Defining the mechanistic binding of viral particles to a multi-modal anion exchange resin.

A multi-tiered approach to determine the binding mechanism of viral clearance utilizing a multi-modal anion exchange resin was applied to a panel of four viral species that are typically used in validating viral clearance studies (i.e., X-MuLV, MVM, REO3, and PrV). First, virus spiked buffer-only experiments were conducted to evaluate the virus's affinity for single mode and multi-modal chromatography resins under different buffer conditions in a chromatography column setting. From these results we hypothesize that the mechanisms of binding of the viruses involve binding to both the hydrophobic and anionic functional groups. This mechanistic view agreed with the general surface characteristics of the different virus species in terms of isoelectric point and relative hydrophobicity values. This hypothesized mechanistic binding was then tested with commercially relevant, in-process materials, in which competitive binding occurred between the load components (e.g., viruses, target product, and impurities) and the resin. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1019-1026, 2018.

Evaluating the effect of in-process material on the binding mechanisms of surrogate viral particles to a multi-modal anion exchange resin.

Bacteriophage binding mechanisms to multi-modal anion exchange resin may include both anion exchange and hydrophobic interactions, or the mechanism can be dominated by a single moiety. However, previous studies have reported binding mechanisms defined for simple solutions containing only buffer and a surrogate viral spike (i.e. bacteriophage ΦX174, PR772, and PP7). We employed phage spiked in-process monoclonal antibody (mAb) pools to model binding under bioprocessing conditions. These experiments allow the individual contributions of the mAb, in-process impurities, and buffer composition on mechanistic removal of phages to be studied. PP7 and PR772 use synergetic binding by the positively charged quaternary amine and the hydrophobic aromatic phenyl group to bind multi-modal resin. ΦX174's binding mechanism remains inconclusive due to operating conditions.

Trans-acting oligodeoxythymidine phosphorothioate triester reagents for transient transfection optimized and facilitated by a high-throughput microbioreactor system.

A rapid and cost-effective transient transfection method for mammalian cells is essential for screening biopharmaceuticals in early stages of development. A library of 25 amphipathic trans-acting oligodeoxythymidine phosphorothioate triester (dTtaPS) transfection reagents, carrying positively charged and lipophilic groups, has been constructed for this purpose. High-throughput screening of the library, using an imaging cytometer and an automated microbioreactor system, has led to the identification of dTtaPS10+ as a potent transfection reagent. This reagent efficiently delivers a plasmid encoding enhanced green fluorescent protein in adherent HeLa cells while exhibiting low cytotoxicity. The microbioreactor system has been particularly useful for assessing the ability of dTtaPS10+ to deliver a plasmid encoding immunoglobulin IgG1 in a fed-batch serum-free suspension CHO cell culture; dTtaPS10+ -mediated transfection resulted in the production of IgG1 in yields comparable to or better than those obtained with commercial lipid-based transfection reagents under similar conditions. The ability of dTtaPS10+ to deliver plasmids is essentially unaffected by the presence of a silicone-based antifoaming reagent, which is commonly used in bioreactor cell cultures. The transfection efficiency of lyophilized dTtaPS10+ -plasmid complexes has been significantly restored upon aqueous reconstitution when compared to that achieved while using commercial transfection reagent complexes under similar conditions. The results of all experiments underscore the potential of dTtaPS10+ for transient transfection of plasmids into adherent cells and fed-batch serum-free suspension CHO cells and rapid screening of reagents in a microbioreactor system.

The step-wise framework to design a chromatography-based hydrophobicity assay for viral particles.

A high-salt, hydrophobic interaction chromatography (HIC) method was developed to measure the relative hydrophobicity of a diverse set of solutes. Through the careful control of buffer pH and salt concentration, this assay was then used to ascertain for the first time the relative hydrophobicity values of three different bacteriophage, four mammalian viruses, and a range of biotech medicinal proteins as benchmarked to protein standards previously characterized for hydrophobicity.

A step-wise approach to define binding mechanisms of surrogate viral particles to multi-modal anion exchange resin in a single solute system.

Multi-modal anion exchange resins combine properties of both anion exchange and hydrophobic interaction chromatography for commercial protein polishing and may provide some viral clearance as well. From a regulatory viral clearance claim standpoint, it is unclear if multi-modal resins are truly orthogonal to either single-mode anion exchange or hydrophobic interaction columns. To answer this, a strategy of solute surface assays and High Throughput Screening of resin in concert with a scale-down model of large scale chromatography purification was employed to determine the predominant binding mechanisms of a panel of bacteriophage (i.e., PR772, PP7, and ϕX174) to multi-modal and single mode resins under various buffer conditions. The buffer conditions were restricted to buffer environments suggested by the manufacturer for the multi-modal resin. Each phage was examined for estimated net charge expression and relative hydrophobicity using chromatographic based methods. Overall, PP7 and PR772 bound to the multimodal resin via both anionic and hydrophobic moieties, while ϕX174 bound predominantly by the anionic moiety. Biotechnol. Bioeng. 2017;114: 1487-1494. © 2017 Wiley Periodicals, Inc.

