Characterization of ALK1-Fc fusion protein aggregates derived from extra-cellular domain disulfide bond rearrangements

Aggregation of protein biotherapeutics has consequences not only for diminished protein production, but also for increased immunogenicity of protein drugs. However, the mechanisms of protein aggregation vary depending on the protein and the expression system utilized, making it difficult to elucidate the conditions that promote their increased aggregation. The mechanisms behind non-native aggregation of recombinant IgG protein therapeutics from mammalian expression systems have been extensively studied. In an effort to better understand the mechanisms behind the aggregation of glycosylated fusion proteins produced in CHO cells, we have examined the high molecular weight (HMW) form of an activin receptor-like kinase 1 Fc fusion protein (ALK1-Fc). Size-exclusion chromatography (SEC) with multi-angle laser light scattering (MALLS) and SDS-PAGE indicate two populations of aggregate: 1) non-disulfide linked, higher-order aggregates, and 2) disulfide-linked dimer, trimer, tetramer, and larger oligomers. CD spectroscopy suggests that the largest aggregated species have increased levels of non-native structure, while the smallest aggregated species maintain secondary structure similar to the native monomer. Peptide map analysis of the different SEC fractions showed decreased levels of O-linked glycosylation, higher amounts of high-mannose N-linked oligosaccharide structures, and overall less sialylation as the size of the aggregates increases. According to surface plasmon resonance, none of the disulfide-linked aggregate species appeared to bind BMP9—a high-affinity, physiologically relevant binding partner.;Disulfide-linked aggregate species were found to associate through the extra-cellular domain (ECD) receptor rather than the Fc portion of the molecule. Multiple, mispaired ECD disulfide bonds were identified for the disulfide-linked dimer. Only the ECD disulfide bonds were observed to participate in the rearrangements that lead to aggregate formation. Removing N-linked glycosylation on the ECD appears to encourage disulfide-linked aggregation. Elucidation of the specific mechanisms behind disulfide-linked aggregates may lead to the design of constructs less-prone to aggregation, as well as design of processes and formulations that limit aggregate formation, thereby resulting in increased production.