Adenovirus Purification Research Articles

Peptide ligands for the affinity purification of adenovirus from HEK293 and vero cell lysates

Adenovirus (AdVs) is the viral vector of choice in vaccines and oncolytic applications owing to its high transduction activity and inherent immunogenicity. For decades, AdV isolation has relied on ultracentrifugation and ion-exchange chromatography, which are not suitable to large-scale production and struggle to deliver sufficient purity. Immunoaffinity chromatography resins of recent introduction feature high binding capacity and selectivity, but mandate harsh elution conditions (pH 3.0), afford low yield (< 20%), and provide limited reusability. Seeking a more efficient and affordable alternative, this study introduces the first peptide affinity ligands for AdV purification. The peptides were identified via combinatorial selection and in silico design to target hexons, the most abundant proteins in the adenoviral capsid. Selected peptide ligands AEFFIWNA and TNDGPDYSSPLTGSG were conjugated on chromatographic resins and utilized to purify AdV serotype 5 from HEK293 and Vero cell lysates. The peptide-functionalized resins feature high binding capacity (> 1010 active virions per mL at the residence time of 2 min), provide high yield (> 50%) and up to 100-fold reduction of host cell proteins and DNA. Notably, the peptide ligands enable gentle elution conditions (pH 8) that prevent the “shedding” of penton and fiber proteins, thus affording intact adenovirus particles with high cell-transduction activity. The study of the peptide ligands by surface plasmon resonance and molecular docking and dynamics simulations confirmed the selective targeting of hexon proteins and elucidated the molecular-level mechanisms underlying binding and release. Collectively, these results demonstrate the strong promise of peptide ligands presented herein for the affinity purification of AdVs from cell lysates.

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Adenovirotherapy delivering cross-hybrid IgGA Fc engineering PD-L1 inhibitors for enhanced cancer immunotherapy

Preparation and identification of a single domain antibody specific for adenovirus vectors and its application to the immunoaffinity purification of adenoviruses

Adenovirus belongs to the family of Adenoviridae. As a vaccine carrier, it has high safety and stimulates the body to produce cellular immunity and humoral immunity. This study prepared an adenoviral vector-specific single-domain antibody for use in adenovirus identification and purification. We successfully constructed a single domain antibody phage display library with a capacity of 1.8 × 109 by immunizing and cloning the VHH gene from Bactrian camel. After the second round of biopanning, clones specific for adenovirus were screened using phage ELISA. Twenty-two positive clones were obtained, and two clones with the highest binding affinity from ELISA were selected and named sdAb 5 and sdAb 31 for further application. The recombinant single-domain antibody was solublely expressed in E. coli and specifically bound to adenoviruses rAd26, ChAd63 and HAd5 in ELISA and live cell immunofluorescence assays. We established an effective method for immunoaffinity purification of adenovirus by immobilizing the single domain antibody to Sepharose beads, and it may be used to selectively capture adenoviruses from cell culture medium. The preparation of the adenovirus-specific single-domain antibody lays a foundation for the one-step immunoaffinity purification and identification of adenoviruses.

Comparison of the performance of anion exchange membrane materials for adenovirus purification using laterally-fed membrane chromatography

Viruses are an emerging class of biotherapeutics used to treat a variety of diseases. Ion-exchange membrane chromatography is commonly used for virus purification, with the Mustang Q (Pall) and Sartobind Q (Sartorius) being two of the most widely used strong anion-exchange membranes. These membranes are made from different materials and have different pore sizes, and while previous studies have attempted to compare their performance in various virus purification applications, those studies have been hindered by the additional fact that these membranes are sold in different module formats. Thus, in order to establish a direct comparison of the capabilities and purification performance of anion exchange membrane materials, we first fabricated equivalent laterally-fed membrane chromatography (LFMC) devices containing either 1 mL of Mustang Q membrane or 1 mL of Sartobind Q membrane and then compared their performance based on residence times, the elution patterns of pre-purified biomolecules, and the recovery and purity of adenovirus from a cell lysate. Under the same linear gradient elution conditions, purified adenovirus eluted approximately one membrane volume earlier for the Sartobind Q membrane; a similar result was obtained during purification of adenovirus from infected suspension 293 cells. While the total recovery of adenovirus was the same for both membranes (~60%), the Sartobind Q membrane provided a much greater removal of DNA impurities in the virus containing elution fractions.

