Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western.

Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post-IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high-throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10 µL of sample volume, and consists of minimal sample handling and hands-on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.

Development of a robust cell-based potency assay for a coxsackievirus A21 oncolytic virotherapy.

Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn't represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput. To address these points, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. In this manuscript, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method. This assay development strategy can provide guidance for the development of robust cell-based potency methods for OVs and other infectious viral products.

Improved mRNA affinity chromatography binding capacity and throughput using an oligo-dT immobilized electrospun polymer nanofiber adsorbent.

Increased demand for mRNA-based therapeutics and improved in vitro transcription (IVT) yields have challenged the mRNA purification platform. Hybridization-affinity chromatography with an immobilized oligo-deoxythymidilic acid (oligodT) ligand is often used to capture mRNA through base pairing with the polyadenylated tail. Commercially available oligodT matrices include perfusive cross-linked poly(styrene-divinylbenzene) 50 µm POROS™ chromatography resin beads and convective polymethacrylate CIMmultus® monolithic columns consisting of 2 µm interconnected channels. POROS™ columns may be limited by poor mass transfer for larger mRNAs and slow flowrates, while monoliths can operate at higher flowrates but are limited by modest binding capacity. To enable both high flowrates and binding capacity for mRNA of all lengths, prototype chromatography media was developed by Cytiva using oligodT immobilized electrospun cellulose nanofibers (Fibro™) with a 0.3-0.4 µm pore size. In this work, four polyadenylated mRNAs ranging from ∼1900-4300 nucleotides were used to compare the dynamic binding capacity (DBC) of Fibro™, POROS® and CIMmultus® columns as a function of residence time and binding buffer compositions. Fibro™ improved the DBC ∼2-4-fold higher than CIMmultus® and ∼2-13-fold higher than POROS™ across all residence times, mRNA length, and binding matrix compositions tested. CIMmultus® DBC was least dependent on residence time and mRNA size, while both Fibro™ and POROS™ DBC increased at slower flowrates and with shorter mRNA. Surprisingly, inverse size exclusion (ISE) experiments showed that POROS™ was not limited by diffusion and POROS™ along with CIMmultus® demonstrate higher mRNA permeation however the Fibro™ prototype is not in the final configuration. Lastly, IVT reaction products were subjected to purification and oligodT elution product yield, quality, and purity were consistent across the three matrices investigated. These results highlight the benefits of high DBC and equivalent product profiles offered by the oligodT Fibro™ prototype compared to commercially available oligodT media.

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250 L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5 L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids Using Capillary Western.

A prototype strain of Coxsackievirus A21 (CVA21) is being evaluated as an oncolytic virus immunotherapy. CVA21 preferentially lyses cells that upregulate the expression of intercellular adhesion molecule 1, which includes some types of tumor cells. CVA21 has an icosahedral capsid structure made up of 60 protein subunits encapsidating a viral RNA genome with a particle diameter size of 30 nm. Rapid and robust analytical methods to quantify CVA21 total, empty, and full virus particles are important to support the process development, meet regulatory requirements, and validate manufacturing processes. In this study, we demonstrate the detection of all four CVA21 capsid proteins, VP1, VP2, VP3, and VP4, as well as VP0, a surrogate for empty particles, using in-house-generated antibodies. An automated and quantitative capillary Western blot assay, Simple Western, was developed using these antibodies to quantify CVA21 total particles through VP1, empty particles through VP0, relative ratio of empty to full particles through VP0 and VP4, and the absolute ratio of empty to total particles through VP0 and VP1. Finally, this Simple Western method was used to support CVA21 cell culture and purification process optimization as a high-throughput analytical tool to make rapid process decisions.

Binding of Coxsackievirus A21 procapsids to immobilized glutathione depends on cell culture conditions during infection.

A prototype strain of Coxsackievirus A21 (CVA21) is under clinical evaluation as an oncolytic virus immunotherapy. To improve scalability of the manufacturing process, an affinity chromatography purification method was developed using immobilized glutathione resin that captured infectious CVA21 virions from cell culture harvests with high recovery and impurity clearance. Unexpectedly, the binding of empty CVA21 procapsids depended on production cell culture conditions during infection including temperature, presence of serum in the media, and production cell line. At 37 °C and 2% serum during infection, procapsids flowed-through while infectious virions bound and were recovered at >95% yield in the chromatography elution. However, at sub-physiological temperature or after removal of serum at infection, both procapsids and mature virions bound and co-eluted from the immobilized glutathione ligand. This work may improve the understanding of CVA21 capsid assembly and presents an efficient purification method that may be applied to picornaviruses that interact with intracellular GSH.

Application of cation exchange chromatography in bind and elute and flowthrough mode for the purification of enteroviruses.

