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Bispecific antibodies expressed and assembled from a single upstream culture require the correct balance and pairing of four different heavy and light chains (HC and LC). The increased potential for chain-mispaired species challenges the downstream purification of this new format. While clearance of HC-mispaired species, including homodimers and half-antibodies, has been assessed, removal of LC mispairs requires a more stringent approach. Here, we report two case studies in which separation is achieved, as well as the structural basis of these separations: (A) In the first case, a main species with a positively charged patch in the correctly formed variable fragment (Fv) is disrupted when paired with the wrong LC. This LC-mispaired variant binds more weakly to a cation exchange resin and can be washed off in a chromatography step. (B) A second molecule whose LC mispair introduces a negative-charge patch and hydrophobic patch in close proximity, presenting increased binding to a multimodal anion exchange resin. This LC-mispaired variant can be retained on the column under conditions in which the bispecific is recovered. In both case studies, the molecular structural analysis by protein surface properties models correlated well with the chromatography experiments. The comprehensive interpretation of experimental and computational results has provided a better understanding of strategies and potential applications for predicting the downstream purification of complex molecules.

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H5N1 highly pathogenic avian influenza virus (HPAIV) infections pose a significant threat to human health, with a mortality rate of around 50%. Limited global approval of H5N1 HPAIV vaccines, excluding China, prompted the need to address safety concerns related to MDCK cell tumorigenicity. Our objective was to improve vaccine safety by minimizing residual DNA and host cell protein (HCP). We developed a downstream processing method for the cell-based H5N1 HPAIV vaccine, employing CaptoTM Core 700, a multimodal resin, for polishing. Hydrophobic-interaction chromatography (HIC) with polypropylene glycol as a functional group facilitated the reversible binding of virus particles for capture. Following the two-step chromatographic process, virus recovery reached 68.16%. Additionally, HCP and DNA levels were reduced to 2112.60 ng/mL and 6.4 ng/mL, respectively. Western blot, high-performance liquid chromatography (HPLC), and transmission electron microscopy (TEM) confirmed the presence of the required antigen with a spherical shape and appropriate particle size. Overall, our presented two-step downstream process demonstrates potential as an efficient and cost-effective platform technology for cell-based influenza (H5N1 HPAIV) vaccines.

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Real-time in-line broad protein content monitoring in biomanufacturing downstream unit operations enables the ability to optimize and afford consistent protein recovery. Protein determination from 2 to 400 mg/mL is demonstrated herein via real-time dual-wavelength LED photometric sensor configured at 280 and 310 nm. The figures of merit of this approach include measurement accuracy within the common acceptance criteria of 100 % ± 5 with negligible bias across the linear dynamic ranges. This work expands the utility of an LED based photometric sensor for biopharmaceutical process analytical technology (PAT) applications. It is also congruent with process digitalization and automation industry 4.0 concepts underpinned by Quality by Design (QbD) principles.

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During the manufacturing of therapeutic antibodies, effective Protein A chromatography as initial column step is crucial to simplify the remaining purification effort for subsequent polishing steps. This is particularly relevant for molecules with high impurity content so that desired product purity can be attained. The present study demonstrates beneficial effects on impurity removal when applying kosmotropic salts, e.g., sodium sulfate or sodium chloride, in the elution phase. Initially, a screen using negative linear pH gradient elution evaluated the impact of the kosmotropic salts in comparison to no additive and chaotropic urea using three mAbs and three common resins. Retaining acceptable yield, the kosmotropic salts improved resolution of monomer and impurities and reduced the contents of process-related host cell proteins and DNA as well as of product-related low and high molecular weight forms, despite some resin- and mAb-dependent variations. Moreover, a decrease in hydrolytic activity measured by a new assay for polysorbase activity was observed. In contrast, urea was hardly effective. The findings served to establish optimized step elution conditions with 0.25 M of sodium sulfate for a challenging mAb with complex format (bispecific 2 + 1 CrossMab) displaying high relative hydrophobicity and impurity levels. With yield and purity both in the range of 90 %, the contents of all impurity components were reduced, e.g., low molecular weight forms by two-fold and polysorbase activity by four-fold. The study indicates the potential of kosmotropic salts to establish efficient and comprehensive impurity separation by Protein A for facilitated downstream processing and economic manufacturing of complex antibodies.

