during the storage period.

3.3  Cell bank storage systems should be stable, backed up (connected to backup generators) and validated prior to use.

3.4  Cell bank content should be managed according to GMP and GSP (good storage practice) requirements. Moreover, cell bank storage systems should be connected to monitoring and alarm systems that were successfully validated as part of a computerized systems/software validation program.

There are two kinds of cell banks that are used for clinical trials and industrial use:

3.4.1   A master cell bank (MCB) is used for create working cell banks and for long term storage. Usually stored cell cultures are diluted after thawing.

MCBs should be continuously monitored and periodically tested for cell identification, viability, purity, phenotype, genotype, stability and storage tube identity and labeling.

3.4.2   A working cell bank (WCB) is used for short- and medium-term cell storage for cell culture trials, studies or research and for use in fermentation on a day-to-day basis. WCBs should be periodically tested for cell viability and identification, and should be maintained in a validated state.

3.5  Using a biological laminar air flow hood, a frozen cell tube (after gradual thawing), colony or lyophilisate is transferred into an Erlenmeyer flask containing the required volume of the appropriate sterile medium. The flask is incubated over night in a shaker incubator at the required incubation temperature (and shaking speed) defined in Table 1 until the required turbidity level is achieved.

Using aseptic techniques in a biological laminar air flow hood, the required inoculum volume is transferred into the sterile bioreactor for the upstream fermentation stage.

4.   Growth substances

4.1  Water

Water purity grades recommended  for media preparation are purified water (PW) for bacteria and yeasts, PW and highly purified water (HPW) for plant cells, and HPW and water for injection (WFI) for mammalian cell fermentations.

Water systems should be of sanitary design, pharmaceutical grade, be validated before use and continuously monitored.

4.2  Air

Air is an oxygen source for cell growth because it contains ~21% O2 and therefore it should be supplied and dissolved in the media during fermentation. Moreover, instrument air is supplied to pressurize the fermentation process equipment components and for pneumatic operation purposes.

Process air that comes into contact with a process vessel or equipment inner surface, media or product, should be dry and particulate-, microorganism- and oil-free.

An oil-free compressed air system should be pharmaceutical grade, should be validated before use and continuously monitored.

4.3  Carbon

Carbohydrates, glucose, glycerol, lactose, etc., are basic substances essential for cell growth and proliferation.

Carbohydrates cannot be used for animal cell media.

4.4  Nitrogen

Amino acids, ammonia, yeast extracts, etc., are basic substances essential for cell growth and proliferation.

4.5  Macro elements

Salts that contain elements such as Na, Cl, Mg, S or K.

4.6  Micro elements

Salts that are required in low quantities for fermentation.

4.7  Serum

Use of large quantities of serum as a growth media is usually forbidden. Serum-free media can be purchased or developed for specific fermentation process needs, and may be used as a substitute serum. Serum is very rich in essential substances and is suitable for growth of most mammalian cell lines. Fetal calf and calf sera are usually characterized by “lot-to-lot” variations and may contain endotoxins, viruses, hormones, etc.

Before use, the serum should be analyzed, at least, for sterility (including mycoplasma detection), growth curves, cloning efficiency and visually (microscopic examination).

4.8  Osmotic pressure maintaining agents

4.9  Vitamins

4.10  Buffers

Buffers are added to media to avoid pH fluctuations during fermentation.

4.11  Anti-foam agents

Often for large scale fermentation processes and as a result of high sheer mixing forces, foam may be created. Silicone or vegetable oils and other anti-foam agents should be added, in addition to anti-foaming mechanical components that can be installed in the bioreactor.

(For more information, refer to the Hebrew article on fluid mixing technology in the biotechnology, pharmaceutical, cosmetics and food industries.)

 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, which is growing cells on a solid substrate.

6.2  Submerged fermentation, which is cells submerged in media and separated to batch, fed batch, continuous and/or perfusion fermentation.

6.2.1   Batch fermentation is a non-continuous process. No substrate and/or cells are added to the bioreactor during the entire fermentation process (except for air, anti-foam and pH-adjustment solutions).

6.2.2   Fed batch fermentation is a non-continuous process. Part of the media substrates are fed into the bioreactor during the fermentation process (carbon and nitrogen sources, etc.) in addition to air, anti-foam and pH-adjustment solutions.

