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.