N-glycosylation Site Occupancy Heterogeneity
We analyzed three batches of R27T purification products and purification intermediates. We defined proteins separated by hydrophobicity over four column steps into main target (R27T) and intermediate (shoulder) peaks (Figure S1). Each peak was analyzed by SDS-PAGE and IEF with Rebif, a single-glycosylated rhIFN-β, as a reference (Figure 1A). The main R27T peaks revealed a protein of ~25−26 kDa with an isoelectric point (pI) of ~5.3−6.9 and a smear pI of 7.5, 8.0 and 8.3 on IEF gels, while Rebif ran as a 22.5 kDa product with major pI values of 8.0 and 8.3. Intermediate peaks separated from major peaks yielded a broad medium-sized band that ran between the R27T and Rebif bands, and its pI bands were more basic than those of R27T, and some were within the main pI range of Rebif, indicating a mixture of single- and double-glycosylated proteins. We attempted to quantitate species with one or two glycosylation sites to analyze glycosylation site occupancy heterogeneity. We used a microchip assay and capillary IEF (cIEF) to separate and quantitate species based on size and pI differences (Figure 1B, 1C). We were able to correlate the size with glycosylation occupancy, although there was considerable size variation (~15.2 kDa for no glycosylation, 21.9−34.8 kDa for one glycosylation, and 41.6−45.7 kDa for two glycosylation, Table S1) compared with qualitative analysis due to limitations of the capillary analysis methods with glycoproteins (Engel et al., 2015). There were also clear differences in pI (~7.19−7.97 for one glycosylation and 5.67−7.09 for two glycosylation). Based on size differences, we found that R27T was made up of 94% of the double glycosylated form and 6% of the single-glycosylated form (Figure 1B). For the R27T intermediate, the ratio was 5%, 77%, and 18% non-glycosylated, single-glycosylated, and double-glycosylated, respectively. By contrast, the Rebif reference protein displayed 100% single-glycosylated form. These results were supported by cIEF, although there was a slight difference in ratios (94.1% double-glycosylation and 1.9% single-glycosylation for R27T, and 84.5% single-glycosylation and 12.2% double-glycosylation for the R27T intermediate), because of more direct effect of number of sialic acid rather than number of glycan chain. (Figure 1C). The inclusion of the 6% single-glycosylated form as a macroheterogeneity with the 94% double-glycosylation form of R27T did not have a significant influence on the specific activity as discussed in our previous study (Song et al., 2014). Thus, the presence of 6% single-glycosylated R27T could be considered a control factor for more homogeneous molecular entity and an example of macroheterogeneity, but such products would not fail to meet the quality criteria.