Biopharmaceuticals refer to medicinal / therapeutic products that are either manufactured using living organisms or semi-synthesized from biological sources. These are essentially complex biological macromolecules, having high molecular weights, or cell-based products, which are not directly extracted from native biological sources, rather are produced using biotechnology tools and methods. The following figure provides an illustrative summary of the various types of biopharmaceuticals.
Since biopharmaceuticals are produced using living organisms, they require various prokaryotic and eukaryotic systems, such as bacteria, yeasts, insect cells and mammalian cells, for their manufacturing.
Further, considering the fact that biopharmaceuticals are essentially structural analogs of various biomolecules found in the human body, they are highly specific and have fewer side effects, as compared to conventional pharmacological molecules. These therapies are also deemed to possess the potential to target and eradicate the cause of a disease at the genetic level. The Biopharmaceutical Contract Manufacturing Market is anticipated to grow at a CAGR of around 9.6%, till 2035, according to Roots Analysis.
Expression Systems for Biopharmaceuticals
As mentioned earlier, the processes associated with the manufacturing of biopharmaceuticals are complex and require highly sterile and aseptic conditions. This can be attributed to the fact that the production of biopharmaceuticals requires living expression systems. Usually, the desired gene, such as human insulin gene, when inserted into the plasmid of the host cell uses transcriptional and translational machinery of the host to express itself. It is worth mentioning that in vitro gene expression requires a suitable host for the production of a specific gene product. Presently, several expression systems are available for manufacturing of biologics; these include (in alphabetic order) insect, mammalian, microbial and plant expression systems. It is also important to note that the use of different systems is associated with their own set of culturing requirements, advantages, and drawbacks.
The figure below provides an overview of the various expression systems used for the production of biopharmaceuticals.
Mammalian versus Microbial Expression Systems
The table presents the differences between mammalian and microbial expression systems.
|Mammalian Expression Systems
|Microbial Expression Systems
|Ease of working with cells
|Their fragile nature makes these systems difficult to handle
|Comparatively easier to work with / handle
|Ease of culturing cells
|Culturing of mammalian cells is a difficult process and requires expertise
|Comparatively easier to culture and do not require highly skilled personnel
|Transfection is primarily done via liposome mediated transfection, electroporation and microinjection
|Transfection is done mainly through heat shock method
|Post-translation modifications occur within the cell
|Post-translational modification is required to be done in an additional step after the release of protein / product
|Preservation of native structure
|Antibody produced will be relatively closer to its native structure
|Antibodies can be expressed; however, their similarity to native structure is low
|Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, and the WI-38 and MRC-5 cell lines (derived from fetal cells)
|Bacterial expression systems: Escherichia coli, Salmonella typhimurum, Vibrio cholerae and Bacillus brevis
Yeast expression systems: Saccharomyces cerevisiae and Pichia pastoris
Source: Roots Analysis
Manufacturing Process of Biopharmaceuticals
The production process of biologics can be categorized into two major stages, namely upstream and downstream processing. Upstream processing includes the production and maintenance of the working microbial expression systems, whereas downstream processing comprises of the various chemical and physical separation steps required to isolate and purify the product from the culture mixture.
The following figure highlights the various stages of the manufacturing process of a biopharmaceutical product.
Upstream processing refers to the entire process of product development, beginning from isolation of the working cell bank, incubation under appropriate conditions, and expansion of the cell culture for the synthesis of the desired biopharmaceutical product. It is worth mentioning that the upstream process chosen for a particular biologic is greatly dependent on the various characteristics of the product, such as selection of host cell lines, culture media and the appropriate bioreactor system used. Steps involved in upstream processing of biopharmaceuticals are formulation of the fermentation media, media sterilization and inoculum development.
Fermentation is the final stage of the manufacturing process and involves the synthesis of the desired product within the microbial expression systems. Fermentation processes are typically of two types (based on oxygen requirements), namely aerobic (in the presence of oxygen) and anaerobic (in the absence of oxygen). The fermentation process is usually modified to suit the oxygen requirements of the microorganisms used. Fermentation processes can also be categorized into batch, continuous / perfusion and fed-batch operations (based on the strategy used to feed the culture and culture medium into the fermenter). During batch operations, the culture medium and seed culture is added to the fermenter at the beginning of the process, after which the system is closed and only oxygen or pH adjusting agents are added. Alternatively, in a continuous system, fresh medium is added in an uninterrupted manner throughout the operating time of the reactor. Further, spent media (containing microorganisms and products) is removed from the system at the same rate at which fresh medium (containing inoculum) is added. A fed-batch system is a combination of the aforementioned processes. In this method, fresh medium (containing inoculum) is added at regular intervals, however, harvesting takes place towards the end of the operation.
Post harvesting, additional steps are required to isolate microorganisms and remove impurities, such as contaminating cell proteins, nucleic acids, endotoxins and residual processing reagents, via centrifugation, filtration and chromatography. These techniques collectively form the basis for downstream processing and are usually performed on large volumes of complex biological mixtures. These operations are intended to extract, concentrate and purify the resultant product(s). During this process, components of fermentation mixture are separated based on various parameters, such as molecular size, electric charge, solubility and binding affinities. Steps involved in downstream processing are centrifugation, filtration, chromatography and fill / finish.
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