Exploring the linkage between cell culture process parameters and downstream processing utilizing a plackett-burman design for a model monoclonal antibody.

Linkage of upstream cell culture with downstream processing and purification is an aspect of Quality by Design crucial for efficient and consistent production of high quality biopharmaceutical proteins. In a previous Plackett-Burman screening study of parallel bioreactor cultures we evaluated main effects of 11 process variables, such as agitation, sparge rate, feeding regimens, dissolved oxygen set point, inoculation density, supplement addition, temperature, and pH shifts. In this follow-up study, we observed linkages between cell culture process parameters and downstream capture chromatography performance and subsequent antibody attributes. In depth analysis of the capture chromatography purification of harvested cell culture fluid yielded significant effects of upstream process parameters on host cell protein abundance and behavior. A variety of methods were used to characterize the antibody both after purification and buffer formulation. This analysis provided insight in to the significant impacts of upstream process parameters on aggregate formation, impurities, and protein structure. This report highlights the utility of linkage studies in identifying how changes in upstream parameters can impact downstream critical quality attributes. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:163-170, 2017.

Adapting viral safety assurance strategies to continuous processing of biological products.

There has been a recent drive in commercial large-scale production of biotechnology products to convert current batch mode processing to continuous processing manufacturing. There have been reports of model systems capable of adapting and linking upstream and downstream technologies into a continuous manufacturing pipeline. However, in many of these proposed continuous processing model systems, viral safety has not been comprehensively addressed. Viral safety and detection is a highly important and often expensive regulatory requirement for any new biological product. To ensure success in the adaption of continuous processing to large-scale production, there is a need to consider the development of approaches that allow for seamless incorporation of viral testing and clearance/inactivation methods. In this review, we outline potential strategies to apply current viral testing and clearance/inactivation technologies to continuous processing, as well as modifications of existing unit operations to ensure the successful integration of viral clearance into the continuous processing of biological products. Biotechnol. Bioeng. 2017;114: 21-32. © 2016 Wiley Periodicals, Inc.

Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure.

Monoclonal antibody (mAb) drugs constitute the largest class of protein therapeutics currently on the market. Correctly folded protein higher order structure (HOS), including quinary structure, is crucial for mAb drug quality. The quinary structure is defined as the association of quaternary structures (e.g., oligomerized mAb). Here, several commonly available analytical methods, i.e., size-exclusion-chromatography (SEC) FPLC, multi-angle light scattering (MALS), circular dichroism (CD), NMR and multivariate analysis, were combined and modified to yield a complete profile of HOS and comparable metrics. Rituximab and infliximab were chosen for method evaluation because both IgG1 molecules are known to be homologous in sequence, superimposable in Fab crystal structure and identical in Fc structure. However, herein the two are identified to be significantly different in quinary structure in addition to minor secondary structure differences. All data collectively showed rituximab was mostly monomeric while infliximab was in mono-oligomer equilibrium driven by its Fab fragment. The quinary structure differences were qualitatively inferred from the less used but more reproducible dilution-injection-SEC-FPLC curve method. Quantitative principal component analysis (PCA) was performed on NMR spectra of either the intact or the in-situ enzymatic-digested mAb samples. The cleavage reactions happened directly in NMR tubes without further separation, which greatly enhanced NMR spectra quality and resulted in larger inter- and intra-lot variations based on PCA. The new in-situ enzymatic digestion method holds potential in identifying structural differences on larger therapeutic molecules using NMR.

Bioreactor process parameter screening utilizing a Plackett-Burman design for a model monoclonal antibody.

Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality. Therefore, changes proposed to currently validated processes usually require justification and are reported to the US FDA for approval. Recently, design-of-experiments-based approaches have been explored to rapidly and efficiently achieve this goal of optimized yield with a better understanding of product and process variables that affect a product's critical quality attributes. Here, we present a laboratory-scale model culture where we apply a Plackett-Burman screening design to parallel cultures to study the main effects of 11 process variables. This exercise allowed us to determine the relative importance of these variables and identify the most important factors to be further optimized in order to control both desirable and undesirable glycan profiles. We found engineering changes relating to culture temperature and nonessential amino acid supplementation significantly impacted glycan profiles associated with fucosylation, ß-galactosylation, and sialylation. All of these are important for monoclonal antibody product quality.

Fermentanomics informed amino acid supplementation of an antibody producing mammalian cell culture.

Fermentanomics, or a global understanding of a culture state on the molecular level empowered by advanced techniques like NMR, was employed to show that a model hybridoma culture supplied with glutamine and glucose depletes aspartate, cysteine, methionine, tryptophan, and tyrosine during antibody production. Supplementation with these amino acids prevents depletion and improves culture performance. Furthermore, no significant changes were observed in the distribution of glycans attached to the IgG3 in cultures supplemented with specific amino acids, arguing that this strategy can be implemented without fear of impact on important product quality attributes. In summary, a targeted strategy of quantifying media components and designing a supplementation strategy can improve bioprocess cell cultures when enpowered by fermentanomics tools.