Oncolytic virus purification with periodic counter-current chromatography.

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and(iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

3D-printed ordered bed structures for chromatographic purification of enveloped and non-enveloped viral particles

In the last few decades we have witnessed a rapid advance in the design of new biotherapeutic products using viral particles. The current limitations of manufacturing these particles are often associated with their purification processes. Additive manufacturing opened the possibility of printing 3D porous chromatographic structures to be used in the bio-separation field. Here, we report the production and use of 3D-printed cellulose chromatographic columns functionalized with two different ligands: diethylaminoethyl (DEAE) and hydroxyapatite for the purification of oncolytic adenovirus and lentiviral vectors. Porous beds with ordered channel structures can be manufactured to achieve separations that are efficient and tailored to the target products. While common chromatographic beads are based on non-ordered bed structures, in this work we explore the use of a triply periodic minimal surface, the Schoen Gyroid, to create bed structures with a channel size of 300 µm in diameter. The dynamic binding capacity (DBC) obtained for non-enveloped oncolytic adenovirus using the 3D-printed DEAE column (1.9 × 1010 viral genomes per millilitre) was consistent with the reported values in the literature for this virus particle. We also demonstrate that by using these 3D-printed chromatographic supports, oncolytic adenoviruses were successfully purified with a recovery yield of 69 ± 6%, while maintaining their size and shape as observed by electron microscopy analysis. For lentiviral vectors, a DBC of 2 × 109 physical particles per millilitre was achieved for the DEAE column. Moreover, these enveloped viruses were purified using this 3D-printed column with a recovery yield of 57% of transducing units. These findings suggest, with advancements, 3D printing technologies applied in the viral vector manufacturing field could improve downstream process efficiency.

Integrated development of enzymatic DNA digestion and membrane chromatography processes for the purification of therapeutic adenoviruses

High titer and purity levels are key requirements for virus-based cancer and gene therapy biopharmaceuticals. However, the task of removing enough host-cell DNA from therapeutic viruses in order to comply with the FDA’s 10 ng/dose limit is particularly challenging. In a previous study, we demonstrated the advantages of using laterally-fed membrane chromatography (LFMC) for adenovirus purification. Although this approach achieved >90% DNA removal, significant amounts of DNA remained in the product due to the poor performance of a pre-LFMC DNA digestion step. In the present study, we attempt to improve upon this outcome by employing an integrated approach to process development that examines the interactions between different downstream steps (i.e., clarification, enzymatic DNA digestion, and membrane chromatography (MC)). First, we identified the most efficient process sequence involving a clarification step followed by a DNA digestion step, as well as three endonucleases (Benzonase®, Denarase®, and Turbonuclease™) that perform similarly with respect to DNA digestion. Next, a factorial design of experiments (DOE) study was used to evaluate how enzyme (Benzonase and Denarase) concentration and incubation time impact the resulting DNA and virus concentrations. Since Denarase showed slightly better efficiency, it was used along with a subset of the DOE conditions to evaluate the removal of DNA via MC with Sartobind Q 96-well filter plates. The lowest amount of DNA per dose was achieved using MC in conjunction with feed that had been digested with 10 U/mL of Denarase for 4 h; as such, this approach was used to prepare lysates, which were then purified using an LFMC device containing 1 mL of Sartobind Q membrane. This process enabled a virus-recovery rate of 73% and residual DNA levels of 77 ng/dose. Ultimately, the proposed integrated process development approach resulted in an approximately 80-fold improvement in DNA removal.

