Purification of a recombinant oncolytic virus from clarified cell culture media by anion exchange monolith chromatography.

The use of viral vectors for vaccine, gene therapy, and oncolytic virotherapy applications has received increased attention in recent years. Large-scale purification of viral vector-based biotherapeutics still presents a significant technical challenge. Chromatography is the primary tool for the purification of biomolecules in the biotechnology industry; however, the majority of chromatography resins currently available have been designed for the purification of proteins. In contrast, convective interaction media monoliths are chromatographic supports that have been designed and successfully utilized for the purification of large biomolecules, including viruses, viruslike particles, and plasmids. We present a case study on the development of a purification method for recombinant Newcastle disease virus directly from clarified cell culture media using strong anion exchange monolith technology (CIMmultus QA, BIA Separations). Resin screening studies showed at least 10 times higher dynamic binding capacity of CIMmultus QA compared to traditional anion exchange chromatography resins. Design of experiments was used to demonstrate a robust operating window for the purification of recombinant virus directly from clarified cell culture without any further pH or conductivity adjustment of the load material. The capture step was successfully scaled up from 1 mL CIMmultus QA columns to the 8 L column scale and achieved a greater than 30-fold reduction in process volume. Compared to the load material, total host cell proteins were reduced by more than 76%, and residual host cell DNA by more than 57% in the elution pool, respectively. Direct loading of clarified cell culture onto a high-capacity monolith stationary phase makes convective flow chromatography an attractive alternative to centrifugation or TFF-based virus purification procedures.

Identification of compendial nonionic detergents for the replacement of Triton X-100 in bioprocessing.

We have systematically investigated six compendial nonionic detergents as potential replacements for Triton ×-100 in bioprocessing applications. Use of compendial raw materials in cGMP bioprocessing is advantageous for a variety of reasons including material specifications developed to meet stringent pharmaceutical product quality requirements, regulatory familiarity and comfort, and availability from vendors experienced supplying the biopharmaceutical industry. We first examine material properties of the detergents themselves including melting point and viscosity. Process performance and product contact in real-world bioprocess applications are then investigated. Lastly, we test the detergents in virus inactivation (VI) experiments with recombinant proteins and adeno-associated virus. Two of the detergents tested, PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides, showed favorable properties that make them attractive for use as potential Triton X-100 replacements. Process performance testing indicated negligible impact of the detergents on product yield, purity, and activity compared to a control with no detergent. Importantly, both PEG 9 Lauryl Ether and PEG 6 Caprylic/Capric Glycerides demonstrated very fast VI kinetics with complete inactivation of XMuLV observed in less than 1 min at a target 1% detergent concentration. Potential advantages and disadvantages of both candidate detergents for use in cGMP bioprocessing are summarized and discussed.

Safety risk management for low molecular weight process-related impurities in monoclonal antibody therapeutics: Categorization, risk assessment, testing strategy, and process development with leveraging clearance potential.

Process-related impurities (PRIs) derived from manufacturing process should be minimized in final drug product. ICH Q3A provides a regulatory road map for PRIs but excludes biologic drugs like monoclonal antibodies (mAbs) that contain biological PRIs (e.g. host cell proteins and DNA) and low molecular weight (LMW) PRIs (e.g., fermentation media components and downstream chemical reagents). Risks from the former PRIs are typically addressed by routine tests to meet regulatory expectations, while a similar routine-testing strategy is unrealistic and unnecessary for LMW PRIs, and thus a risk-assessment-guided testing strategy is often utilized. In this report, we discuss a safety risk management strategy including categorization, risk assessment, testing strategy, and its integrations with other CMC development activities, as well as downstream clearance potentials. The clearance data from 28 mAbs successfully addressed safety concerns but did not fully reveal the process clearance potentials. Therefore, we carried out studies with 13 commonly seen LMW PRIs in a typical downstream process for mAbs. Generally, Protein A chromatography and cation exchange chromatography operating in bind-and-elute mode showed excellent clearances with greater than 1,000- and 100-fold clearance, respectively. The diafiltration step had better clearance (greater than 100-fold) for the positively and neutrally charged LMW PRIs than for the negatively charged or hydrophobic PRIs. We propose that a typical mAb downstream process provides an overall clearance of 5,000-fold. Additionally, the determined sieving coefficients will facilitate diafiltration process development. This report helps establish effective safety risk management and downstream process design with robust clearance for LMW PRIs.

Refolding and purification of cGMP-grade recombinant human neurturin from Escherichia coli inclusion bodies.

