Upstream & Downstream Processing
The production of active pharmaceutical ingredients (API) of biopharmaceuticals typically starts with generation of the API via living cells known as the upstream process, followed by purification of the target API, or the downstream process.
The initial steps of the upstream process start with milliliter quantities of mammalian or microbial cells engineered to produce a specific protein molecule. Using several cultivation steps in a controlled environment within a bioreactor, these cell cultures are grown to a volume of several thousand liters to produce several kilograms of the desired API.
Mammalian Cell Culture:
Mammalian cell culture is used to produce large, complex proteins such as monoclonal antibodies. Utilization of mammalian cells allows for modification of the API, similar to those which may occur in human cells (e.g. glycosylation). Production processes with mammalian cell cultures take several days to weeks. Growth of these cells requires a complex and well-balanced mixture of nutrients as well as relatively gentle growth conditions regarding stirring and aeration.
Microbial organisms can grow much faster with cultivation times of several days or even hours. Smaller sized molecules such as peptides, enzymes, antibody fragments and protein scaffolds can be produced very efficiently. Microbial organisms are usually very robust and can grow to high cell densities generally several orders of magnitude beyond mammalian cell cultures. Microbial organisms typically have some limitations concerning the size of the protein and “human-like” modification of the molecules.
Following the generation of the protein or antibody molecule, isolation and purification from the complex mixture of host cell proteins, cells, cell debris, nutrients and waste materials must now take place.
Purification begins with separating the cells from the media. In most mammalian cultures, the protein is secreted into the media, thus the supernatant must be collected for further purification. In microbial production, the protein can also accumulate within the cells, therefore the cells need to be disrupted and the cell debris separated from the protein of interest. Insoluble products including unfolded, pure proteins sticking together, known as inclusion bodies must be refolded to achieve their native and active conformation. Precipitation steps based on temperature, pH, or chemical interaction can be used for removal of process related impurities.
The solution containing the protein of interest is purified using different techniques. Chromatographic separation steps are widely used; they are based on separation principles by charge, size or hydrophobicity of the protein, or by specific affinity to the chromatographic resin. A wide range of filtration techniques are used to separate the protein from the remaining solids, remove impurities and exchange buffers.
We at Boehringer Ingelheim BioXcellence™ have extensive expertise with a variety of different host cell and strain types, including cell culture and microbial organisms (bacteria and yeasts), producing a range of molecule types such as monoclonal antibodies, recombinant glycosylated and non-glycosylated proteins, peptides, antibody fragments, Fc-Fusion proteins, protein scaffolds and plasmid DNA. We offer platform solutions for monoclonal antibodies and tailor-made solutions based on a large toolbox of manufacturing techniques.
Our decades of experience in development, transfer and scale-up of biological processes is combined with modelling tools and thorough characterization of the processes and state-of-the-art analytical methods allow us to offer custom solutions for a wide range of molecules, efficient process development, and a fast and secure supply of material along the whole life cycle of a given API.