Figure 1. Batch Fermentation vs Fed Batch Fermentation

6.2.3   Continuous fermentation

Fresh media is added to the bioreactor at the same rate spent media is being removed from the bioreactor. Cells are kept in the logarithmic proliferation phase longer, in comparison to other fermentation techniques, which is ideal for biological materials or products that are being produced by the cells in the logarithmic phase of proliferation.

6.2.4   Perfusion fermentation

Cells are immobilized on beads or discs, or filtered, retained in the bioreactor throughout the fermentation process and are not removed from the bioreactor during removal of spent media, as is done in continuous fermentation (higher biological mass yield).

7.  In-process controls

Critical parameters throughout 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 the defined validated limits during fermentation, separation, purification and filling processes.

7.1   Biological parameters

Culture purity (at every stage, starting from the inoculum stage) must be verified by microscopic examination, as should agar rich medium and selective medium (petri dish incubation) throughout the entire fermentation process, in addition to cells and product concentrations, microbial contamination testing, etc.

7.2   Physical parameters

Temperature, pressure, flow rates (gases and/or liquids), agitation speeds, foam detection, viscosity, biological mass concentration weight/volume, turbidity, etc.

7.3   Chemical parameters

pH, dissolved gases concentration (O2, CO2), redox potential, gas concentrations, etc.

8. Sterilization

8.1   Most media types are sterilized using heat. Animal cell media, which is usually sensitive to high temperatures, is usually sterilized by sterile filtration.

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

8.3   Instrumentation elements that come 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 time 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 at upstream stages, due to the risk-based approach.

9. Qualification and validation

9.1   Utilities validation

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

9.2   Fermentation equipment validation

Fermentation equipment such as bioreactors, pumps, fermentors and vessels should be validated for installation, operational and performance qualification (IQ/OQ/PQ).

9.3   Fermentation, separation and purification systems validation

Equipment should be validated for cleaning validation and clean hold time.

9.4   Analytical equipment and systems validation

Equipment and systems should be validated for Installation and Operational Qualification (IQ/OQ) as well as for computerized systems validation (CSV); if the system includes software, then also according to EU Annex 11, US FDA 21 CFR Part 11 and GAMP 5 requirements.

(For more information, refer to the article on CE/FDA compliant computerized systems and software validation.)

9.5   Analytical methods validation

Analytical methods used for detection of product, impurities, cleaning agents and microorganisms should be validated.

9.6   CIP (Clean In Place) skids validation

CIP skids should be validated for installation, operational and performance qualification (IQ/OQ/PQ), including spray device qualification of spray devices installed on equipment.

9.7   Control panel and HMI validation

Equipment and skids control panels and HMIs will be validated for computerized systems validation (CSV) according to EU Annex 11, US FDA 21 CFR Part 11 and GAMP 5 requirements.

(For more information, refer to the article on CE/FDA compliant computerized systems 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/PQ).

9.9   Sterile hold time validation

The sterile state of equipment during storage should be validated if the equipment will not be sterilized prior to batch production.

9.10  Cleaning validation

Cleaning processes for equipment such as bioreactors, vessels, chromatography columns, ultra filtration equipment, small equipment instrumentation and others should be validated.

(For more information, refer to the article on cleaning validation.)

9.11  Clean hold time validation

The cleaned equipment storage period and conditions after cleaning should be validated.

(For more information, refer to the article on cleaning validation.)

9.12  Growth promotion validation

Growth promotion factors in media should be validated using cell viability and growth rate calculations.

9.13  Media shelf life storage validation

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

9.14  Product storage validation

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

9.15  Cell bank storage systems

Cryostats, freezers, refrigerators and freezing chambers should be validated for installation, operational and performance qualification (IQ/OQ/PQ).

(For more information, refer to the article on CE/FDA compliant computerized systems and software validation.)

9.16   Cell bank alarms and computerized monitoring systems should be validated for computerized systems validation (CSV) according to EU Annex 11,  US FDA 21 CFR Part 11 and GAMP 5.

9.17  Product manufacturing procedures 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 shipments and supply chain equipment should be validated for installation, operational and performance qualification (IQ/OQ,PQ) for transport systems as well as for transportation routes.

(For more information, refer to the article on Good storage and distribution practice, cold chain safety and validation.)

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