Neurturin is a potent neurotrophic factor that has been investigated as a potential therapeutic agent for the treatment of neurodegenerative diseases, including Parkinson's disease, and, more recently, for the treatment of type II diabetes. However, purification of neurturin for clinical applications has been hampered by its low solubility in aqueous solutions. Here we describe the development of a scalable manufacturing process for recombinant neurturin from E. coli. inclusion bodies. Neurturin was refolded from solubilized inclusion bodies by fed-batch dilution refolding with a titer of 90 mg per liter refold and a refold yield of 89%. A two-step purification process using cation exchange and hydrophobic interaction chromatography, followed by formulation using tangential flow filtration resulted in an overall process yield of about 56 mg purified neurturin per liter refold. Solubility of neurturin during the purification process was maintained by the addition of 15% (w/v) glycerol to all buffers. For clinical applications and parenteral administration glycerol was replaced by 15% (w/v) sulfobutyl ether-beta-cyclodextrin (i.e. Captisol) in the drug substance formulation buffer. The final purified product had low or undetectable levels of product-related impurities and concentrations of process-related contaminants such as host cell proteins, host cell DNA, endotoxins and Triton X-100 were reduced more than 10,000-fold or below the limit of detection. Bioactivity of purified recombinant neurturin was demonstrated in a cell-based assay by activation of the MAPK signaling pathway.

Developing an Anion Exchange Chromatography Assay for Determining Empty and Full Capsid Contents in AAV6.2.

Adeno-associated virus (AAV) vectors are clinically proven gene delivery vehicles that are attracting an increasing amount of attention. Non-genome-containing empty AAV capsids are by-products during AAV production that have been reported to potentially impact AAV product safety and efficacy. Therefore, the presence and amount of empty AAV capsids need to be characterized during process development. Multiple methods have been reported to characterize empty AAV capsid levels, including transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), charge detection mass spectrometry (CDMS), UV spectrophotometry, and measuring capsid and genome copies by ELISA and qPCR. However, these methods may lack adequate accuracy and precision or be challenging to transfer to a quality control (QC) lab due to the difficulty of implementation. In this study, we used AAV serotype 6.2 (AAV6.2) as an example to show the development of a QC-friendly anion exchange chromatography (AEX) assay for the determination of empty and full capsid percentages. The reported assay requires several microliters of material with a minimum titer of 5 × 1011 vg/mL, and it can detect the presence of as low as 2.9% empty capsids in AAV6.2 samples. Additionally, the method is easy to deploy, can be automated, and has been successfully implemented to support testing of various in-process and release samples.

Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli.

Secretion of heterologous proteins into Escherichia coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings, and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxinH35L (ATH35L) was selected as an antigen for vaccine development against Staphylococcus aureus infections. It was successfully secreted into culture medium of E. coli by using bacterial signal peptides linked to the N-terminus of the protein. In order to improve the level of secreted ATH35L, we designed a series of novel signal peptides by swapping individual domains of modifying dsbA and pelB signal peptides and tested them in a fed-batch fermentation process. The data showed that some of the modified signal peptides improved the secretion efficiency of ATH35L compared with E. coli signal peptides from dsbA, pelB and phoA proteins. Indeed, one of the novel signal peptides improved the yield of secreted ATH35L by 3.5-fold in a fed-batch fermentation process and at the same time maintained processing at the expected site for signal peptide cleavage. Potentially, these new novel signal peptides can be used to improve the secretion efficiency of other heterologous proteins in E. coli. Furthermore, analysis of the synthetic signal peptide amino acid sequences provides some insight into the sequence features within the signal peptide that influence secretion efficiency.

Development and scale-up of a commercial fed batch refolding process for an anti-CD22 two chain immunotoxin.

We describe the development and scale-up of a novel two chain immunotoxin refolding process. This work provides a case study comparing a clinical manufacturing process and the commercial process developed to replace it. While the clinical process produced high quality material, it suffered from low yield and high yield variability. A systematic approach to process development and understanding led to a number of improvements that were implemented in the commercial process. These include a shorter inclusion body recovery process, limiting the formation of an undesired deamidated species and the implementation of fed batch dilution refolding for increased refold titers. The use of a combination of urea, arginine and DTT for capture column cleaning restored the binding capacity of the capture step column and resulted in consistent capture step yields compared to the clinical process. Scalability is shown with data from 250 L and 950 L scale refolding processes. Compared to the clinical process it replaces, the commercial process demonstrated a greater than fivefold improvement in volumetric productivity at the 950 L refolding scale.

Congenital exposure to Plasmodium falciparum antigens: prevalence and antigenic specificity of in utero-produced antimalarial immunoglobulin M antibodies.

Congenital Plasmodium falciparum malaria in newborns is uncommon in sub-Saharan Africa. A significant number of infants, however, become infected or exposed to malarial antigens either in utero or at delivery and have the potential to produce antimalarial antibodies and memory cells before their first natural infection. In Yaounde, Cameroon, parasite-specific immunoglobulin M (IgM) was detected in 14% of cord blood samples. The IgM antibodies reacted with a wide range of asexual-stage antigens, with each newborn having its own unique pattern of IgM reactivity. PCR-based detection and genotyping of cord blood parasites found that the prevalence, total number of parasite genotypes, and complexity of infection were higher in newborns who had produced antimalarial IgM than those who had not. Maternal placental malaria and anemia were associated with the production of P. falciparum-specific IgM by the fetus. The effect of early immune priming on acquisition of immunity by infants is unknown and merits further investigation, since a significant proportion of Cameroonian newborns developed a humoral response to malaria before birth.