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2023

Nejc Pavlin, Urh Černigoj, Mojca Bavčar, Tjaša Plesničar, Jan Mavri, Martin Zidar, Matevž Bone, Urška Kralj Savič, Tadej Sever, and Aleš Štrancar

Electrophoresis. 2023; 1– 11

High-performance liquid chromatography (HPLC)-based analytical assays are used to effectively monitor purity and quantity of plasmid DNA (pDNA) throughout the purification process. However, the phenomenon of physical entrapment of open circular (OC) isoforms pDNA inside narrow channels of chromatographic support decreases its accuracy and precision and the effect increases with pDNA size. The purpose of the study was to develop a chromatographic method for accurate analytical separation between isoforms of <16 kbp pDNA using weak anion exchanging monolithic column with large (6 µm) convective channels. Purified samples of 4.7 and 15.4 kbp large pDNA with known isoform composition were prepared and their isoforms separated in ascending salt gradient. Both OC and supercoiled (SC) isoforms were baseline separated at a flow rate below 0.5 mL min−1 in a guanidinium chloride (GdnCl) gradient with a ≥95% OC pDNA elution recovery. However, these chromatographic conditions increased 2 times the peak width for linear (LIN) pDNA isoform compared to the results using monoliths with 1.4 µm channel size. If other chaotropic agents, such as urea or thiocyanate (SCN), were added to Gdn ions, the elution volume for LIN isoform decreased. Optimization of combined GdnCl/GdnSCN gradient for pDNA elution resulted in a simple and robust chromatographic method, where OC–LIN and LIN–SC pDNA (up to 15 kbp size) were separated with resolution above 1.0 and above 2.0, respectively. The accessibility and general acceptance of anion exchange chromatography for pDNA analytics give the newly developed method a great potential for in-process control monitoring of pDNA production processes.

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Irena Trbojević-Akmačić, Frano Vučković,Tea Pribić, Marija Vilaj, Urh Černigoj, Jana Vidič, Jelena Šimunović, Agnieszka Kępka, Ivana Kolčić, Lucija Klarić, Mislav Novokmet, Maja Pučić-Baković, Erdmann Rapp, Aleš Štrancar, Ozren Polašek, James F. Wilson and Gordan Lauc 

Communications Biology volume 6, Article number: 312 (2023)

Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans’ role in their distinct physiological functions.

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Lucija Rebula, Andrej Raspor, Mojca Bavčar, Aleš Štrancar and Maja Leskovec

Journal of Chromatography B, Volume 1217, 15 February 2023

Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.
In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.

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Hana Jug, Natalija Hosta, Mojca Tajnik Sbaizero, Aleš Štrancar, Daniela Brodesser, Bianca Tisch, Theresa Heger, Markus Wolschek, Joachim Seipelt and Manfred Reiter

BioProcess International, 9 February 2023

Newcastle disease is an extremely infectious condition among domesticated poultry and other avian species. Its high morbidity and mortality rates among infected birds give the disease significant economic importance. Thus, many commercially available vaccines based on live or inactivated virions are used globally to protect against Newcastle disease infection.
The causative agent is Newcastle disease virus (NDV), which belongs to the Paramyxoviridae family. NDV is a single-stranded, negative sense, enveloped RNA virus of avian origin that is highly attenuated in humans and other primates because of strong host-range restriction. Attenuated NDV has been evaluated as a vector for vaccines against SARS-CoV-2, Ebola, H5N1 influenza, West Nile, and simian immunodeficiency viruses. Oncolytic NDV vectors also hold much promise for immunotherapies against various cancers.
Whether for vaccines or viral-vector therapies, NDV particles must meet certain criteria for yield, purity, and concentration. Previously, we have described a highly effective process for purification of influenza virus produced in Vero cells. Based on CIMmultus SO3 (sulfonate) monolith columns, the process yielded excellent recovery and impurity removal and enabled good manufacturing practice (GMP) scale-up to produce clinical-grade material. Here, we describe our adaptation of the process to NDV purification and thus demonstrate the broad applicability of SO3 monolith technology.

