Control of glycoforms in IgG

#This invention relates to processes for controlling glycoforms in recombinantly produced IgG.

#Monoclonal antibodies (IgG isotypes) are produced using a variety of expression systems. The majority of the expression systems used for production of monoclonal antibodies are mammalian and include host systems such as chinese hamster ovary (CHO), hybridoma and myeloma cells or their derivatives.

#Antibodies significantly differ from other recombinant proteins in their glycosylation patterns. Glycosylation tends to be highly conserved in IgG molecules with a single N-linked biantennary structure at Asn297, which is buried between the CH2 domains, forming extensive contacts with the amino acid residues within CH2. Typically, in recombinantly produced IgG, there is heterogeneous processing of the core oligosaccharide structures attached at the Asn297 site and the IgGs exist in multiple glycoforms. In contrast, non-IgG proteins produced in CHO cells may have multiple N-linked glycosylation sites. Examples are interferon-γ (IFN-γ) which has two glycosylation sites at Asn25 and Asn97 and human tissue plasminogen activator (t-PA) which has 3 sites at Asn117, Asn184 and Asn448.

#FIG. 1 is a graph of experimental results demonstrating the effect of culture temperature on NGHC formation.

#FIG. 2 is a graph of experimental results demonstrating the effect of culture temperature and batched medium osmolality on NGHC formation.

#FIG. 3 is a graph of a statistical regression of experimental results predicting the effect of culture temperature and batched medium osmolality on NGHC formation.

#All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.

#The present invention provides a method to control the level of IgG non-glycosylated heavy chain (NGHC) in a cell culture process producing a recombinant monoclonal antibody. By adjustment of the culture temperature, adjustment of the batched medium osmolality or adjustment of both temperature and batched medium osmolality of a cell culture such as a production culture, the level of NGHC can be decreased or increased. Batched medium osmolality of a final seed culture used to inoculate a production culture can optionally be adjusted to match that of the production culture.

#In one embodiment of the invention, NGHC can be decreased by increasing the temperature of the cell culture. Likewise, NGHC can be increased by decreasing the temperature of the cell culture. Preferred temperatures for the process are within a range of about 33° C. to about 35° C.