Preparation of Protein A Membranes Using Propargyl Methacrylate-Based Copolymers and Copper-Catalyzed Alkyne-Azide Click Chemistry.

The development of convective technologies for antibody purification is of interest to the bioprocessing industries. This study developed a Protein A membrane using a combination of graft polymerization and copper(I)-catalyzed alkyne-azide click chemistry. Regenerated cellulose supports were functionalized via surface-initiated copolymerization of propargyl methacrylate (PgMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMEMA300), followed by a reaction with azide-functionalized Protein A ligand. The polymer-modified membranes were characterized using attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR), gravimetric analysis, and permeability measurements. Copolymer composition was determined using the Mayo-Lewis equation. Membranes clicked with azide-conjugated Protein A were evaluated by measuring static and dynamic binding (DBC10) capacities for human immunoglobulin G (hIgG). Copolymer composition and degree of grafting were found to affect maximum static binding capacities, with values ranging from 5 to 16 mg/mL. DBC10 values did not vary with flow rate, as expected of membrane adsorbers.

Preparation of Protein A Membrane Adsorbers Using Strain-Promoted, Copper-Free Dibenzocyclooctyne (DBCO)-Azide Click Chemistry.

Protein A chromatography is the preferred unit operation for purifying Fc-based proteins. Convective chromatography technologies, like membrane adsorbers, can perform the purification rapidly and improve throughput dramatically. While the literature reports the preparation of Protein A membrane adsorbers utilizing traditional coupling chemistries that target lysine or thiol groups on the Protein A ligand, this study demonstrates a new approach utilizing copper-free dibenzocyclooctyne (DBCO)-azide click chemistry. The synthetic pathway consists of three main steps: bioconjugation of Protein A with a DBCO-polyethylene glycol (PEG) linker, preparation of an azide-functionalized membrane surface, and click reaction of DBCO-Protein A onto the membrane surface. Using polyclonal human immunoglobulins (hIgG) as the target molecule, Protein A membranes prepared by this synthetic pathway showed a flowrate-independent dynamic binding capacity of ~10 mg/mL membrane at 10% breakthrough. Fitting of static binding capacity measurements to the Langmuir adsorption isotherm showed a maximum binding (qmax) of 27.48 ± 1.31 mg/mL and an apparent equilibrium dissociation constant (Kd) of value of 1.72 × 10-1 ± 4.03 × 10-2 mg/mL. This work represents a new application for copper-less click chemistry in the membrane chromatography space and outlines a synthetic pathway that can be followed for immobilization of other ligands.

Comparative Evaluation of Commercial Protein A Membranes for the Rapid Purification of Antibodies.

Protein A chromatography is ubiquitous to antibody purification. The high specificity of Protein A for binding the Fc-region of antibodies and related products enables unmatched clearance of process impurities like host cell proteins, DNA, and virus particles. A recent development is the commercialization of research-scale Protein A membrane chromatography products that can perform capture step purification with short residence times (RT) on the order of seconds. This study investigates process-relevant performance and physical properties of four Protein A membranes: Purilogics Purexa™ PrA, Gore® Protein Capture Device, Cytiva HiTrap™ Fibro PrismA, and Sartorius Sartobind® Protein A. Performance metrics include dynamic binding capacity, equilibrium binding capacity, regeneration-reuse, impurity clearance, and elution volumes. Physical properties include permeability, pore diameter, specific surface area, and dead volume. Key results indicate that all membranes except the Gore® Protein Capture Device operate with flow rate-independent binding capacities; the Purilogics Purexa™ PrA and Cytiva HiTrap Fibro™ PrismA have binding capacities on par with resins, with orders of magnitude faster throughput; and dead volume and hydrodynamics play major roles in elution behavior. Results from this study will enable bioprocess scientists to understand the ways that Protein A membranes can fit into their antibody process development strategies.

Computational framework for the techno-economic analysis of monoclonal antibody capture chromatography platforms.

We developed a computational framework that integrates commercial software components to perform customizable technoeconomic feasibility analyses. The use of multiple software packages overcomes the shortcomings of each to provide a detailed simulation that can be used for sensitivity analyses and optimizations. In this paper, the framework was used to evaluate the performance of monoclonal antibody capture processes. To this end, the simulation framework incorporated dynamic models for the affinity chromatography step that were validated with experimental breakthrough curves. The results were integrated with an Intelligen SuperPro Designer process simulation for the evaluation of key performance indicators of the operations. As proof of concept, the framework was used to perform a sensitivity analysis and optimization for a case study in which we sought to compare membrane and resin chromatography for disposable and reusable batch capture platforms. Two membranes and one resin were selected for the capture media, which yielded six process alternatives to compare. The objective functions were set to be cost of goods, process time, and buffer utilization. The results of the optimization of these process alternatives were a set of operating conditions that display tradeoffs between competing objectives. From this application exercise, we conclude that the framework can handle multiple variables and objectives, and it is adaptable to platforms with different chromatography media and operating modes. Additionally, the framework is capable of providing ad hoc analyses for decision making in a specific production context.

High-capacity multimodal anion-exchange membranes for polishing of therapeutic proteins.

This contribution reports on a study using Purexa™-MQ multimodal anion-exchange (AEX) membranes for protein polishing at elevated solution conductivities. Dynamic binding capacities (DBC10 ) of bovine serum albumin (BSA), human immunoglobulins, and salmon sperm DNA (ss-DNA) are reported for various salt types, salt concentrations, flowrates, and pH. Using 1 mg/ml BSA, DBC10 values for Purexa™-MQ were >90 mg/ml at conductivities up to 15 mS/cm. The membranes maintained a high, salt-tolerant BSA DBC10 of 89.8 ± 2.7 (SD) over the course of 100 bind-elute cycles. Polishing studies with acidic and basic monoclonal antibodies at >2 kg/L loads showed that Purexa™-MQ had higher clearance of host cell proteins and aggregate species at high conductivity (13 mS/cm) and in the presence of phosphate than other commercial AEX media. Purexa™-MQ also had a high ss-DNA DBC10 of 50 mg/ml at conductivities up to 15 mS/cm, markedly outperforming other commercial products. In addition to the effectiveness of Purexa™-MQ for protein polishing at elevated solution conductivities, its unusually high binding capacity for ss-DNA indicates potential applications for plasmid DNA purification.