Publications
2021
- How does chromatography affect RNA stability?
- Can HPLC be used for RNA stability assessment?
- Is precipitation a good approach for small-scale RNA purification?
- How do HPLC, bioanalyser, AGE, and other methods compare?
Thermal and freeze-thaw stability of a model RNA construct purified by various methods is evaluated by a range of analytical methods. Results indicate a stark contrast in stability, and discuss applicability of analytical methods for stability assessment.
Matevž Korenč, Nina Mencin, Jasmina Puc, Janja Skok, Kristina Šprinzar Nemec, Anže Martinčič Celjar, Pete Gagnon, Aleš Štrancar, Rok Sekirnik
Cell & Gene Therapy Insights 2021; 7(9), 1207–1216
Abstract:
One of the major challenges of mRNA based vaccines has been their requirement for distribution and storage at extremely low temperatures, indicating that exposure of mRNA to suboptimal physico-chemical conditions can result in degradation and loss of potency; it is unclear whether this is due to instability of mRNA drug substance, or LNP-encapsulated mRNA, or both. In this study we compare the stability of model mRNA drug substance (eGFP, 995 nt) prepared by affinity chromatography with the stability of mRNA purified by precipitation. We show that both purification methods lead to highly pure mRNA drug substance, however, mRNA purified by chromatography remains stable for 28 days at 37°C, whereas mRNA purified by precipitation is subject to significant degradation under the same storage conditions. We conclude that chromatography eliminates elements and/or conditions with adverse impact on the quality of mRNA to a greater extent than precipitation method and that choosing appropriate purification strategy is crucial not only to achieve target purity but also to obtain a stable product with retained integrity.
Pete Gagnon, Blaž Goričar, Sara Drmota Prebil, Hana Jug, Maja Leskovec, Aleš Štrancar
BioProcess J, 2021; 20
Abstract:
Separation of empty and full AAV8 capsids was achieved during their elution from a weak anion exchanger with an ascending pH gradient at low conductivity. Experimental data suggest elution was mediated by loss of positive charge from the exchanger. The method produced a full capsid peak with fewer empty capsids than elution of a strong anion exchanger with a salt gradient. Elution of the weak exchanger by sodium chloride gradients or by pH gradients in the presence of sodium chloride gave inferior separation performance. Pre-elution of empty capsids with a pH step allowed full capsids to be eluted by salt without compromising separation. Loading at intermediate pH prevented empty capsid binding and enabled step elution of full capsids in a physiological buffer environment.
Nerea Zabaleta, Wenlong Dai, Urja Bhatt, Cécile Hérate, Pauline Maisonnasse, Jessica A. Chichester, Julio Sanmiguel, Reynette Estelien, Kristofer T. Michalson, Cheikh Diop, Dawid Maciorowski, Nathalie Dereuddre Bosquet, Mariangela Cavarelli, Anne-Sophie Gallouët, Thibaut Naninck, Nidhal Kahlaoui, Julien Lemaitre, Wenbin Qi, Elissa Hudspeth, Allison Cucalon, Cecilia D. Dyer, M. Betina Pampena, James J. Knox, Regina C. LaRocque, Richelle C. Charles, Dan Li, Maya Kim, Abigail Sheridan, Nadia Storm, Rebecca I. Johnson, Jared Feldman, Blake M. Hauser, Vanessa Contreras, Romain Marlin, Raphaël Ho Tsong Fang, Catherine Chapon, Sylvie van der Werf, Eric Zinn, Aisling Ryan, Dione T. Kobayashi, Ruchi Chauhan, Marion McGlynn, Edward T. Ryan, Aaron G. Schmidt, Brian Price, Anna Honko, Anthony Griffiths, Sam Yaghmour, Robert Hodge, Michael R. Betts, Mason W. Freeman, James M. Wilson, Roger Le Grand, Luk H. Vandenberghe
Cell Host & Microbe, Volume 29, Issue 9, 2021, Pages 1437-1453
Abstract:
The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and can be deployed easily. Here, AAVCOVID-1, an adeno-associated viral (AAV), spike-gene-based vaccine candidate demonstrates potent immunogenicity in mouse and non-human primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. The AAVCOVID vector has no relevant pre-existing immunity in humans and does not elicit cross-reactivity to common AAVs used in gene therapy. Vector genome persistence and expression wanes following injection. The single low-dose requirement, high-yield manufacturability, and 1-month stability for storage at room temperature may make this technology well suited to support effective immunization campaigns for emerging pathogens on a global scale.