A novel method to purify adenovirus based on increasing salt concentrations in buffer

With the rapid development of gene therapy, gene-based medicine with adenovirus as vectors has become a new method for disease treatment. However, there are still enormous challenges in the large-scale production of adenoviruses for clinical use. Recent reports show that ion-exchange chromatography (IEC) is an effective tool for the isolation and purification of adenovirus. However, during the separation and purification, host cell protein and DNA, as well as serum from the culture medium, can non-specifically occupy numerous binding sites of the chromatography packings, thereby reducing the binding between the adenovirus and packing media. We here report a novel method for highly efficient purification of adenoviruses by increasing the salt concentrations of the samples to be ultrafiltrated by tangential flow filtration, the diafiltration buffer, and the samples for IEC purification. This method could significantly remove a large amount of serum proteins and host cell proteins, increase the amount of sample loaded on the IEC column, and improve the binding of the adenovirus samples to the packing media. A purity of > 95% could be obtained after one chromatography operation, and the number of purification steps and the amount of used packing media were reduced. The method is simple, economical, and efficient, and has excellent applications.

Purification of the Recombinant Adenovirus by Cesium Chloride Gradient Centrifugation.

Cesium chloride gradient centrifugation is the most widely used method for purification of recombinant adenovirus. This protocol describes the entire process, from the preparation and clarification of crude viral lysate to the formulation and storage of purified virus.

Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components

A variety of Good Manufacturing Practice (GMP) compliant processes have been reported for production of non-replicating adenovirus vectors, but important challenges remain. Most clinical development of adenovirus vectors now uses simian adenoviruses or rare human serotypes, whereas reported manufacturing processes mainly use serotypes such as AdHu5 which are of questionable relevance for clinical vaccine development. Many clinically relevant vaccine transgenes interfere with adenovirus replication, whereas most reported process development uses selected antigens or even model transgenes such as fluorescent proteins which cause little such interference. Processes are typically developed for a single adenovirus serotype – transgene combination, requiring extensive further optimization for each new vaccine.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. We compare performance of a range of commercially available ion exchange media, including what we believe to be the first published use of a novel media for adenovirus purification (NatriFlo® HD-Q, Merck). We demonstrate the need for minimal process individualization for each vaccine, and that the product fulfils regulatory quality expectations. Cell-specific yields are at the upper end of those previously reported in the literature, and volumetric yields are in the range 1 × 1013 – 5 × 1013 purified virus particles per litre of culture, such that a 2–4 L process is comfortably adequate to produce vaccine for early-phase trials. The process is readily transferable to any GMP facility with the capability for mammalian cell culture and aseptic filling of sterile products.

Purification of therapeutic adenoviruses using laterally-fed membrane chromatography

Viruses are an emerging class of biotherapeutics with the potential to treat cancer, genetic, cardiovascular and other diseases. Their commercial success depends on cost-effective purification processes. Membrane chromatography processes offer great potential for virus purification, however conventional devices are known to suffer from issues related to resolution and sample dilution. Laterally-fed membrane chromatography (LFMC) overcomes these issues by minimizing the dead-volume and establishing a uniform flow distribution pattern within the device. While the efficiency of LFMC has been previously demonstrated for protein purification studies, here we present a direct comparison of the performance of a LFMC device and a conventional radial flow device (both containing 1 mL of the same strong anion-exchange membrane) for the purification of adenoviruses. Using a gradient elution strategy, a 74% virus recovery with 4% of residual amount of total protein and 1% residual amount of total DNA was obtained using the LFMC device; for the radial flow device, only 20% virus recovery was achieved for a similar level of purity. Using a stepwise elution strategy, close to 100% virus recovery with 10% residual protein and DNA was achieved with both devices. However, the product was at least 50% more dilute with the radial flow device. Overall, this study demonstrates the considerable benefits of using LFMC over conventional membrane chromatography devices for purifying virus-based biotherapeutics.

