In silico immunogenicity assessment of E. coli derived HCPs
The immunogenicity of most abundant E. coli derived HCPs that were estimated to have an abundance levels of at least 10 ng/mg based on SDS-PAGE and LC-MS data were assessed using the in silico tools described earlier (Bailey-Kellogg et al.). The proteins where multiple high affinity epitopes (top 5%) and associated with a cluster were identified and shown in Table 3. A relative ranking of the epitopes on the immunogenicity scale is displayed in Figure 8. The sequence-based risk (subjective risk) of the most abundant six HCPs (levels estimated to be > 20 ng/mg) is considered low with few non-autoreactive Clustimers displaying relatively low Clustimer scores on the scale. A few other E. coli HCPs with a higher predicted risk as evident by higher number of high binding and high affinity eiptopes were present at much lower levels and hence considered low risk (de Zafra, Quarmby, Francissen, Vanderlaan, & Zhu-Shimoni; F. Q. Wang, Richardson, & Shameem, 2015). It should be noted that the residual proteins are relatively small with MW ranges from 7.4 kDa to 48.2 kDa, and the pI ranges from 5.17 to 7.24 (top 6). Although considered low risk, this information was supplied to process chemists to further develop a purification method to de-risk the potential impact from these residual proteins.