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2022

Domen Pregeljc, Janja Skok, Tina Vodopivec, Nina Mencin, Andreja Krušič, Jure Ličen, Kristina Šprinzar Nemec, Aleš Štrancar & Rok Sekirnik

Biotechnology and Bioengineering, December 2022

The COVID‐19 pandemic triggered an unprecedented rate of development of messenger ribonucleic acid (mRNA) vaccines, which are produced by in vitro transcription reactions. The latter has been the focus of intense development to increase productivity and decrease cost. Optimization of in vitro transcription (IVT) depends on understanding the impact of individual reagents on the kinetics of mRNA production and the consumption of building blocks, which is hampered by slow, low‐ throughput, end‐point analytics. We implemented a workflow based on rapid at‐line high pressure liquid chromatography (HPLC) monitoring of consumption of nucleoside triphosphates (NTPs) with concomitant production of mRNA, with a sub‐3 min read‐ out, allowing for adjustment of IVT reaction parameters with minimal time lag. IVT was converted to fed‐batch resulting in doubling the reaction yield compared to batch IVT protocol, reaching 10 mg/ml for multiple constructs. When coupled with exonuclease digestion, HPLC analytics for quantification of mRNA was extended to monitoring capping efficiency of produced mRNA. When HPLC monitoring was applied to production of an anti‐reverse cap analog (ARCA)‐capped mRNA construct, which requires an approximate 4:1 ARCA:guanidine triphosphate ratio, the optimized fed‐ batch approach achieved productivity of 9 mg/ml with 79% capping.

The study provides a methodological platform for optimization of factors influencing IVT reactions, converting the reaction from batch to fed‐batch mode, determining reaction kinetics, which are critical for optimization of continuous addition of reagents, thus in principle enabling continuous manufacturing of mRNA.

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Rok Žigon, Mojca Tajnik Sbaizero, Ivana Petrović Koshmak, Veronika Fujs, Maja Leskovec & Aleš Štrancar

Cell & Gene Therapy Insights 2022; 8(10), 1315–1328

Manufacture and purification of recombinant adeno-associated viruses (rAAV) require development and optimization of processes to ensure the best possible quality of the final rAAV product. To do so, different strategies in upstream can be used to achieve the highest possible viral titer and lowest amount of impurities, both of which further influence downstream. Second challenge involves removal of cell debris where different pre-treatments can be utilized. In the next step, optimized capture of rAAV on a cation-exchange chromatography should be developed to remove impurities and achieve a high recovery of rAAV. In the end, several chromatographic options are available to remove empty and defected capsids, so only functional viruses can be isolated. Here, the process of manufacturing and purification of rAAV has been designed using monolithic columns to achieve this important goal of preparing rAAV for the use in gene therapy.

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Nina Mencin, Dona Štepec, Alja Margon, Jana Vidič, Darko Dolenc, Tina Simčič, Sara Rotar, Rok Sekirnik, Aleš Štrancar, Urh Černigoj

Separation and Purification Technology, Volume 304, 1 January 2023

Oligo-deoxythymidilic acid (OdT) probes conjugated to solid-phase supports exhibit high affinity for poly-adenylated mRNA (target) through high-affinity base-pairing interactions. Here we report the development of a OdT-functionalized chromatographic monolith for purification of polyadenylated mRNA and development of purification methods to support large-scale purification of mRNA-based therapeutics. We report the development of a chromatographic assay based on a synthetic oligo-deoxyadenylic acid chain of 10 or 20 nucleotides (OdA10 and OdA20) as a surrogate for polyadenylated mRNA, which was used for optimization of the OdT affinity column (i.e. the amount and structure of OdT immobilized and monolithic channel size). OdA hybridization to OdT monoliths correlated well with the amount of immobilized OdT, while an in-depth analysis revealed that hybridization yield decreased with increasing size of the target, temperature and probe surface coverage. OdA hybridization kinetics was flow rate-independent, confirming convection-based mass transport within the monolith’s channels. The demonstrated steep adsorption isotherms enable chromatographic capture of even highly diluted OdA-containing molecules. Dynamic binding capacity for model mRNA was independent of OdT length and amount of immobilized OdT probes above a critical threshold but was highly influenced by the composition of the binding buffer and mRNA residence time. We demonstrated the scalability of the mRNA purification process with OdT monoliths from 0.1 mL to at least 800 mL bed volume, paving the way for manufacturing processes on OdT monoliths with 40 L bed volume.

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Janja Skok, Polona Megušar, Tina Vodopivec, Domen Pregeljc, Nina Mencin, Matevž Korenč, Andreja Krušič, Anže Martinčič Celjar, Nejc Pavlin, Jana Krušič, Matthias Mueller, Kevin McHugh, Aleš Štrancar, and Rok Sekirnik

Chemie Ingenieur Techik, October 2022

The COVID-19 pandemic triggered an unprecedented surge in development of mRNA-based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single-use bioreactors are traditionally used for cell-based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell-free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed-batch IVT approach, and demonstrate the feasibility of continuous fed-batch production, paving the way towards continuous manufacturing of mRNA.