In this study, the AAVCOVID candidates were produced at larger scale via standard AAV production processes by Novartis Gene Therapies, following their stablished protocol with only minimal modifications to adjust to the AAVrh32.33 technology. Briefly, AC1 and AC3 were produced via three plasmid transfection. After cell lysis and lysate clarification, tangential flow filtration (TFF) was conducted to achieve volume reduction. The TFF retentate was next enriched for AAV particles on a cation exchange chromatography column (Sartorius BIA Separations). The eluate was concentrated, and buffer exchanged through an additional TFF step, before CsCl ultracentrifugation to separate genome containing versus empty AAV particles. Finally, formulation was achieved through TFF before bulk drug substance was filtered.
Rafael G. Ferreira, Neal F. Gordon, Rick Stock, Demetri Petrides
Processes 2021, 9(8), 1430
Abstract:
The COVID-19 pandemic has motivated the rapid development of numerous vaccines that have proven effective against SARS-CoV-2. Several of these successful vaccines are based on the adenoviral vector platform. The mass manufacturing of these vaccines poses great challenges, especially in the context of a pandemic where extremely large quantities must be produced quickly at an affordable cost. In this work, two baseline processes for the production of a COVID-19 adenoviral vector vaccine, B1 and P1, were designed, simulated and economically evaluated with the aid of the software SuperPro Designer.
In the purification process the retentate from the ultrafiltration step was subjected to anion-exchange chromatography (AEX), operating in capture mode, to remove protein and DNA impurities. A strong anion-exchange column with a monolithic structure (CIMmultus QA from Sartorius BIA Separations) is used in this step due to its high binding capacity for VPs.
Both processes were sized to produce 400 M/yr vaccine doses. The media and facility-dependent expenses were found to be the main contributors to the operating cost. The results indicate that adenoviral vector vaccines can be practically manufactured at large scale and low cost.
Tomas Kostelec, Rok Sekirnik, Anže Martinčič Celjar, Kristina Šprinzar Nemec, Andreja Gramc Livk, Pete Gagnon, Aleš Štrancar
BioProcess International, June 2021
Abstract:
COVID-19 has focused a spotlight on the ability of mRNA technology to accelerate vaccine development and approval. That same technology can hasten development and approval of other therapeutic classes, including cancer immunotherapy, protein replacement, and gene therapy. Fulfilling those opportunities imposes significant challenges on process developers and manufacturers to improve existing processes. Scale-up to produce millions of doses (tens of kilograms) compounds those challenges. Furthermore, every step of the journey requires high-performance analytical methods, to ensure patient safety and maximize productivity.
