CoV2Research - May2020 - 29

broadly reactive to the array of HCPs in this clarified,
conditioned media. The predictably lower coverage
by 2D WB compared to silver stain clearly demonstrates the superiority of AAE in terms of sensitivity.
The poor specifiity of 2D WB as evidenced by 48%
non-specific binding to a non-cross-reacting control
antibody leads to the conclusion that 2D WB is of no
predictive value in determining how the ELISA using
that antibody will quantitatively react to the more
limited array of HCPs in downstream samples. In
addition to the focus on coverage to the entire proteome that in theory might be present in a final drug
substance, orthogonal methods should determine
reactivity to actual downstream HCPs. Downstream
reactivity data along with qualification of the ELISA
for accuracy and specificity as evidenced by dilution
linearity and spike recovery analysis will determine if
an assay is fit for purpose.

employing AAE analysis, biopharmaceutical companies have a method with sufficient sensitivity to
evaluate individual HCPs that persist through various purification schemes. The extraction and concentration of downstream HCP by AAE will also
eliminate greater than 99.9% of the drug substance.
Removal of drug substance greatly improves the
ability to identify individual HCPs by 2D PAGE and
Mass Spectrometry analysis. Cygnus is now using
AAE as a simple, lower cost liquid chromatography
step for 2D-LC-MS/MS analysis of HCPs.
The question of "generic" or "platform" antibodies
and assays versus "process-specific" methods has
been a theoretical argument based on the assumption that generic and platform antibodies might
miss certain process-specific HCPs, and thus a process-specific antibody ought to be superior. With
AAE we now have the ability to look at downstream
HCPs to determine objectively if a process-specific
assay is necessary and superior to a well-developed
generic or platform method.

While coverage to the total HCP mixture present in a
cell culture harvest stream by orthogonal methods
has been traditionally requested by regulators, the
most significant advantage of AAE is its ability to detect and demonstrate reactivity to individual, downstream HCPs. It is the HCPs that persist through a given purification process that are the most important
with respect to patient safety and drug efficacy. By

About the authors
Eric Bishop and Ken Hoffman are at Cygnus Technologies.

Additional Resources
"What are HCPs? Why do we care?"

https://www.cygnustechnologies.com/resources/videos/index/category/videos/

CoV2Research - May2020

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