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High-throughput process development has become a standard practice in the biopharmaceutical industry to enable time, cost, and material savings. In downstream biopharmaceutical process development, miniaturized, parallelized chromatography columns, known as RoboColumn, have become the standard for process development, as RoboColumn have shown generally comparable performance to bench and manufacturing scale columns. However, RoboColumn have yet to be widely implemented in process validation and characterization, where many multifactor experiments are typically executed, and there is a strong value proposition for performing high-throughput experiments. The hesitancy to utilize RoboColumn in process validation arises from scale differences that result in exacerbated peak broadening at RoboColumn scale relative to traditional bench or manufacturing scales. Thus, to support reliable application of RoboColumn in process validation, the present study provides a comprehensive investigation to understand how scale differences affect chromatographic performance by comparing RoboColumn, bench, and manufacturing scales using seven different production processes covering three different antibody formats, five different resin types, and three chromatographic modes of operation. RoboColumn chromatographic performance was compared at target and off-target conditions to emulate scale-down model qualification and multifactor studies, respectively. RoboColumn demonstrated good comparability at both target and off-target process conditions. To further demonstrate an understanding of comparability, a study was performed to show a rare case in which product quality offsets may occur as a result RoboColumn scale differences. By showing scale comparability and an understanding of potential offsets, this work demonstrates that RoboColumn can be used in any stage of process development, including process validation and characterization.

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Precipitation can be used for the removal of impurities early in the downstream purification process of biologics, with the soluble product remaining in the filtrate through microfiltration. The objective of this study was to examine the use of polyallylamine (PAA) precipitation to increase the purity of product via higher host cell protein removal to enhance polysorbate excipient stability to enable a longer shelf life. Experiments were performed using three monoclonal antibodies (mAbs) with different properties of isoelectric point and IgG subclass. High throughput workflows were established to quickly screen precipitation conditions as a function of pH, conductivity and PAA concentrations. Process analytical tools (PATs) were used to evaluate the size distribution of particles and inform the optimal precipitation condition. Minimal pressure increase was observed during depth filtration of the precipitates. The precipitation was scaled up to 20L size and the extensive characterization of precipitated samples after protein A chromatography showed >75% reduction of host cell protein (HCP) concentrations (by ELISA), >90% reduction of number of HCP species (by mass spectrometry), and >99.8% reduction of DNA. The stability of polysorbate containing formulation buffers for all three mAbs in the protein A purified intermediates was improved at least 25% after PAA precipitation. Mass spectrometry was used to obtain additional understanding of the interaction between PAA and HCPs with different properties. Minimal impact on product quality and <5% yield loss after precipitation were observed while the residual PAA was <9 ppm. These results expand the toolbox in downstream purification to solve HCP clearance issues for programs with purification challenges, while also providing important insights into the integration of precipitation-depth filtration and the current platform process for the purification of biologics.

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Nonwoven membranes are highly engineered fibrous materials that can be manufactured on a large scale from a wide range of different polymers, and their surfaces can be modified using a large variety of different chemistries and ligands. The fiber diameters, surface areas, pore sizes, total porosities, and thicknesses of the nonwoven mats can be carefully controlled, providing many opportunities for creative approaches for the development of novel membranes with unique properties to meet the needs of the future of downstream processing. Fibrous membranes are already finding use in ultrafiltration, microfiltration, depth filtration, and, more recently, in membrane chromatography for product capture and impurity removal. This article summarizes the various methods of manufacturing nonwoven fabrics, and the many methods available for the modification of the fiber surfaces. It also reviews recent studies focused on the use of nonwoven fabric devices in membrane chromatography and provides some perspectives on the challenges that need to be overcome to increase binding capacities, decrease residence times, and reduce pressure drops so that eventually they can replace resin column chromatography in downstream process operations.

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The formation of inclusion bodies (IBs) during expression of recombinant therapeutic proteins using E. coli is a significant hurdle in producing high-quality, safe, and efficacious medicines. The improved understanding of the structure-function relationship of the IBs has resulted in the development of novel biotechnologies that have streamlined the isolation, solubilization, refolding, and purification of the active functional proteins from the bacterial IBs. Together, this overall effort promises to radically improve the scope of experimental biology of therapeutic protein production and expand new prospects in IBs usage. Notably, the IBs are increasingly used for applications in more pristine areas such as drug delivery and material sciences. In this review, we intend to provide a comprehensive picture of the bio-processing of bacterial IBs, including assessing critical gaps that still need to be addressed and potential solutions to overcome them. We expect this review to be a useful resource for those working in the area of protein refolding and therapeutic protein production.

