The present invention provides a manufacturing method for polypeptides that are produced in
insect cells using a baculoviral expression
system. In one example, the
insect cell culture is supplemented with a lipid mixture immediately prior to infection (e.g., one hour prior to infection). The polypeptides are isolated from the
insect cell culture using a method that employs anion exchange or mixed-mode
chromatography early in the purification process. This process step is useful to remove insect-
cell derived endoglycanases and
proteases and thus reduces the loss of desired polypeptide due to
enzymatic degradation. In another example, mixed-mode
chromatography is combined with dye-ligand
affinity chromatography in a continuous-flow manner to allow for
rapid processing of the insect-
cell culture liquid and capture of the polypeptide. In yet another example, a polypeptide is isolated from an
insect cell culture liquid using a process that combines hollow
fiber filtration, mixed-mode
chromatography and dye-ligand affinity in a single
unit operation producing a polypeptide solution that is essentially free of endoglycanase and proteolytic activities. In a further example, the isolated polypeptides are glycopeptides having an insect specific
glycosylation pattern, which are optionally conjugated to a modifying group, such as a
polymer (e.g., PEG) using a
glycosyltransferase and a modified
nucleotide sugar.