Development of a high throughput protein a well‐plate purification method for monoclonal antibodies

Abstract

We have developed a new high throughput method for the purification of monoclonal antibodies from harvested cell culture fluid for analytical characterization. This method uses Protein A resin in a 96 well-plate format with protein loading sufficient to perform multiple analyses per well. Resin and buffer conditions were optimized to obtain aggregate and charge variant comparability with three preparative Protein A purified monoclonal antibodies. We are able to successfully demonstrate comparability for aggregate within 0.25% based upon size-exclusion chromatography. Acidic species were found to be within 2% from the preparative purified control based upon cation-exchange chromatography, 5% based upon capillary zone electrophoresis, and 3% based upon imaged capillary isoelectric focusing. Glycan distribution was analyzed and was within 1% of the preparative purified controls. A tryptic digest was performed and all peaks in the preparative purified control were found in the first elution from the well-plate format.

Current advances in the development of high-throughput purification strategies for the generation of therapeutic antibodies

“…As a result of this, HTP methods can act as an input for many analytical tools, with protein A or other affinity-based purification often used in filter plates. Hopp et al screened four affinity resins with three elution buffers, four salt concentrations and three neutralization buffers to deliver 500 μg of purified antibody at 2 mg/ml [33]. The purified antibodies were found to be suitable for aggregate screening using size exclusion chromatography, charge heterogeneity determination using IEX and isoelectric High-throughput purification strategies for the generation of therapeutic antibodies Review focusing, N-glycan analysis and tryptic peptide mapping.…”

“…ProSep A Plate [33,36] Poros A Plate [33] Protein A, protein G Magnetic beads [25] MabSelect and MabSelect SuRe Plate [28,30,33,36] MabSelect and MabSelect SuRe…”

Cross-Interaction Chromatography: A Rapid Method to Identify Highly Soluble Monoclonal Antibody Candidates

“…Thus, solubility is one biophysical property that is important to the success of formulation development. In contrast to the high-throughput screening methods available for purification, activity, and affinity characterization of antibodies (5)(6)(7)(8)(9)(10)(11), there are few methods available for characterizing the solubility properties of proteins at small scale. Indeed, characterization of the solubility properties of proteins has traditionally relied on larger quantities of protein that are not routinely available at the screening stage of antibody discovery.…”

Adaptation of the pore diffusion model to describe multi-addition batch uptake high-throughput screening experiments

“…While the multi-addition experimental technique has been successfully utilized to achieve target loading densities for investigation of elution behavior [28,29], the focus of this work is to quantify the uptake kinetics of the successive protein addition steps. Experimental methods will be described in greater detail in a separate work [30].…”