Defining and preventing the mechanisms responsible for disulphide bond reduction of monoclonal antibodies during bioprocessing

Mammalian cells (particularly Chinese hamster ovary) have become the dominant system for the production of IgG based therapeutic antibodies due to their ability to produce properly folded and assembled heterologous proteins as well as their capability for post translational modifications. A prerequisite for the function of such IgG antibodies is their correct assembly into defined quaternary structures, composed of two heavy chain (HC) and two light chain (LC) polypeptides that form disulphide bonded HC2LC2 molecules. Folding, assembly and post-translational modifications occur within the ER where the molecule is subjected to rigorous quality control before being transported to the Golgi. IgG polypeptides sequentially interact with a range of molecular chaperones, foldases and oxioreductases present within the ER complex. The LC polypeptide can be secreted as both monomer and disulphide bonded dimer. Conversely, HC polypeptide secretion requires the presence of LC for secretion, the absence of which results in the HC polypeptide being retained in an unfolded state within the ER before being trafficked to the proteasome for degradation. Biochemical and physiological stimuli have been reported to impose stress on the ER and lead to the accumulation of unfolded or misfolded proteins within the cell. In addition, contrary to previously reported results, data generated at Lonza have shown the presence of HC monomer and dimer in culture broths exposed to low oxygen conditions at harvest. The presence of free LC and HC monomers and dimers is undesirable and defining the mechanism(s) by which these are derived and approaches to avoid the presence of these is required. There is also a need to understand the relationship between changes in physiochemical parameters in the bioreactor and the molecular mechanisms allowing the secretion of misfolded/unfolded proteins from the ER and the influence of cell culture supernatant on the structure and stability of fully secreted antibody. Proposed work programme. The student will use a combination of molecular and cell biology techniques to develop methods to prevent disulphide bond reduction in both IgG1 and IgG4 subtypes. Initial studies will use cell lines (and product) that yield disulphide bond reduction products. These cell lines will be used to establish a laboratory scale test system that can provide material for analysis. The student will then identify (a) intra-cellular factors that cause the secretion of misfolded/unfolded proteins under different physiochemical conditions (i.e. factors that enable the secretion of misfolded/unfolded proteins by overcoming the ERAD pathway) and (b) key factors in cell culture processes that yield cell culture supernatant that is more susceptible to disulphide bond reduction/shuffling (i.e. define those reductions/shuffling processes that occur in the supernatant post-secretion). This information will be used to identify specific markers within cell culture supernatant that can determine the propensity of antibodies to dissociate prior to initiation of harvest and downstream processing. The outcomes of the PhD will be: 1.Establishment of a molecular model of the key intra-cellular factors that cause the secretion of mis-folded proteins (free HC/LC) under reducing conditions (are there differences between IgG1 and IgG4 subtypes, how does this differ between host cell lines?). 2.Identification of reducing components in cell culture supernatant that catalyze the reduction of disulphide bonds. 3.Identification of factors in cell culture processes that yield supernatant more susceptible to disulphide bond reduction/shuffling (e.g. media, dissolved oxygen tension, temperature, pH, filtration, centrifugation, total cellular protein). 4.Identification of markers within cell culture supernatant that determine the propensity of antibodies to dissociate prior to initiation of harvest and downstream processing steps (e.g. apoptosis, activity of reducing enzymes.