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Immunoglobulins of complex formats possess great potential for increased biopharmaceutical efficacy. However, challenges arise during their purification as the removal of numerous product-related impurities typically requires several expensive chromatographic steps. Additionally, many complex antibody formats have a high hydrophobicity which impairs the use of conventional mixed mode chromatography. In the present study, both of these challenges were addressed through the development of an innovative mixed mode resin with 2-amino-4methylpentanoic acid ligands that combines weak cation exchange with moderate hydrophobic interactions. Supported by high throughput partition coefficient screens for identification of preferable pH and salt concentration ranges in bind and elute mode, this mixed mode resin successfully demonstrated efficient impurity separation from an extremely hydrophobic bispecific antibody with a single unit operation. High purity (>97%) was obtained as a result of significant reduction of product-related impurities as well as process-related host cell proteins (>3 log scale), while maintaining satisfactory recovery (70%). This also supports that highly hydrophobic antibody formats can be efficiently purified using a resin with moderate hydrophobic characteristics. Studies involving additional antibodies possessing different formats and a wide range of hydrophobicity confirmed the broad applicability of the new resin. In view of its high selectivity and robust operating ranges, as well as the elimination of the need for an additional column step, the novel resin enables simplified downstream processing and economic manufacturing of complex antibody formats.

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The effective fractionation of structural components of abundantly available lignocellulosic biomass is essential to unlock its full biorefinery potential. In this study, the feasibility of humic acid on the pretreatment of Kentucky bluegrass biomass in alkaline condition was assessed to separate 70.1% lignin and hydrolyzable biocomponents. The humic acid-assisted delignification followed by enzymatic saccharification yielded 0.55 g/g of reducing sugars from 7.5% (w/v) pretreated biomass loading and 16 FPU/g of cellulase. Yeast fermentation of the biomass hydrolysate produced 76.6% (w/w) ethanol, which was subsequently separated and concentrated using direct contact membrane distillation. The hydrophobic microporous flat-sheet membrane housed in a rectangular-shaped crossflow module and counter-current mode of flow of the feed (hot) and distillate (cold) streams yielded a flux of 11.6 kg EtOH/m2/24 h. A modular, compact, flexible, and eco-friendly membrane-integrated hybrid approach is used for the first time to effectively valorize Kentucky bluegrass biomass for sustainable production of biofuel.

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Detergent-mediated virus inactivation (VI) provides a valuable orthogonal strategy for viral clearance in mammalian processes, in particular for next-generation continuous manufacturing. Furthermore, there exists an industry-wide need to replace the conventionally employed detergent Triton X-100 with eco-friendly alternatives. However, given Triton X-100 has been the gold standard for VI due its minimal impact on protein stability and high inactivation efficacy, inactivation by other eco-friendly detergents and its impact on protein stability is not well understood. In this study, the sugar-based detergent commonly used in membrane protein purification, n-dodecyl-ß- d-maltoside was found to be a promising alternative for VI. We investigated a panel of detergents to compare the relative VI efficacy, impact on therapeutic quality attributes, and clearance of the VI agent and other impurities through subsequent chromatographic steps. Detergent-mediated inactivation and protein stability showed comparable trends to low pH inactivation. Using experimental and modeling data, we found detergent-mediated product aggregation and its kinetics to be driven by extrinsic factors such as detergent and protein concentration. Detergent-mediated aggregation was also impacted by an initial aggregation level as well as intrinsic factors such as the protein sequence and detergent hydrophobicity, and critical micelle concentration. Knowledge gained here on factors driving product stability and VI provides valuable insight to design, standardize, and optimize conditions (concentration and duration of inactivation) for screening of detergent-mediated VI.

