At this stage, the process therefore has shaped up into eight unit operations. First, an initial ultrafiltration/ diafiltration
(UF/DF) concentrates the crude material and removes a portion of the low molecular weight impurities. This is followed by
S/D virus inactivation. The first major purification step is Protein A chromatography. This is followed by HIC chromatography.
A second UF/DF is then needed to reduce the salt concentration for the subsequent mixed-mode chromatography. The seventh unit
operation is nanofiltration for virus removal. The process is completed with a final UF/DF step into the final formulation.
Individual unit operations, especially the chromatography steps, would benefit from additional characterization and optimization.
Nonetheless, this process has been successfully scaled up, and is being used to produce material for preclinical and early
EA2 is a unique, engineered Fc-fusion protein, and as a result, cannot be purified following a fully platformed process. However,
its purification process, as initially envisioned, was to take steps from various platform processes targeted toward the affinity
and pseudo-affinity binding sites on the EA2 molecule. However, what we learned is that with a molecule this complex, none
of the three potential steps functioned as expected. The process that was developed was quite different from that initially
envisioned, for a truly unexpected purification.
Our thanks to the following scientists for their contributions to this paper: Dr. Hong Li, Dr. Peychii Lee, and Munir Nahri
at Laureate Pharma, Downstream Process Development: the Analytical Development group, and Eric Leblanc at Enobia Pharma.
DOUGLAS W. REA is senior specialist of downstream process development, MICHIEL E. ULTEE, PhD, is senior director of process sciences, and SHARON X. CHEN, PhD, is senior scientist of analytical and downstream process development, all at Laureate Pharma, Inc., East Princeton, NJ, 609.919.3395,
Thomas P. Loisel, PhD, is group leader of biology at Enobia Pharma, Inc.
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