Pete Gagnon, Maja Leskovec, Sara Drmota Prebil, Rok Žigon, Maja Štokelj, Andrej Raspor, Sebastijan Peljhan, Ales Štrancar
Journal of Chromatography A, 2021
Abstract:
Separation of empty and full adeno-associated virus capsids by multimodal metal affinity chromatography was investigated using a positively charged metal affinity ligand. A subpopulation of empty capsids eluted first, followed by full capsids, and later by more empty capsids and debris. Empty and full capsid composition of chromatography fractions was evaluated by cesium chloride density gradient centrifugation followed by stratigraphic flow analysis of the centrifuge tube contents, monitored by intrinsic fluorescence. Columns charged with barium, calcium, magnesium, zinc, manganese, and ferric ions gave similar results with respect to capsid separation. Charging with cupric ions maintained resolution between early-eluting empty capsids and full capsids but caused them to elute at lower conductivity. Empty and full capsids were fractionated with Tris-borate gradients, sodium chloride gradients, and magnesium chloride gradients. Recovery of full serotype 9 capsids was 100% with complete elimination of empty capsids. All metal ions bound contaminant subsets that required sodium hydroxide for removal. Columns charged with ferric iron and manganese bound more contaminants than all other metals. Columns charged with calcium, magnesium, barium, and copper bound the least. Contaminant binding on zinc-charged columns was intermediate between the two groups.
Artaches A. Kazarian, Wesley Barnhart, Iain D.G. Campuzano, Jeremy Cabrera, Theodore Fitch, Jason Long, Kelvin Sham, Bin Wu, Justin K. Murray
Journal of Chromatography A,Volume 1634, 2020
Abstract:
The current study investigates a method for purification of the G-quadruplex secondary structure, naturally formed by a guanine-rich 21-mer oligonucleotide strand using a monolithic convective interaction media quaternary amine (CIM-QA) column under ion-exchange conditions. The monolithic support was initially evaluated on a preparative scale against a highly efficient TSKgel SuperQ-5PW ion-exchange support designed for oligonucleotide purification. The CIM analogue demonstrated clear advantages over the particle based support on the basis of rapid separation times, while also affording high purity of the G-quadruplex. Various parameters were investigated including the type of mobile phase anion, cation, pH and injection load to induce and control quadruplex formation, as well as enhance chromatographic separation and final purity. Potassium afforded the most prominent quadruplex formation, yet sodium allowed for the highest resolution and purity to be achieved with a 30 mg injection on an 8 ml CIM-QA monolithic column. This method was applied to purify in excess of 300 mg of the quadruplex, with excellent retention time precision of under 1% RSD. Native mass spectrometry was utilized to confirm the identity of the intact G-quadruplex under non denaturing conditions, while ion-pairing reversed-phase methods confirmed the presence of the single stranded oligonucleotide in high purity (92%) under denaturing conditions.
The key advantage of the purification method enables isolation of the G-quadruplex in its native state on a milli-gram scale, allowing structural characterization to further our knowledge of its role and function. The G-quadruplex can also be subsequently denaturated at elevated temperature causing single strand formation if additional reactions are to be pursued, such as annealing to form a duplex, and evaluation in in vitro or in vivo studies.
Michael Winkler, Mikhail Goldfarb, Shaojie Weng, Jeff Smith, Susan Wexelblat, John Li, Alejandro Becerra, Sandra Bezemer, Kevin Sleijpen, Aleš Štrancar, Sara Primec, Romina Zabar, April Schubert, Akunna Iheanacho, and David Cetlin
BioProcess International, April 2021
Abstract
Over the past decade, adenoassociated virus (AAV) vectors have become established as leading gene-delivery vehicles. In 2017, the pipeline for gene therapies included 351 drugs in clinical trials and 316 in preclinical development. As those candidates advance, significant efforts are being made in process development and manufacturing for viral vectors, with the overall goal of reducing process impurities while maintaining the highest possible process yield.
Sartorius BIA Separations has developed and commercialized CIMmultus QA monoliths, which have been cited in several AAV downstream processes for their ability to separate empty and full virus particles effectively. Monolithic supports represent a unique type of stationary phase for liquid chromatography, bioconversion, and solid-phase synthesis. Aside from increased processing speed, monolithic flow-through pores (channels) also provide easy access for large molecules, which supports both purification and depletion of nanoparticles such as plasmid DNA (pDNA) molecules and AAV particles.