Clinical-Grade Oncolytic Adenovirus Purification Using Polysorbate 20 as an Alternative for Cell Lysis.

Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic ap-proach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach. Triton X-100 is commonly used in cell lysis buffer prepara-tions. The addition of this surfactant in the list of substances with the very high concern of the Registra-tion, Evaluation, Authorization and Restriction of Chemicals (REACH) regulation promoted the research for effective alternatives.Methods: In this work, a purification strategy for oncolytic adenovirus compatible with phase I clinical trials, using an approved surfactant – Polysorbate 20 was developed. The proposed downstream train, composed by clarification, concentration using tangential flow filtration, intermediate purification with anion exchange chromatography, followed by a second concentration and a final polishing step was evaluated for both Triton X-100 and Polysorbate 20 processes. The impact of cell lysis with Polysorb-ate20 and Triton X-100 for each downstream step was evaluated in terms of product recovery and impu-rities removal. Overall, 61 ± 4% of infectious viral particles were recovered. Depletion of host cell pro-teins and ds-DNA was 99.9% and 97.1%, respectively.Results & Conclusion: The results indicated that Polysorbate 20 can be used as a replacement for Triton X-100 during cell lysis with no impact on product recovery, potency, and purity. Moreover, the devel-oped process is scalable and able to provide a highly purified product to be used in phase I and II clinical trials.

182 - Industrialization of AdenoVirus production and purification with the iCELLis® 500 single-use bioreactor

Diabetes is a major global health problem with the WHO reporting over 422 million affected persons and 3.7 million diabetes related deaths annually worldwide. Orgenesis is developing a cell therapy approach that genetically modifies a patient's own liver cells to secrete insulin. To bring this approach into pharmaceutical development, Orgenesis and Pall have combined their respective expertise to develop a manufacturing strategy for large scale adenoviral vector production in the packed-bed iCellis® 500 single-use bioreactor together with the development of a downstream purification industrialized process. In this study, we describe scale-up of the optimized adenovirus serotype 5 production process that was developed using a predictive small scale iCellis® Nano bioreactor. By optimizing culture and infection parameters such as HEK293 cell seeding density, multiplicity of infection, time of infection, day of harvest and media circulation parameters, yield was increased from 1.6E9 to 1.4E10 infectious virus particles per cm2 fixed bed. This yield makes the iCellis® bioreactor a promising scalable technology for the production of adenovirus products. The current density gradient based purification method for adenovirus is a time consuming, inefficient and non-manufacturing process. Here, an optimized rapid AEX membrane chromatography step was used for purification. Following depth and sterile filtration, clarified harvest is processed over a Mustang Q membrane in bind/elute mode. Eluted material is immediately concentrated and buffer exchanged into the final formulation buffer. Final product is then sterile filtered and vialed for potency studies. Purified adenovirus hPDX-1 was fully functional and successfully transduced the target liver derived cells. The next step of the study will incorporate the viral trans-differentiation step into the cGMP autologous patient cell expansion process in Pall's Xpansion® 200 bioreactor.

An artistic Approach for Purification of Adeno‐Associated Virus and Adenovirus

Since “virus‐like” particles were first discovered in adenovirus (AdV) preparations, adeno‐associated virus (AAV) has been characterized and developed in the last 50 years as a potent viral vector to deliver genes in vitro in cultured cells and in vivo in animal models. Numerous investigations have revealed its attracting features including nonpathogenicity, efficient transduction and stable expression, thus laying foundations for recombinant AAV for use as one of the most successful gene delivery vehicles. Although there are a few of traditional techniques used for purification of the viruses, one rapid and facile procedure for AAV purification remains to be developed. Here we present a novel approach using combination of techniques that has been developed as products for purification of AAV viruses and adenoviruses as well. The whole procedure of the approach takes about 3 h to produce viral solutions of AAV or adenovirus with a titer up to 1013 genomic copies or 1010 transducing units per ml. Coomassie staining reveals that the purified AAV solution exhibits only three bands of capsid proteins. Besides, there are no any toxic compounds or reagents used in this procedure, so that the high‐titer, ultrapure viral solutions obtained by the novel technology can meet the quality requirements for most research purposes and may be also qualified for preclinical or clinical trials.

