Several protein therapeutics exist in different isoforms and with a variety of post-translational modifications, such as glycosylation, that differentiate their therapeutic activity. The various isoforms and glycoforms are frequently hard to separate and characterize, thereby limiting our ability to isolate individual proteins with precise therapeutic power. Chromatofocusing has provided a means to separate protein isoforms based on differences in isoelectric point. Traditional chromatofocusing, which employs ion exchange or multimodal adsorbents, requires extensive chromatographic optimization and usage of buffers in order to achieve the desired separation. We therefore propose a more efficient version of chromatofocusing, based on the combination of a novel buffer system and software for controlling the chromatographic gradients (PiSep) with peptide-based affinity adsorbents. We have demonstrated our technology through separation of the four IgG subclasses of human polyclonal IgG. To this end, we have coupled a dual-gradient (decreasing pH - increasing ionic strength) protocol with an IgG-binding adsorbent functionalized with our peptide ligand HWRGWV. The competing effects of decreasing pH, which promotes protein elution by electrostatic repulsion, and increasing salt concentration, which promotes protein retention by hydrophobic interaction, offered an efficient separation of human IgG 1 - 4 subclasses in a single chromatographic run. Notably, this fractionation has been achieved using both purified IgG and an IgG-rich fraction of human plasma as feed samples. The comparison with traditional multimodal adsorbents (CaptoAdhere and Capto MMC) indicates the superior performance of our system as compared to traditional chromatofocusing approaches.

Chromatofocusing of IgG isoforms using peptide-based affinity adsorbents and dual ionic strength-pH gradients

253rd National Meeting (2017)

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