Sebastijan Peljhan, Maja Štokelj, Sara Drmota Prebil, Pete Gagnon and Aleš Štrancar
Cell & Gene Therapy Insights, March 2021
Abstract:
Ultracentrifugation (UC) is a well-known technique for fractionating adeno-associated virus (AAV) capsids according to their density, which is mainly a function of their encapsidated DNA mass. Empty capsids represent the lowest density subpopulation. Full capsids represent the highest density subpopulation, sometimes accompanied by partially full capsids of intermediate density. Fractions can be collected after sedimentation for analysis but the practice is laborious and discourages application of multiple monitoring techniques that might provide deeper insights into sample composition. Anion exchange chromatography (AEC) also achieves fractionation of empty and full capsids for many AAV serotypes. The degree of separation varies among serotypes and does not correlate strictly with UC. This is not surprising since separation by AEC is highly influenced by capsid surface charge, which is independent of the amount of DNA packaged within the capsids. Chromatography methods however present a significant analytical advantage in the ease of monitoring the column effluent, including with multiple detectors. UV absorbance at 260 nm and 280 nm permits estimation of empty and full capsid proportions in any given peak. Intrinsic fluorescence enables estimation of relative areas of empty capsid peaks and full capsid peaks. Light scattering does the same and permits the further determination of capsid size and mass. In this report, we merge UC with an HPLC monitoring array to simultaneously analyze dual wavelength UV, intrinsic fluorescence, and light scattering through cesium chloride density gradient strata. Limitations of each monitoring method are discussed. UC results are compared with chromatography profiles to highlight distinction between separation methods. Practical application of results for final product characterization is considered, along with potential to support development of better purification processes.
Attachments
Pete Gagnon, Maja Leskovec, Blaz Goricar, and Aleš Štrancar
BPI, December 17, 2020
Abstract:
With its first licensed therapeutic now marketed worldwide, adeno-associated virus (AAV) has become a preferred vector for gene therapy. However, unlocking its full potential still poses challenges, many of which are associated with purification. The first involves the transition from upstream to downstream processes. AAV-bearing lysates are laden with debris that foul filtration media and limit or prevent concentration. Another challenge involves reduction of soluble host-cell DNA, which is complicated by its strong association with nucleoproteins. A third involves elimination of empty capsids. Currently, ultracentrifugation meets that need, but scale-up issues make chromatographic alternatives attractive. A fourth challenge involves the need for rapid, accurate, and revealing analytical results to guide process development, support validation, document control, and enable reproducibility of manufacturing processes. The following article shares experimental data showing how those challenges can be addressed to advance the evolution of gene therapy with AAV.
Attachments
Pete Gagnon, Blaz Goricar, Nina Mencin, Timotej Zvanut, Sebastijan Peljhan, Maja Lescovec and Ales Strancar
Pharmaceutics. 2021 Jan 17;13(1):113
Abstract:
HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.
2020
by Maribel Rios, Aleš Štrancar, J. Michael Hatfield and Pete Gagnon
BioProcess International, 2020
Abstract:
Adenoassociated viral (AAV) vectors have become synonymous with gene therapy delivery. However, because they are produced in such small quantities and because their upstream processes carry comparatively large amounts of host-cell DNA and other impurities, AAV purification can be challenging. Several researchers have applied different chromatographic strategies, but no universal method has been adopted in the biopharmaceutical industry.
This eBook features a discussion among several industry experts that explores challenges specific to AAV purification, shedding light on whether current strategies and separation technologies are up to the task. The conversation traverses issues relating to material handling at the upstream–downstream interface, removal of host-cell DNA, chromatographic separation of empty and full capsids, and a lack of fast and robust in-process analytics for downstream processes. Participants also explore whether the rise of AAV-based treatments will require downstream scientists to shift away from the antibody-centered conceptions of chromatography that have grown alongside the biotherapeutics industry.