Novel High-throughput Approach for Purification of Infectious Virions.

Viruses are extensively studied as pathogens and exploited as molecular tools and therapeutic agents. Existing methods to purify viruses such as gradient ultracentrifugation or chromatography have limitations, for example demand for technical expertise or specialized equipment, high time consumption, and restricted capacity. Our laboratory explores mutations in oncolytic reovirus that could improve oncolytic activity, and makes routine use of numerous virus variants, genome reassortants, and reverse engineered mutants. Our research pace was limited by the lack of high-throughput virus purification methods that efficiently remove confounding cellular contaminants such as cytokines and proteases. To overcome this shortcoming, we evaluated a commercially available resin (Capto Core 700) that captures molecules smaller than 700 kDa. Capto. Core 700 chromatography produced virion purity and infectivity indistinguishable from CsCl density gradient ultracentrifugation as determined by electron microscopy, gel electrophoresis analysis and plaque titration. Capto Core 700 resin was then effectively adapted to a rapid in-slurry pull-out approach for high-throughput purification of reovirus and adenovirus. The in-slurry purification approach offered substantially increased virus purity over crude cell lysates, media, or high-spin preparations and would be especially useful for high-throughput virus screening applications where density gradient ultracentrifugation is not feasible.

Optimization and scale-up of cell culture and purification processes for production of an adenovirus-based tuberculosis vaccine

Background Tuberculosis (TB) is the second leading cause of death by infectious disease worldwide. About 1.4 million people die of TB each year. Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. AdAg85A adenovirus, a new promising tuberculosis vaccine candidate, has been studied with mouse, guinea pig, goat and cow animal models, and was shown to be effective against Mycobacterium tuberculosis (Mtb) infection [1]. A phase I trial on Ad5Ag85A adenovirus was also conducted, which demonstrated that AdAg85A adenovirus was safe and highly immunogenic [2]. To further evaluate the efficacy of this vaccine and reduce the cost of this promising vaccine candidate, a feasible and cost-effective large-scale cell culture production process had to be developed for manufacturing large quantities of AdAg85A adenovirus required for further clinical trials. Furthermore, the process had to be designed to meet all requirements for industrialization and commercialization of this vaccine candidate. Here we report our study on optimization of cell culture conditions, scale up of AdAg85A adenovirus production in 60L bioreactor and purification of the AdAg85A adenovirus at different scales. The optimized conditions for AdAg85A adenovirus production and purification were transferred to a GMP facility for manufacturing of AdAg85A adenovirus for further clinical trials. Materials and methods Four commercial serum-free cell culture media (SFM4HEK-293 and SFM4Transfx-293 from HyClone; Adenovirus Expression Medium (AEM) and CD 293 from Life Technologies), were evaluated for supporting the growth of HEK293SF-3F6 cell in suspension and also for the production of AdAg85a adenovirus in 125 mL shake flask cultures under various experimental conditions. The production of the AdAg85A adenovirus was then scaled up to 3L controlled bioreactor under the optimized conditions obtained from the shake flask experiment, further validated in a 60L bioreactor. Purification of the AdAg85A adenovirus was accomplished through many different steps. Some of the critical steps include cell lysis, benzonase treatment, Q-Sepharose HP anion exchange chromatography for capture of adenovirus/purification, Capto Core 700 multimodal chromatography for polishing, concentration and diafiltration into formulation buffer. The purification processes were also scaled up from 3L to 60 L production scale.

