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2023

Ivana Petrović Koshmak, Hana Jug, Katja Vrabec, Ana Mavri, Valentina Novak, Petra Dekleva, Veronika Fujs, Maja Leskovec and Aleš Štrancar

Electrophoresis 2023; 1–11

Adenoviruses are well-known viral vectors that have been previously used in gene therapy and as a vaccine-delivery vehicle for humans and animals. During the COVID-19 pandemic, it gained renewed attention, but at the same time, it raised concerns due to side effects observed with some of the resulting vaccines administered to patients. It has been indicated that these side effects might be attributed to impurities present in the final product. Therefore, constant enhancement of the vaccine purity and further improvement of impurity detection methods are needed. In this work, we showcase an example of industry-relevant adenovirus bioprocess optimization. Our data show the effect of upstream parameters on the bioburden introduced to the downstream process. We provide an example of process optimization using a combination of the PATfix analytical method, ddPCR, infectivity, total DNA, and total protein analyses to optimize cell density, multiplicity of infection, and length of production. Additionally, we provide data illustrating the robustness of the convective interaction media quaternary amine monolithic chromatography step. This anion exchange strategy was shown to remove over 99% of protein and DNA impurities, including those unable to be addressed by tangential flow filtration, while maintaining high adenovirus recoveries.

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Kaja Bažec, Mirjam Krašna, Andrej Mihevc, Maja Leskovec, Aleš Štrancar, Mojca Tajnik Sbaizero

Electrophoresis. 2023; 1–10

Adeno-associated virus (AAV) vectors are crucial tools for gene therapy applications. As AAVs are administered in vivo, stringent purity requirements must be met, necessitating the development of various downstream processing strategies in accordance with regulatory guidelines. In this context, we focus on the non-affinity serotype-independent recombinant AAV (rAAV) capture step, which involves the use of Convective Interaction Media (CIM) cation-exchange SO3 monoliths. We analyzed differentially pretreated viral samples obtained from the Sf9 cell line and applied these samples to the capture SO3 chromatography step. We conducted screening experiments using CIM SO3 0.05 mL monolithic 96-well plates with buffers of varying pH, sodium chloride concentrations, and the inclusion of poloxamer 188, aiming to select the optimal binding mobile phase. Dynamic binding capacity was defined for different pretreatments and the optimal conditions were subsequently retested using the industrial purification CIMmultus line. The results demonstrated a high overall vector recovery (51%) and a significant reduction in impurities (99.98% for protein reduction and 99.25% for DNA reduction) using the selected capture step parameters, thereby confirming the successful optimization of the rAAV capture step in the downstream process using monoliths.

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Maja Leskovec, Andrej Raspor, Veronika Fujs, Andrej Mihevc, Aleš Štrancar

Electrophoresis. 2023; 1–9

Preferential exclusion chromatography (PXC) sometimes described as hydrophobic interaction chromatography is a well-known, but not widely used technique for purification of Adeno-associated viruses. It employs high molarity of preferentially excluded cosolvent (salt in our case). The downside of this method is that high molarity of salt can lead to aggregation and precipitation of different compounds from the sample. In the case of viruses that are excreted to medium, the concentration of impurities is much lower compared to cell lysates, and PXC can be used as a first chromatographic, serotype independent step to concentrate and purify adeno-associated virus (AAV). Here, we explored PXC for adherent and suspension harvests using monolithic chromatographic columns (CIMmultus). Suspension extracellular adeno-associated virus, serotype 9 (AAV9) harvest had more impurities compared to adherent harvest, therefore it required higher input regarding method development. Final conditions for suspension harvest included higher molarity of binding salt and using more open channel format of chromatographic column (6 µm channel size). Vector genome analysis with droplet digital polymerase chain reaction (ddPCR) revealed 84% and 97% recovery for suspension and adherent AAV9 harvest, respectively. After PXC capture step, adherent AAV9 was purified by already described ion exchange techniques. Overall process vector genome recovery, from clarified harvest to anion exchange elution fraction, was 54% measured by ddPCR. Residual host cell DNA was measured at 40 ng per 1E13 vector genome, and empty AAV was below 5% in final anion exchange chromatography fraction.

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Troy Rogerson,  Guoling Xi,  Amanda Ampey,  Jon Borman,  Sally Jaroudi,  Dan Pappas,  Thomas Linke

Electrophoresis. 2023; 1– 11.

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). 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.

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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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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

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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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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2021

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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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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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.

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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.

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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.

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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.

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Petrović T, Alves I, Bugada D, Pascual J, Vučković F, Skelin A, Gaifem J, Villar-Garcia J, Vicente MM, Fernandes Â, Dias AM, Kurolt IC, Markotić A, Primorac D, Soares A, Malheiro L, Trbojević-Akmačić I, Abreu M, Sarmento E Castro R, Bettinelli S, Callegaro A, Arosio M, Sangiorgio L, Lorini LF, Castells X, Horcajada JP, Pinho SS, Allegri M, Barrios C, Lauc G.

Glycobiology. 2020 Nov.

Abstract:

A large variation in the severity of disease symptoms is one of the key open questions in COVID-19 pandemics. The fact that only a small subset of people infected with SARS-CoV-2 develop severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the IgG glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of inter-individual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting Nacetylglucosamine (GlcNAc) in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing. To our knowledge, this is the first study exploring IgG N-glycome variability in COVID-19 severity.

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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.

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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.

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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.

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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.

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Keywords: AAV, AAV6.2, Chromatography, Anion exchange chromatography (AEC), Empty capsids, AUC, High-throughput

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