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Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality. The downstream processing of this class of antibodies is therefore of crucial importance in ensuring that these products can be obtained with high purity and yield. Due to the various fundamental structural similarities between bsAbs and monoclonal antibodies (mAbs), many of the current bsAb downstream purification methodologies are based on the established purification processes of mAbs, where affinity, charge, size, hydrophobicity and mixed-mode-based purification are frequently employed. Nevertheless, the downstream processing of bsAbs presents a unique set of challenges due to the presence of bsAb-specific byproducts, such as mispaired products, undesired fragments and higher levels of aggregates, that are otherwise absent or present in lower levels in mAb cell culture supernatants, thus often requiring the design of additional purification strategies in order to obtain products of high purity. Here, we outline the current major purification methods of bsAbs, highlighting the corresponding solutions that have been proposed to circumvent the unique challenges presented by this class of antibodies, including differential affinity chromatography, sequential affinity chromatography and the use of salt additives and pH gradients or multistep elutions in various modes of purification. Finally, a perspective towards future process development is offered.

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The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and is being evaluated. The development of an efficient downstream purification process (DSP) enables the vaccine to be advanced to clinical trials. The DSP must eliminate impurities, either process- or product-related, to yield a sufficient product with high purity, potency and quality. To acquire critical information on process restrictions and qualities, the application of in-line monitoring is vital and should significantly impact the process yield, product quality and economy of the entire process. Here, we describe an in-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining the concentrations of metabolites, nutrients and a host cell protein using the automatic chemistry analyzer, Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adopting this technique during the purification process improves the process yield and the product quality and enhances the economy of the entire downstream process for biotechnology and bio pharmaceutical products.

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Endotoxins are the major contributors to the pyrogenic response caused by contaminated pharmaceutical products, formulation ingredients, and medical devices. Recombinant biopharmaceutical products are manufactured using living organisms, including Gram-negative bacteria. Upon the death of a Gram-negative bacterium, endotoxins (also known as lipopolysaccharides) in the outer cell membrane are released into the lysate where they can interact with and form bonds with biomolecules, including target therapeutic compounds. Endotoxin contamination of biologic products may also occur through water, raw materials such as excipients, media, additives, sera, equipment, containers closure systems, and expression systems used in manufacturing. The manufacturing process is, therefore, in critical need of methods to reduce and remove endotoxins by monitoring raw materials and in-process intermediates at critical steps, in addition to final drug product release testing. This review paper highlights a discussion on three major topics about endotoxin detection techniques, upstream processes for the production of therapeutic molecules, and downstream processes to eliminate endotoxins during product purification. Finally, we have evaluated the effectiveness of endotoxin removal processes from a perspective of high purity and low cost.

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Recombinant human myelin basic protein (rhMBP) produced in the milk of transgenic cows was found exclusively associated with milk caseins. This hindered its direct determination without extensive sample pretreatment. Here, a label-free potentiometric immunosensor was developed and validated for the determination of rhMBP. An ion flux was generated under zero-current based on surface blocking of the polymeric membrane ion-selective electrode by anti-hMBP antibody and tetrabutylammonium bromide as a marker ion. The immunosensor was successfully employed in the quantitative determination of hMBP in the range of 0.10-20.00 µg/mL with a limit of detection of 50.00 ng/mL. The applicability of the passive ion flux immunosensor for determination of target analyte in complex matrices was investigated. Downstream purification of rhMBP from the milk of transgenic cows was achieved using cation exchange chromatography, immobilized metal affinity chromatography, and immunoaffinity chromatography. The specificity of the immunosensor along with matrix effect of milk proteins were demonstrated. Results obtained using the rhMBP immunosensor were further cross-validated using an orthogonal testing protocol assembled of RP-HPLC and SE-HPLC. It should be noted that the proposed ion flux immunosensor provided a feasible and specific tool for monitoring rhMBP concentration/purity, immunogenic activity, and stability. Such approach provides an attractive economic alternative to sophisticated biosensors required for in-process quality control of biopharmaceutical products.

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Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterized by GAA deficiency in the cell lysosomes (Raben et al., Curr Mol Med. 2002; 2:145-166). The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines (GAA, mAb, and Null) capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co-eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:666-676, 2017.

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Anion exchange (AEX) is a common downstream purification operation for biotechnology products manufactured in cell culture such as therapeutic monoclonal antibodies (mAbs) and Fc-fusion proteins. We present a head-to-head comparison of the viral clearance efficiency of AEX adsorbers and column chromatography using the same process fluids and comparable run conditions. We also present overall trends from the CDER viral clearance database. In our comparison of multiple brands of resins and adsorbers, clearance of three model viruses (PPV, X-MuLV, and PR772) was largely comparable, with some exceptions which may reflect run conditions that had not been optimized on a resin/membrane specific basis.

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