P. Gagnon, B. Goričar, Š. Peršič, U. Černigoj, A. Štrancar
Cell & Gene Therapy Insights 2020; 6(7), 1035–1046
Abstract:
One of the barriers to development of industrial purification platforms for large mRNA has been an inadequate selection of high-performing capture-purification tools. Hybridization-affinity uses a polythymidine (Oligo dT) ligand to base-pair with the polyadenine tail of mRNA. It can be used for capture but it cannot discriminate dsRNA (double-stranded) from ssRNA (single-stranded) and it supports only brief cleaning with 100 mM sodium hydroxide. Traditional anion exchangers elute only mRNA smaller than about 500 bases unless the columns are heated to 50–70°C. Hydrophobic interaction chromatography (HIC) and reverse phase chromatography (RPC) separate ssRNA from dsRNA and short transcripts, but their sensitivity to fouling by proteins and aggregates makes them better suited for polishing than for capture. Better capture options are needed to meet the needs of large clinical trials, scale-up, and manufacture of vaccines. Beyond that, a new spectrum of gene therapy treatments await. This article introduces two new capture options that both eliminate dsRNA, DNA, and proteins in a wash step, then provide high-resolution polishing of ssRNA in an elution gradient at ambient temperature. One represents a new class of anion exchangers. The other exploits hydrogen bonding. Both support prolonged exposure to 1 M sodium hydroxide. Easy transition to either HIC or RPC provides high-resolution orthogonal polishing.
2019
Calef Sánchez-Trasviña, Marco Rito-Palomares, and José González-Valdez
Advances in Polymer Technology, Volume 2019, December 12 2019, 10 pages
Abstract
PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.
Wang Chunlei, Mulagapati Sri Hari Raju, Chen Zhongying, Du Jing, Zhao Xiaohui, Xi Guoling, Chen Liyan, Linke Thomas, Gao Cuihua, Schmelzer Albert, Liu Dengfeng
Molecular Therapy Methods & Clinical Development, Volume 15, September 26 2019, Pages 257-263
Abstract
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.
Keywords: AAV, AAV6.2, Chromatography, Anion exchange chromatography (AEC), Empty capsids, AUC, High-throughput
Sofiya Fedosyuk, Thomas Merritt, Marco Polo Peralta-Alvarez, Susan J. Morris, Ada Lam, Nicolas Laroudie, Anilkumar Kangokar, Daniel Wright, George M. Warimwe, Phillip Angell-Manning, Adam J. Ritchie, Sarah C. Gilbert, Alex Xenopoulos, Anissa Boumlic, Alexander D. Douglas
Vaccine (2019).
Published online 30 April 2019.
A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. There is a need for rapid production platforms for small GMP batches of non-replicating adenovirus vectors for early-phase vaccine trials, particularly in preparation for response to emerging pathogen outbreaks. Such platforms must be robust to variation in the transgene, and ideally also capable of producing adenoviruses of more than one serotype. It is also highly desirable for such processes to be readily implemented in new facilities using commercially available single-use materials, avoiding the need for development of bespoke tools or cleaning validation, and for them to be readily scalable for later-stage studies.
Here we report the development of such a process, using single-use stirred-tank bioreactors, a transgene-repressing HEK293 cell – promoter combination, and fully single-use filtration and ion exchange components. We demonstrate applicability of the process to candidate vaccines against rabies, malaria and Rift Valley fever, each based on a different adenovirus serotype.
Keywords: Simian adenovirus, GMP, Clinical trials, Single-use, Biomanufacturing, Bioreactor, Purification
Dr. Xiaotong Fu, Dr. Wei-Chiang Chen, C. Argento, R. Dickerson, P. Clarner, V. Bhatt, G. Bou-Assaf, Dr. M. Bakhshayeshi, Dr. Xiaohui Lu, Dr. S. Bergelson, Dr. J. Pieracci
Human Gene Therapy (2019)
Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids—viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography (AEX-HPLC) assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability to both the transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC) methods used in empty-to-full capsid ratio quantification, yet providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes (vg)/ml.