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Ana Ferjančič Budihna, Nejc Pavlin, Anže Martinčič Celjar, Andreja Gramc Livk and Aleš Štrancar

BioProcess International eBook, September 14, 2022

Robust and precise chromatographic analytical methods are key for the efficient development of the mRNA production process.

Three different analytical methods, which utilize three different column chemistries, are embedded in a ready-to-use PATfix™ analytical platform to support mRNA process development and product quantification and characterization.

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Tingting Cui, Kareem Fakhfakh, Hannah Turney, Gülin Güler-Gane, Aleksandra Toloczko, Martyn Hulley, Richard Turner

American Institute of Chemical Engineers, September 2022

In recent years, mRNA-based therapeutics have been a fast-growing new class of biologics that can, in principle, encode any protein(s) directly in patients to treat various diseases. mRNA vaccines have been proven to work efficiently, have high potency, and can be rapidly developed and deployed, which is critical for a quick responses in the case of a pandemic. Such agile development is enabled by rapid synthesis of RNA in vitro using recombinant enzymes rather than relying on lengthy and complex cell culture processes. mRNA exhibits physical and chemical properties differing from protein-based therapeutics. It is highly negatively charged and the hydroxyl group makes mRNA less stable and more susceptible to hydrolysis and nucleophilic cleavage. This novel work shares comprehensive studies carried out to compare the performance of various mRNA purification strategies by considering its scalability and critical quality attributes. In addition, the paper provides insights on how to establish a scalable mRNA purification process that consists of ultrafiltration/diafiltration and chromatography steps with good recoveries. Alternative Oligo(dT) based columns were further explored aiming to improve total process recovery. With Oligo(dT) as a capture step, overall recoveries of 70% can be achieved for mRNAs studied here that encode anti-influenza immunoglobulin G monoclonal antibodies.

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Katarina Markovič, Maja Cemazar, Gregor Sersa, Radmila Milačič and Janez Sčančar

Journal of Analytical Atomic Spectrometry

Ceruloplasmin (Cp) is the major copper-carrying (Cu) protein in human plasma. Due to copper's important physiological functions and its role in various diseases, there is a need to quantify the concentration bound to Cp and the exchangeable form of Cu. In the present work, conjoint liquid chromatography (CLC) on short-bed convective interaction media (CIM) monolithic disks was used to separate the Cu bound to low molecular mass (LMM) species, and the Cu bound to Cp and albumin (HSA) in human serum. Two immunoaffinity CIMmic albumin depletion (α-HSA) disks and one CIMmic weak anion-exchange diethylaminoethyl (DEAE) disk were assembled in a single housing, forming a CLC monolithic column. By applying isocratic elution with a 50 mmol L−1 MOPS buffer (pH 7.4) in the first 3 min, followed by gradient elution with 1 mol L−1 NH4Cl (pH 7.4) in the next 9 min, HSA was retained by the α-HSA disk, allowing subsequent separation of the LMM-Cu from the Cu bound to the Cp on the DEAE disk. Further elutio with 0.5 mol L−1 acetic acid in the next 4 min rinsed the HSA from the α-HSA disk. The separated Cu species were quantified by post column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS), while the elution profile of the proteins was followed by UV detection at 278 nm. Quantitative column recoveries were obtained. Good repeatability of the measurement was achieved for Cu-Cp (±1%), while for Cu-HSA and Cu-LMM species the repeatability of the measurements was slightly worse, due to the much lower Cu concentrations (±6% and ±9%, respectively). The developed method required only 20 μL of a 15-times diluted sample. Low limits of detection for the Cu-Cp, Cu-HSA and Cu-LMM species (6.1, 5.3 and 3.3 ng mL−1 Cu, respectively) were obtained. The technique was successfully applied in the determination of Cu-Cp, Cu-HSA and a fraction that most probably corresponds to the Cu-LMM species in the human serum of healthy individuals, kidney transplant patients and cancer patients.

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Nina Mencin, Andreja Krušic, Jure Ličen, Sebastijan Peljhan, Jana Vidič, Urh Černigoj, Tomas Kostelec, Aleš Štrancar and Rok Sekirnik

BioProcess International's Special Report, June 2022

Abstract:

Messenger RNA (mRNA) emerged as a powerful therapeutic tool for treatments in gene therapy, oncology, and infectious diseases, as recently demonstrated by vaccines against Covid-19. mRNA is produced by an enzymatic reaction that can be rapidly designed and scaled-up, and the platform is highly adaptable to different targets. One of the greatest challenges in mRNA production is the removal of process-related impurities stemming from in vitro transcription (IVT) reaction, such as residual nucleotide triphosphates, DNA template, enzymes, abortive transcripts.

