High‐efficiency affinity precipitation of multiple industrial mAbs and Fc‐fusion proteins from cell culture harvests using Z‐ELP‐E2 nanocages

Abstract

Affinity precipitation using Z-elastin-like polypeptide-functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high-yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A-D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two-step model to investigate the protein interactions that occur during mAb-nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb-nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A-D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc-containing proteins with minimal optimization of process parameters.

Enhanced filtration performance usingfeed‐and‐bleedconfiguration for purification of antibody precipitates

“…The estimated HCP removal is 910-fold for the combination of the dewatering and washing steps, which is similar to, if not better than, the HCP reduction after a typical platform Protein A chromatography capture step. 11 The capacity of the bench-scale apparatus is 7.2 g mAb/day using the three membrane modules (one for dewatering and two for washing) each with 0.01 m 2 area. Expected buffer consumption is 0.9 L/g mAb.…”

“…1,2 There is also growing interest in the use of precipitation for the initial purification (capture) of monoclonal antibody (mAb) products due to the significant increase in product titer that have been achieved over the past two decades. 3,4 mAbs have been successfully precipitated using cold ethanol and CaCl 2, 5 polyethylene glycol (PEG), 6 PEG and CaCl 2, 7 CaCl 2 and caprylic acid, 8 PEG and low pH 9 , zinc chloride (ZnCl 2 ) and PEG, 10 and elastin-like polypeptides (ELP) 11,12 among others.…”

Overcoming challenges of improving legacy biopharmaceutical processes

“…Other technologies such as affinity precipitation and aqueous two‐phase systems (ATPS) have also been investigated as alternative downstream processing technologies to chromatographic purification. For example, affinity precipitation has been viewed as a cost‐effective and scalable alternative to affinity chromatography, but there are still some issues to overcome for this technology, such as co‐precipitation of contaminants and the use of harsh chemicals in affinity precipitation 16–19 . ATPS may be another attractive technology which performs multiple tasks for the extraction and purification of biological products using phase‐forming solvents 20–23 .…”

Enhancing Protein A performance in mAb processing: A method to reduce and rapidly evaluate host cell DNA levels during primary clarification

“…A peptide or biopolymer featuring a Protein A epitope is introduced to the cell culture and binds the desired mAb, subsequently inducing precipitation. Upon isolation and washing of the precipitate, the mAb can be released into solution by pH modulation, allowing further purification …”

“…Upon isolation and washing of the precipitate, the mAb can be released into solution by pH modulation, allowing further purification. 16,17 Other methods to overcome the challenges chromatin/DNA presents to Protein A chromatography involve modifying the operating conditions for Protein A chromatography itself. One strategy used chromatofocusing using a pH gradient through the Protein A column to reduce HCP coelution with the target mAb.…”