This is a complex process of biologics as it involves proteins which are large molecules variable in structure and sensitive to environmental conditions. It is a four step process: production of a master cell line, growth of cells producing proteins, isolation and purification of proteins from cells and then preparing the biologic for patients. The manufacturing process can consume years and cost hundreds of millions of dollars.
Using R&D Specifications:
Initial production during this phase is limited to small scale manufacturing. Usually an injection or infusion in respect of biotechnology medicine and its final formulation and physical form is produced. Going by the data generated large scale production is planned. The scale-up and manufacturing adheres to CGMP guidelines in respect of safety and purity.
Common Cell Lines:
A large number of biotechnology products are proteins produced by cells grown in a culture media. Chinese hamster ovary (CHO) cells, non-secreting (NOS) cells and E. coli are cell lines involved in production of biotherapeutics with emphasis on monoclonal antibodies. CHO and NSO cells help to synthesize proteins like human cells do. Since they can produce and grow forever, they are called immortal cell lines. These cell lines are generally regarded as safe (GRAS) for producing therapeutic proteins. NSO cells have an advantage to produce antibodies but do not secrete or make any of their own antibody protein. Other suitable cells can also be used but, the selection of such cell lines will depend on the expertise of the company’s research team. Such selection will hinge on the properties of the cells and regulatory requirements.
The scale-Up of a cell culture is complex and time consuming. Several months can elapse before a product is obtained. Production of a biotech product is divided into two parts: upstream and downstream. Upstream processes constitutes production of protein product by using cells like microbial, insect or mammalian in culture growth. Downstream processes help in recovery, purification, formulation and packaging of the product.
This is a process where researchers create and engineer to formulate a protein product. Once the desired cell line is achieved it is cryopreserved i.e. frozen in a large number of vials to create a CELL BANK. It is then removed and thawed from the cell bank for cell culture in a flask containing a small amount of growth media as little as 5ml. The media is the provider of nutrients and the environment for the cell to survive.
Scale-Up is achieved by a gradual transfer of growing cells into larger containers having larger media volumes, and these are constantly dividing due to favorable growth environment. More cells are present with each step and hence more protein product is generated.
This sets the stage for quicker growth of cells and thereby production of substantial protein product. Assays or other testing methods are used by scientists to measure CELL VIABILITY and concentration, product concentration and activity at each scale up stage for better monitoring.
Advanced laboratory environment helps to control physical environment for cell to grow. This is done manually in the nascent steps and helps to optimize growth parameters like temperatures, PH, nutrient concentration and oxygen level. Since this is a large and automated culture it can be grown in bioreactors. FERMENTATION and manufacturing stages helps technicians to monitor contamination, bacteria, yeast or other microorganisms. Contamination ruins an entire batch and so technicians maintain ASEPTIC conditions during this phase.
Quality Control and Quality Assurance:
These departments quality control (QC) and quality assurance (QA) are responsible to monitor scale-up activities, leading to manufacture and product development. QC department maintains product standards by assuring product quality during development and marketing stages. Quality assurance determine quality objectives.
Isolation of the protein product from the cells is one of major objectives of this phase. INTRACELLULAR PROTEINS found in the cells require special techniques for extraction and purification. The cell is burst open so that it releases the protein product for purification, and isolates it from various components of the cell. Proteins which are extracted away from the cells (EXTRACELLULAR PROTEINS) are easier to isolate.
CLARIFICATION is the next step after harvesting the protein product. Here proteins are separated from cellular debris. A protein solution is applied to a series of chromatography columns and hence a pure protein product is derived. The process of COLUMN CHROMATOGRAPHY that separates proteins is dependent on physical and chemical properties like size, shape or change i.e. + or -. Further purification helps in removing residual DNA, and deactivates any viral particles.
Confirmed testing protocols help to verify the process of isolation and purification. The protein product thus obtained as per R&D specification is then packaged for use by physicians and patients.