Enhancing the therapeutic activity of hyperimmune IgG against chikungunya virus using FcγRIIIa affinity chromatography.

Introduction: Chikungunya virus (CHIKV) is a re-emerging mosquito transmitted alphavirus of global concern. Neutralizing antibodies and antibody Fc-effector functions have been shown to reduce CHIKV disease and infection in animals. However, the ability to improve the therapeutic activity of CHIKV-specific polyclonal IgG by enhancing Fc-effector functions through modulation of IgG subclass and glycoforms remains unknown. Here, we evaluated the protective efficacy of CHIKV-immune IgG enriched for binding to Fc-gamma receptor IIIa (FcγRIIIa) to select for IgG with enhanced Fc effector functions. Methods: Total IgG was isolated from CHIKV-immune convalescent donors with and without additional purification by FcγRIIIa affinity chromatography. The enriched IgG was characterized in biophysical and biological assays and assessed for therapeutic efficacy during CHIKV infection in mice. Results: FcγRIIIa-column purification enriched for afucosylated IgG glycoforms. In vitro characterization showed the enriched CHIKV-immune IgG had enhanced human FcγRIIIa and mouse FcγRIV affinity and FcγR-mediated effector function without reducing virus neutralization in cellular assays. When administered as post-exposure therapy in mice, CHIKV-immune IgG enriched in afucosylated glycoforms promoted reduction in viral load. Discussion: Our study provides evidence that, in mice, increasing Fc engagement of FcγRs on effector cells, by leveraging FcγRIIIa-affinity chromatography, enhanced the antiviral activity of CHIKV-immune IgG and reveals a path to produce more effective therapeutics against these and potentially other emerging viruses.

Mechanistic modeling of the loss of protein sieving due to internal and external fouling of microfilters.

Fed-batch and perfusion cell culture processes used to produce therapeutic proteins can use microfilters for product harvest. In this study, new explicit mathematical models of sieving loss due to internal membrane fouling, external membrane fouling, or a combination of the two were generated. The models accounted for membrane and cake structures and hindered solute transport. Internal membrane fouling was assumed to occur due to the accumulation of foulant on either membrane pore walls (pore-retention model) or membrane fibers (fiber-retention model). External cake fouling was assumed to occur either by the growth of a single incompressible cake layer (cake-growth) or by the accumulation of a number of independent cake layers (cake-series). The pore-retention model was combined with either the cake-series or cake-growth models to obtain models that describe internal and external fouling occurring either simultaneously or sequentially. The models were tested using well-documented sieving decline data available in the literature. The sequential pore-retention followed by cake-growth model provided a good fit of sieving decline data during beer microfiltration. The cake-series and cake-growth models provided good fits of sieving decline data during the microfiltration of a perfusion cell culture. The new models provide insights into the mechanisms of fouling that result in the loss of product sieving. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1323-1333, 2017.

The role of more than 40 years of improvement in protein A chromatography in the growth of the therapeutic antibody industry.

Protein A chromatography has been used as the mAb capture step in the majority of FDA submissions. In this study, the performance of protein A chromatography, as indicated by capacity, operational flow rate, and productivity (rate of mAb production per liter of resin) was examined over its full history to gain insights into the reasons for its consistent use. Protein A productivity and capacity have increased 4.3 and 5.5% a year, respectively, since 1978. In contrast, protein A operational flow rate increased between 1978 and 2001 and then remained constant or declined as further improvements provided only marginal benefits. The productivity of protein A resin and also the mAb bioreactor titer (14% growth) rapidly improved starting in about 1990 to economically provide material for clinical trials. Technology improvement is typically driven by product sales. The sales of protein A resin, as indicated by sales of protein A ligand (21% growth), have closely paralleled the sales of mAbs (20% growth). Both increased rapidly in 2000 after the first major mAb therapeutics were approved and the markets were developed. It is likely that alternatives to protein A chromatography have not been implemented because of the order of magnitude improvement in protein A performance. Protein A membrane adsorbers and monoliths have higher productivity than packed columns due to their short bed heights and high operational flow rates. These devices are not currently practical for large-scale manufacturing but may represent a format for future improvements in protein A productivity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1193-1202, 2016.

Inactivation of viruses using novel protein A wash buffers.

Low pH viral inactivation is typically performed in the eluate pool following the protein A capture step during the manufacturing of monoclonal antibodies and Fc-fusion proteins. However, exposure to low pH has the potential to alter protein quality. To avoid these difficulties, novel wash buffers capable of inactivating viruses while antibodies or Fc-fusion proteins were bound to protein A or mixed mode resins were developed. By equilibrating the column in high salt buffer (2 M ammonium sulfate or 3 M sodium chloride) after loading, the hydrophobic interactions between antibodies and protein A ligands were increased enough to prevent elution at pH 3. The ammonium sulfate was also found to cause binding of an antibody to a mixed mode cation exchange and a mixed mode anion exchange resin at pH values that caused elution in conventional cation and anion exchange resins (pH 3.5 for Capto Adhere and pH 8.0 for Capto MMC), indicating that retention was due to enhanced hydrophobic interactions. The potential of the 2 M ammonium sulfate pH 3 buffer, a 1 M arginine buffer, and a buffer containing the detergent LDAO to inactivate XMuLV virus when used as protein A wash buffers with a 1 hour contact time were studied. The high salt and detergent containing wash buffers provided about five logs of removal, determined using PCR, and complete combined removal and inactivation (> 6 logs), determined by measuring infectivity. The novel protein A washes could provide more rapid, automated viral inactivation steps with lower pool conductivities.