Affinity-based chromatographic isolation of mRNA is robust and simple, lending itself as a useful industrial platform. mRNA constructs typically contain a 3’ polyA tail to increase stability in vivo, thereby enabling affinity purification using oligo-deoxythymidinic acid (Oligo dT) probes covalently coupled to a solid support. Macro-porous polymethacrylate monoliths offer high binding capacity and resolution for mRNA due to the convective nature of interconnected flow-through channels (>1.5 μm) modified with ligands that are easily accessible for mRNA. Typical binding capacity for CIMmultus™ Oligo dT for mRNA is 2-4 mg/mL, depending on construct length and loading concentration of NaCl.

Due to an increasing productivity of IVT reaction protocols, which routinely reach 5-10 mg/mL, elucidation of conditions that increase binding capacity of Oligo dT has been an intense focus of development. CIM® Oligo dT 0.05 mL Monolithic 96-well Plates were used for multi-parallel screening of binding conditions. Binding capacity could be significantly increased if NaCl is replaced with Gu-HCl, with DBC values of >6 mg/mL demonstrated, and scalability of binding capacity shown on CIMmultusTM Oligo dT preparative scale, which spans bed volume range 1 mL – 40 L, thereby theoretically supporting the purification of >200 g mRNA in a single run.

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2021

Rok Sekirnik and Tomas Kostelec

BioProcess International's special report, December 2021

Abstract:

Rapid response to global pandemics requires the manufacture of billions of vaccine doses within months. This short timeline must allow for design and testing of active ingredients, development of production and purification processes, clinical evaluations, regulatory filings, and manufacturing. Existing purification methods often have been adopted from laboratory-scale techniques to allow rapid implementation, and those have provided adequate product quality. But future mRNA development will require optimized production and purification processes.

Chromatography has been a workhorse of biomanufacturing for decades, including for monoclonal antibodies, plasmid DNA, viruses, and other modalities — as well as for supporting analytics. As an emerging therapeutic modality, mRNA production requires the development of new methodologies to suit its peculiar physicochemical profile: large, charged, and relatively unstable. Due to requirements for high purity, these methodologies will be based in large part on chromatography.

This article describes the versatility of chromatography when applied to mRNA production, starting with the purification of the key raw material (plasmid DNA) to final polishing of mRNA drug substance.

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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.

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by Nejc Pavlin, Blaž Bakalar, Janja Skok, Špela Kralj, Andreja Gramc Livk, and Aleš Štrancar

BioProcess International, October 2021

Abstract:

Plasmid DNA (pDNA) has become a crucial component in the production of next generation therapeutics such as messenger RNA (mRNA) and viral vectors.

As companies ramp up their production capabilities and move towards clinical applications, obtaining cGMP grade pDNA has become a production bottleneck, leading to lengthy production delays.

There is a growing market demand for solutions that can streamline the production of cGMP pDNA and help optimize down-stream processes (DSP) for better yields & purity.

The key step in this process is having quantifiably reliable analytics that give rapid results
for process optimization and scale-up, as well as production runs.

Establishing and expanding inhouse pDNA production platform in a quick and efficient manner will be a key differentiator between more and less successful next generation therapeutics projects.

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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.

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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.

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Urh Černigoj, Jana Vidič, Ana Ferjančič, Urša Sinur, Klemen Božič, Nina Mencin, Anže Martinčič Celjar, Pete Gagnon, Aleš Štrancar

Electrophoresis, September 2021. https://doi.org/10.1002/elps.202100210

Abstract:

Elution of strong and weak anion exchangers with sodium chloride gradients is commonly employed for analysis of sample mixtures containing different isomers of plasmid DNA. Gradient elution of a weak anion exchanger (diethylaminoethyl, DEAE) in the presence of guanidine hydrochloride (Gdn) roughly doubles resolution between open-circular (oc) and supercoiled (sc) isomers. It also improves resolution among sc, linear, and multimeric/aggregated forms. Sharper elution peaks with less tailing increase sensitivity about 30%. However, elution with an exclusively-Gdn gradient to 900 mM causes more than 10% loss of plasmid. Elution with a sodium chloride gradient while maintaining Gdn at a level concentration of 300 mM achieves close to 100% recovery of sc plasmid while maintaining the separation improvements achieved by exclusively-Gdn elution. Corresponding improvements in separation performance are not observed on a strong (quaternary amine) anion exchanger. Other chaotropic salts do not produce a favorable result on either exchanger, nor does the inclusion of surfactants or EDTA. Selectivity of the DEAE-Gdn method is orthogonal to electrophoresis, but with better quantification than agarose electrophoresis, better quantitative accuracy than capillary electrophoresis, and resolution approaching capillary electrophoresis.

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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.

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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.

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