Members of the enterovirus genus are promising oncolytic agents. Their morphogenesis involves the generation of both genome-packed infectious capsids and empty capsids. The latter are typically considered as an impurity in need of removal from the final product. The separation of empty and full capsids can take place with centrifugation methods, which are of low throughput and poorly scalable, or scalable chromatographic processes, which typically require peak cutting and a significant trade-off between purity and yield. Here we demonstrate the application of packed bed cation exchange (CEX) column chromatography for the separation of empty capsids from infectious virions for a prototype strain of Coxsackievirus A21. This separation was developed using high throughput chromatography techniques and scaled up as a bind and elute polishing step. The separation was robust over a wide range of operating conditions and returned highly resolved empty and full capsids. The CEX step could be operated in bind and elute or flowthrough mode with similar selectivity and returned yields greater than 70% for full mature virus particles. Similar performance was also achieved using a selection of other bead based CEX chromatography media, demonstrating general applicability of this type of chromatography for Coxsackievirus A21 purification. These results highlight the wide applicability and excellent performance of CEX chromatography for the purification of enteroviruses, such as Coxsackievirus A21.

Reverse-Phase Ultra-Performance Chromatography Method for Oncolytic Coxsackievirus Viral Protein Separation and Empty to Full Capsid Quantification.

Oncolytic virus immunotherapy is emerging as a novel therapeutic approach for cancer treatment. Immunotherapy clinical drug candidate V937 is currently in phase I/II clinical trials and consists of a proprietary formulation of Coxsackievirus A21 (CVA21), which specifically infects and lyses cells with overexpressed ICAM-1 receptors in a range of tumors. Mature Coxsackievirus virions, consisting of four structural virion proteins, (VPs) VP1, VP2, VP3, and VP4, and the RNA genome, are the only viral particles capable of being infectious. In addition to mature virions, empty procapsids with VPs, VP0, VP1, and VP3, and other virus particles are produced in V937 production cell culture. Viral protein VP0 is cleaved into VP2 and VP4 after RNA genome encapsidation to form mature virions. Clearance of viral particles containing VP0, and quantification of viral protein distribution are important in V937 downstream processing. Existing analytical methods for the characterization of viral proteins and particles may lack sensitivity or are low throughput. We developed a sensitive and robust reverse-phase ultra-performance chromatography method to separate, identify, and quantify all five CVA21 VPs. Quantification of virus capsid concentration and empty/full capsid ratio was achieved with good linearity, accuracy, and precision. ClinicalTrials.gov ID: NCT04521621 and NCT04152863.

Rapid Quantification of Monoclonal Antibody Titer in Cell Culture Harvests by Antibody-Induced Z-ELP-E2 Nanoparticle Cross-Linking.

Existing assays for the quantification of monoclonal antibody (mAb) cell culture titer often require expensive instruments or reagents and may be limited by the low-throughput or tedious protocols. Here, we developed a quick and cost-effective alternative assay based on mAb-induced cross-linking with Z-domain-ELP-E2 nanocages functionalized by SpyTag/SpyCatcher conjugation. After mixing mAb samples with a fixed nanoparticle concentration for 10 min, we found that the turbidity, measured by absorbance at 600 nm, exhibited a high-signal-to-background ratio and was proportional to the mAb concentration. A simple logarithmic regression was found to fit ( R2 = 0.99) the turbidity data for mAb concentrations between 100 and 1000 µg/mL. The optimized assay procedure was validated using two industrial mAb cell culture harvests, and a bridging study using Octet biolayer interferometry with Protein A sensors confirmed accurate and reproducible results. The assay procedure can be easily adapted to a high-throughput format for rapid mAb titer screening.

SpyTag/SpyCatcher Functionalization of E2 Nanocages with Stimuli-Responsive Z-ELP Affinity Domains for Tunable Monoclonal Antibody Binding and Precipitation Properties.

E2 nanocages functionalized with Z-domain-elastin-like polypeptide affinity ligands (Z-ELP40) using Sortase A (SrtA) ligation have been shown to be a promising scaffold for purifying monoclonal antibodies (mAbs) based on affinity precipitation. However, the reversible nature of SrtA reaction has been attributed to the low ligation efficiency (<25%) and has significantly limited the practical utility of the technology. Here, we reported an improved conjugation platform using the SpyTag/SpyCatcher pair to form a spontaneous isopeptide bond between SpyTag-E2 and Z-ELP-SpyCatcher fusion proteins of two different ELP chain-lengths. Using this system, E2 ligation efficiencies exceeding 90% were obtained with both 40- and 80-repeat Z-ELP-SpyCatcher fusions. This enabled the production of nanocages fully functionalized with Z-ELP for improved aggregation and mAb binding. Compared to the 50% decorated Z-ELP40-E2 nanocages produced by SrtA ligation, the fully decorated Z-ELP80-Spy-E2 nanocages exhibited a 10 °C lower transition temperature and a 2-fold higher mAb binding capacity. The improved transition property of the longer Z-ELP80 backbone allowed for >90% recovery of Z-ELP80-Spy-E2 nanocages at room temperature using 0.1 M ammonium sulfate after mAb elution. The flexibility of customizing different affinity domains onto the SpyTag-E2 scaffold should expand our ability to purify other non-mAb target proteins based on affinity precipitation.

