By Allison Farrand, PhD; David Schmidt; and Jesse McCool, Ph.D.
Fragment crystallizable (Fc)-fusion proteins are a growing class of biopharmaceuticals that have gained popularity due to the manufacturability benefits and extended drug half-life afforded by the Fc fragment. One such fusion protein, consisting of a protein receptor fused to a human IgG Fc fragment (hereafter referred to as Protein X), was manufactured by Cytovance Biologics. Size Exclusion High-Performance Liquid Chromatography (SE-HPLC) is an analytical method that is routinely performed to determine size-based purity of manufactured lots of Protein X to support product release. Numerous manufacturing runs were performed for Protein X and results from the SE-HPLC analyses indicated that the purity of a recent lot was lower than expected and outside of the established specification. Upon further investigation, it was determined that the low Protein X purity was due to an increase in a poorly resolved, unidentified shoulder species eluting just after the main Protein X peak. Characterization studies were performed to both identify and measure the functionality of this low molecular weight (LMW) impurity and more fully understand if the species was a critical attribute. The results of our analyses suggest that the shoulder species represents a Protein X heterodimer comprised of one full-length monomer and one monomer lacking the receptor sequence. These findings defined a critical quality attribute and led to the closure of a CGMP investigation.