PEGylated L-Chymotrypsinogen A (N-Terminal PEG 20KDa) on BioSep2000 (2)

LC Conditions

Column

BioSep 5 µm SEC-s2000 145 Å, LC Column 300 x 7.8 mm, Ea

Brand

Biosep

Part No

00H-2145-K0

Phase Name

SEC-s2000

Elution Type

Isocratic

Mobile Phase

A: 100mM Phosphate buffer pH 6.8

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

100

0

Flow Rate

1.00 mL/min

Temperature

22°C

Detection

UV-Vis Abs.-Variable Wave.(UV) @ (22°C)

Detection Info

Sample Notes

Application Topic: Monitoring protein PEGylation and purifying PEGylated proteins from their reaction Therapeutic proteins are often PEGylated to increase their serum lifetime; however, such reactions typically generate a heterogeneous product that can be difficult to characterize and purify. PEGylated proteins are usually purified by GFC or RPC after synthesis and the PEGylation reaction is monitored by HPLC to maximize recovery of a singly-modified protein. While reaction conditions are optimized, it is common that proteins can be PEGylated at multiple sites even with N-terminal specific chemistries; thus the need for time course monitoring. While different molecular weight range BioSep columns can be used for monitoring such reactions and purifying PEGylated protein, BioSep 2000 is typically used because it provides optimal separation of molecular weights below 150 Kilodaltons, the range of most PEGs, proteins, and their conjugates. In this example application, protein was reacted using N-terminal favoring conditions (phosphate pH 6.5 with cyanoborohydrate and 5x PEG aldehyde excess). For reaction monitoring, intact protein and reaction timepoints at 0 minutes, 30 minutes, 60 minutes and an overnight reaction were run on a BioSep 2000 at neutral pH conditions (100 mM phosphate buffer, pH 6.8) and chromatograms were overlaid. Based on the GFC results protein is rapidly PEGylated over the course of an hour with concomitant decrease in intact protein. Note that even with using an N-terminal specific PEG reagent, additional sites of PEG modification occur even at the 30 minute timepoint. The overnight timepoint has a majority of the protein in multiple PEGylated forms. Resolution of each component is such that the BioSep 2000 could be used either a monitoring or purification capacity to get high recovery and purity of the desired PEGylated protein.