High-efficiency affinity precipitation of multiple industrial mAbs and Fc-fusion proteins from cell culture harvests using Z-ELP-E2 nanocages.

Affinity precipitation using Z-elastin-like polypeptide-functionalized E2 protein nanocages has been shown to be a promising alternative to Protein A chromatography for monoclonal antibody (mAb) purification. We have previously described a high-yielding, affinity precipitation process capable of rapidly capturing mAbs from cell culture through spontaneous, multivalent crosslinking into large aggregates. To challenge the capabilities of this technology, nanocage affinity precipitation was investigated using four industrial mAbs (mAbs A-D) and one Fc fusion protein (Fc A) with diverse molecular properties. A molar binding ratio of 3:1 Z:mAb was sufficient to precipitate >95% mAb in solution for all molecules evaluated at ambient temperature without added salt. The effect of solution pH on aggregation kinetics was studied using a simplified two-step model to investigate the protein interactions that occur during mAb-nanocage crosslinking and to determine the optimal solution pH for precipitation. After centrifugation, the pelleted mAb-nanocage complex remained insoluble and was capable of being washed at pH ≥ 5 and eluted with at pH < 4 with >90% mAb recovery for all molecules. The four mAbs and one Fc fusion were purified from cell culture using optimal process conditions, and >94% yield and >97% monomer content were obtained. mAb A-D purification resulted in a 99.9% reduction in host cell protein and >99.99% reduction in DNA from the cell culture fluids. Nanocage affinity precipitation was equivalent to or exceeded expected Protein A chromatography performance. This study highlights the benefits of nanoparticle crosslinking for enhanced affinity capture and presents a robust platform that can be applied to any target mAb or Fc-containing proteins with minimal optimization of process parameters.

One-step affinity capture and precipitation for improved purification of an industrial monoclonal antibody using Z-ELP functionalized nanocages.

Protein A chromatography has been identified as a potential bottleneck in the monoclonal antibody production platform, leading to increased interest in non-chromatographic capture technologies. Affinity precipitation using environmentally responsive, Z-domain-elastin-like polypeptide (Z-ELP) fusion proteins has been shown to be a promising alternative. However, elevated temperature and salt concentrations necessary for precipitation resulted in decreased antibody monomer content and reduced purification capacity. To improve upon the existing technology, we reported an enhanced affinity precipitation of antibodies by conjugating Z-ELP to a 25 nm diameter, self-assembled E2 protein nanocage (Z-ELP-E2). The enlarged scale of aggregate formation and IgG-triggered crosslinking through multi-valent binding significantly outperformed traditional Z-ELP-based methods. In the current work, we sought to develop an affinity precipitation process capable of purifying industrial monoclonal antibodies (mAbs) at ambient temperature with minimal added salt. We discovered that the mAb-nanocage complex aggregated within 10 min at room temperature without the addition of salt due to the enhanced multi-valent cross-linking. After precipitating out of solution, the complex remained insoluble under all wash buffers tested, and only resolubilized after a low pH elution. Through optimization of key process steps, the affinity precipitation yield and impurity clearance met or exceeded protein A chromatography performance with 95% yield, 3.7 logs host cell protein reduction, and >5 logs of DNA reduction from mAb cell culture. Because of the operational flexibility afforded by this one-step affinity capture and precipitation process, the Z-ELP-E2 based approach has the potential to be a viable alternative to platform mAb purification.

Ligand-Induced Cross-Linking of Z-Elastin-like Polypeptide-Functionalized E2 Protein Nanoparticles for Enhanced Affinity Precipitation of Antibodies.

Affinity precipitation is an ideal alternative to chromatography for antibody purification because it combines the high selectivity of an affinity ligand with the operational benefits of precipitation. However, the widespread use of elastin-like polypeptide (ELP) capture scaffolds for antibody purification has been hindered by the high salt concentrations and temperatures necessary for efficient ELP aggregation. In this paper, we employed a tandem approach to enhance ELP aggregation by enlarging the dimension of the capturing scaffold and by creating IgG-triggered scaffold cross-linking. This was accomplished by covalently conjugating the Z-domain-ELP (Z-ELP) capturing scaffold to a 25 nm diameter E2 protein nanocage using Sortase A ligation. We demonstrated the isothermal recovery of IgG in the virtual absence of salt due to the significantly increased scaffold dimension and cross-linking from multivalent IgG-E2 interactions. Because IgG cross-linking is reversible at low pH, it may be feasible to achieve a high yielding IgG purification by isothermal phase separation using a simple pH trigger.