PharmTech: How might platform technologies be applied to upstream and downstream processes? Is one easier than the other?
Atkinson (Cook): It is not easier to develop platforms for either upstream or downstream, it is just different. The main difference in developing platform processes for either is that, in most cases, one develops upstream processes for the cell line and the expression system, while downstream processes are tailored to the molecule itself. If the molecules are of a similar type, then the downstream process becomes easy to develop.In terms of difficulty, the cell lines and expression systems are inherently variable, and clone-to-clone variability adds to the complexity. Scale factors are also more difficult to control in cell culture and fermentation. In general, upstream therefore probably poses a slightly greater challenge, assuming that the molecules are in a given class or category.
Moesta (Bristol-Myers): With today's level of know-how in molecular biology and expression, platform technologies are easier to develop for upstream processes. Identification of a preferred strain or cell line for microbial or mammalian expression, combined with a well-developed expression vector, is the first step in establishing a production platform. This step allows for the use of standardized fermentation or cell-culture conditions requiring limited media and feed optimization. The use of platform expression systems and upstream conditions allow for the generation of significant process experience and forms the basis for developing downstream platforms to the extent possible.
It is easiest to develop a standardized process for initial downstream steps (e.g., centrifugation and depth filtration for cell-culture products). For monoclonal antibodies (mAbs), where the Fc protein domain–Protein A interaction can be exploited to capture the protein from clarified cell-culture broths, additional platform steps are possible (e.g., Protein A-based affinity chromatography and viral inactivation and filtration steps). The final purification steps (i.e., polishing) need to be tailored to the particular antibody at hand and usually require individual optimization. For other proteins, downstream processing becomes less amenable to the platform approach. Individual process steps can be standardized, but will need to be pieced together and optimized on a case-by-case basis.
BMS is developing molecules to which we apply platform-based approaches, including antibodies and adnectins. But even when dealing with well-defined classes of proteins, key challenges for establishing production platforms result from unique properties of individual proteins, such as charge heterogeneity, differences caused by post-translational modifications, and stability. These unique properties can impact both the cell's ability to express a correctly folded and stable protein as well as purification of a homogeneous drug substance.
PharmTech: Could a platform for purification accommodate variations between mAbs? Is it possible to develop a purification platform for various classes of products (e.g., mAbs and enzyme products)?
Atkinson (Cook): Platform purification processes must deal with both process- and product-related impurities. With antibody processes, the process-related impurities tend to dominate the development of the platform. Removal of host cell proteins (HCP), in particular, is usually a primary driver.
For the product-related impurities, most antibody processes are usually dominated by the removal of higher-molecular weight aggregates, followed by clipped forms and other charge variants. This is why so many platforms use an affinity step, followed by some combination of ion-exchange and/or mixed-mode separation.