K. Trabelsi, M. Ben Zakour, H. Kallel
Vaccine (2019)
Rabies is a viral zoonosis caused by negative-stranded RNA viruses of the Lyssavirus genus. It can affect all mammals including humans. Dogs are the main source of human rabies deaths, contributing up to 99% of all rabies transmissions to humans. Vaccination against rabies is still the sole efficient way to fight against the disease.
Cell culture vaccines are recommended by World Health Organization (WHO) for pre and post exposure prophylaxis; among them Vero cell rabies vaccines which are used worldwide. In this work we studied the purification of inactivated rabies virus produced in Vero cells grown in animal component free conditions, using different methods. Cells were grown in VP-SFM medium in stirred bioreactor, then infected at an MOI of 0.05 with the LP2061 rabies virus strain. Collected harvests were purified by zonal centrifugation, and by chromatography supports, namely the Capto Core 700 and the monolithic CIM-QA column. Generated data were compared in terms of residual DNA level, host cell proteins (HCP) level and the overall recovery yield.
2018
M. Tajnik Sbaizero, M. Wolschek, M. Reiter, T. Muster, Pete Gagnon and Aleš Štrancar
BioProcess International, 15 October 2018
Influenza is a global respiratory disease with an estimated mortality of up to a half million people per year. The majority of traditional influenza vaccines are still produced in eggs. Downstream processing typically consists of clarification by centrifugation, concentration by ultrafiltration, and purification by ultracentrifugation. Recombinant vaccines are most often purified by chromatography. Chromatographic purification of viruses already has achieved major improvements in recovery and scalability, but it also is important because it enables virus purification to keep pace with important regulatory and manufacturing trends across the field of biopharmaceuticals. One of those trends is process intensification, referring to development of processes that harmonize integration of fewer and more capable steps to achieve higher productivity and reproducibility as well as reduce manufacturing costs.
In this report we describe processes for purification of influenza A and influenza B, both lacking TFF steps, and both using a single chromatography step with a cation exchange monolith on a single-use basis. The choice of process buffers enables final formulation by simple dilution of the product pool. DNA digestion requires two hours. Capture, purification, and formulation are achieved within four hours. Host-cell DNA and host-cell protein (HCP) are reduced more than 99%, and final virus recovery is 80%.
Laura M. Fischer, Michael W. Wolff, Udo Reichl, Vaccine 2017 July 17
The continuously increasing demand for potent and safe vaccines and the intensifying economic pressure on health care systems underlines the need for further optimization of vaccine manufacturing. Here, we focus on downstream processing of human influenza vaccines, investigating the purification of serum free cell culture-derived influenza virus (A/PR/8/34 H1N1) using continuous chromatography. Therefore, quaternary amine anion exchange monoliths (CIM QA) were characterized for their capacity to capture virus particles from animal cells cultivated in different media and their ability to separate virions from contaminating host cell proteins and DNA. The continuous chromatography was implemented as simulated moving bed chromatography (SMB) in a three zone open loop configuration with a detached high salt zone for regeneration.
SMBs exploiting 10% and 50% of the monoliths’ dynamic binding capacity, respectively, allowed the depletion of >98% of the DNA and >52% of the total protein. Based on the hemagglutination assay (HA assay), the virus yield was higher at 10% capacity use (89% vs. 45%). Both SMB separations resulted in a ratio of total protein to hemagglutinin antigen (based on single radial diffusion assay, SRID assay) below the required levels for manufacturing of human vaccines (less than 100 mg of protein per virus strain per dose). The level of contaminating DNA was five-times lower for the 10% loading, but still exceeded the required limit for human vaccines. A subsequent Benzonase treatment step, however, reduced the DNA contamination below 10 ng per dose. Coupled to continuous cultivations for virus propagation, the establishment of integrated processes for fully continuous production of vaccines seems to be feasible.