Separation of nonfucosylated antibodies with immobilized FcγRIII receptors.

Post-translational modifications can dramatically impact protein activity, but identifying such structure:function relationships, as well as capitalizing on functionally enhanced variants, is a significant challenge. Here, affinity chromatography resins that contained immobilized FcγRIII receptors were used to enrich nonfucosylated antibodies 6- to 9-fold, offering what may be a tractable method for both the identification of post-translational modifications that affect function, as well as a means to enrich variants with enhanced activity.

The effect of protein A cycle number on the performance and lifetime of an anion exchange polishing step.

Most mAb platform purification processes consist of an affinity capture step followed by one or two polishing steps. An understanding of the performance linkages between the unit operations can lead to robust manufacturing processes. In this study, a weak-partitioning anion-exchange chromatography polishing step used in a mAb purification process was characterized through high-throughput screening (HTS) experiments, small-scale experiments including a cycling study performed on qualified scale-down models, and large-scale manufacturing runs. When material from a Protein A column that had been cycled <10× was loaded on the AEX resin, early breakthrough of impurities and premature loss of capacity was observed. As the cycle number on the Protein A resin increased, the capacity of the subsequent AEX step increased. Different control strategies were considered for preventing impurity breakthrough and improving AEX resin lifetimes. Depth filtration of the Protein A peak pool significantly improved the AEX resin capacity, robustness, and lifetime. Further, the turbidity of the Protein A pool has the potential for use as an in-process control parameter for monitoring the performance of the AEX step.

Effect of protein and solution properties on the Donnan effect during the ultrafiltration of proteins.

Formulation of protein biopharmaceuticals as highly concentrated liquids can improve the drug substance storage and supply chain, improve the target product profile, and allow greater flexibility in dosing methods. The Donnan effect can cause a large offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins with ultrafiltration membranes. For neutral formulations, the pH will typically increase above the diafiltration buffer pH for basic monoclonal antibodies and decline below the diafiltration buffer pH for acidic Fc-fusion proteins. In this study, new equations for the Donnan effect during the diafiltration and concentration of proteins in solutions containing monovalent and divalent ions were derived. The new Donnan models obey mass conservation laws, account for the buffering capacity of proteins, and account for protein-ion binding. Data for the pH offsets of an Fc-fusion protein and a monoclonal antibody were predicted in both monovalent and divalent buffers using these equations. To compensate for the pH offset caused by the Donnan effect, diafiltration buffers with pH and excipient values offset from the ultrafiltrate pool specifications can be used. The Donnan offset observed during the concentration of an acidic Fc-fusion protein was mitigated by operating at low temperature. It is important to account for the Donnan effect during preformulation studies. The excipients levels in an ultrafiltration pool may differ from the levels in a protein solution obtained by adding buffers into concentrated protein solutions due to the Donnan effect.

Achieving high mass-throughput of therapeutic proteins through parvovirus retentive filters.

Parvovirus retentive filters that assure removal of viruses and virus-like particles during the production of therapeutic proteins significantly contribute to total manufacturing costs. Operational approaches that can increase throughput and reduce filtration area would result in a significant cost savings. A combination of methods was used to achieve high throughputs of an antibody or therapeutic protein solution through three parvovirus retentive filters. These methods included evaluation of diatomaceous earth or size-based prefilters, the addition of additives, and the optimization of protein concentration, temperature, buffer composition, and solution pH. An optimum temperature of 35°C was found for maximizing throughput through the Virosart CPV and Viresolve Pro filters. Mass-throughput values of 7.3, 26.4, and 76.2 kg/m(2) were achieved through the Asahi Planova 20N, Virosart CPV, and Viresolve Pro filters, respectively, in 4 h of processing. Mass-throughput values of 73, 137, and 192 kg/m(2) were achieved through a Millipore Viresolve Pro filter in 4.0, 8.8, and 22.1 h of processing, respectively, during a single experiment. However, large-scale parvovirus filtration operations are typically controlled to limit volumetric throughput to below the level achieved during small-scale virus spiking experiments. The virus spike may cause significant filter plugging, limiting throughput. Therefore newer parvovirus filter spiking strategies should be adopted that may lead to more representative viral clearance data and higher utilization of large-scale filter capacity.

Increasing the capacity of parvovirus-retentive membranes: performance of the Viresolve Prefilter.

Removal of small parvoviruses from highly purified proteins can be performed using normal-flow filters. The entrapment of protein aggregates, denatured proteins and other impurities can cause plugging and a decrease in filter capacity. In the present study a variety of prefilters were investigated for their ability to remove the species that foul Viresolvetrade mark NFP (normal-flow parvovirus) filters. The Viresolvetrade mark Prefilter, which utilizes entrapped diatomaceous earth to hydrophobically bind fouling species, provided a dramatic increase in virus filter capacity for solutions containing human IgG or a variety of monoclonal antibodies. We found that the component of the human IgG stream that bound to the Prefilter, when analysed using SDS/PAGE, isoelectric-focusing, size-exclusion chromatography, CD and ANS (1-anilinonaphthalene-8-sulphonate) titration, consisted of monomeric IgG variants containing more exposed hydrophobic surfaces. The bound component may represent oxidized or otherwise degraded IgG species or a subset of IgG molecules with more hydrophobic antigen-binding surfaces. The results indicate that NFP membranes do not foul solely as a result of entrapment of protein aggregates in the pore structure. The Viresolvetrade mark Prefilter has a high permeability, did not diminish protein yield and provided consistent performance between different media lots, device lots and device scales.