Example 5
The pIFN-α-PS-E107 variant (SEQ ID NO: 14) is taken from the Capto chromatography pool after using Capto chromatography as per manufacturer instructions and 0.2 M glycine is added to the purified form. The pH of the mixture is adjusted to 4.0 with acetic acid. The pIFN-a variant is then concentrated to 8.2 mg/mL using an Amicon Ultra centrifugal filter according to manufacturer's instructions. Once concentrated, 30K linear PEG (PEG can be purchased commercially from NOF America Corporation or EMD Merck for examples) is added in a 8:1 molar ratio of pEG: pIFN-α variant. The PEG/pIFN-α variant mixture is then incubated at 28° C. for about 18 hours. This method results in >95% of the pIFN-α variant being conjugated with PEG after 18 hours of incubation.
The pegylated variant (pIFN-α-PS-E107-PEG30K) can then be purified as follows. A 143 mL SP650S Tosoh column can be used to purify the pegylated variant using a mobile phase of:
A: 30 mM sodium acetate, pH 5.0
B: 30 mM sodium acetate, 5% ethylene glycol, pH 5.0
0 to 100% B over 20 column volumes.