Fourier-transform infrared spectroscopy as a process analytical technology for near real time in-line estimation of the degree of PEGylation in chromatography.

PEGylation of biological macromolecules is a well-established strategy to increase circulation half-life, decrease renal clearance and improve biocompatibility. PEGylation is a process in which polyethylene glycol (PEG) is covalently attached to a target molecule. The production of PEGylated biopharmaceuticals is usually executed by first producing and purifying the base molecule followed by the PEGylation reaction and purification of the modified molecule. Most PEGylated pharmaceuticals are produced by random PEGylation in batch mode and need to be purified as mainly the mono-PEGylated form is the desired drug product. In this work we propose a method to estimate the degree of PEGylation (DOP) of modified protein eluting from a chromatography column in near real-time. extended multiplicative signal correction (EMSC) is used in conjunction with asymmetric least squares (aaLS) to alleviate the influence of a salt gradient during ion exchange chromatography (IEX) on the spectral data. To convert the raw data obtained from spectral data to the actual DOP additional information obtained from off-line measurements is utilized. Once the signal correction is applied to in-line spectral data the DOP can be estimated without further use of off-line analytics. As the prerequisites for the application of this method are relatively easy to obtain it may also find use to speed up process development.