Single-step concentration and purification of adenoviruses by coxsackievirus-adenovirus receptor-binding capture and elastin-like polypeptide-mediated precipitation.

A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the "spiked" PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples.

Large-scale adenovirus and poxvirus-vectored vaccine manufacturing to enable clinical trials.

Efforts to make vaccines against infectious diseases and immunotherapies for cancer have evolved to utilize a variety of heterologous expression systems such as viral vectors. These vectors are often attenuated or engineered to safely deliver genes encoding antigens of different pathogens. Adenovirus and poxvirus vectors are among the viral vectors that are most frequently used to develop prophylactic vaccines against infectious diseases as well as therapeutic cancer vaccines. This mini-review describes the trends and processes in large-scale production of adenovirus and poxvirus vectors to meet the needs of clinical applications. We briefly describe the general principles for the production and purification of adenovirus and poxvirus viral vectors. Currently, adenovirus and poxvirus vector manufacturing methods rely on well-established cell culture technologies. Several improvements have been evaluated to increase the yield and to reduce the overall manufacturing cost, such as cultivation at high cell densities and continuous downstream processing. Additionally, advancements in vector characterization will greatly facilitate the development of novel vectored vaccine candidates.

Biobased monoliths for adenovirus purification.

Adenoviruses are important platforms for vaccine development and vectors for gene therapy, increasing the demand for high titers of purified viral preparations. Monoliths are macroporous supports regarded as ideal for the purification of macromolecular complexes, including viral particles. Although common monoliths are based on synthetic polymers as methacrylates, we explored the potential of biopolymers processed by clean technologies to produce monoliths for adenovirus purification. Such an approach enables the development of disposable and biodegradable matrices for bioprocessing. A total of 20 monoliths were produced from different biopolymers (chitosan, agarose, and dextran), employing two distinct temperatures during the freezing process (-20 °C and -80 °C). The morphological and physical properties of the structures were thoroughly characterized. The monoliths presenting higher robustness and permeability rates were further analyzed for the nonspecific binding of Adenovirus serotype 5 (Ad5) preparations. The matrices presenting lower nonspecific Ad5 binding were further functionalized with quaternary amine anion-exchange ligand glycidyltrimethylammonium chloride hydrochloride by two distinct methods, and their performance toward Ad5 purification was assessed. The monolith composed of chitosan and poly(vinyl) alcohol (50:50) prepared at -80 °C allowed 100% recovery of Ad5 particles bound to the support. This is the first report of the successful purification of adenovirus using monoliths obtained from biopolymers processed by clean technologies.

Robust design of adenovirus purification by two-column, simulated moving-bed, size-exclusion chromatography

A simple, yet efficient, two-column simulated moving-bed (2CSMB) process for purifying adenovirus serotype 5 (Ad5) by size-exclusion chromatography (SEC) is presented and validated experimentally, and a general procedure for its robust design under parameter uncertainty is described. The pilot-scale run yielded a virus recovery of 86 percent and DNA and HCP clearances of 90 and 89 percent, respectively, without any fine tuning of the operating parameters. This performance compares very favorably against that of single-column batch chromatography for the same volume of size-exclusion resin. To improve the robustness of the 2CSMB-SEC process the best set of operating parameters is selected only among candidate solutions that are robust feasible, that is, remain feasible for all parameter perturbations within their uncertainty intervals. This robust approach to optimal design replaces the nominal problem by a worst case problem. Computational tractability is ensured by formulating the robust design problem with only the vertices of the uncertainty region that have the worst effect on the product purity and recovery. The robust design is exemplified on the case where the column volume and interparticle porosity are subject to uncertainty. As expected, to increase the robustness of the 2CSMB-SEC process it is necessary to reduce its productivity and increase its solvent consumption. Nevertheless, the design solution given by our robust approach is the least detrimental of all feasible operating conditions for the 2CSMB-SEC process.