Reviewing the process intensification landscape through the introduction of a novel, multitiered classification for downstream processing.

A demand for process intensification in biomanufacturing has increased over the past decade due to the ever-expanding market for biopharmaceuticals. This is largely driven by factors such as a surge in biosimilars as patents expire, an aging population, and a rise in chronic diseases. With these market demands, pressure upon biomanufacturers to produce quality products with rapid turnaround escalates proportionally. Process intensification in biomanufacturing has been well received and accepted across industry based on the demonstration of its benefits of improved productivity and efficiency, while also reducing the cost of goods. However, while these benefits have been shown empirically, the challenges of adopting process intensification into industry remain, from smaller independent start-up to big pharma. Traditionally, moving from batch to a process intensification scheme has been viewed as an "all or nothing" approach involving continuous bioprocessing, in which the factors of complexity and significant capital costs hinder its adoption. In addition, the literature is crowded with a variety of terms used to describe process intensification (continuous, periodic counter-current, connected, intensified, steady-state, etc.). Often, these terms are used inappropriately or as synonyms, which generates confusion in the field. Through a detailed review of current state-of-the-art systems, consumables, and process intensification case studies, we herein propose a defined approach in the implementation of downstream process intensification through a standardized nomenclature and viewing it as distinct independent levels. These can function separately as intensified single-unit operations or be built upon by integration with other process steps allowing for simple, incremental, cost-effective implementation of process intensification in the manufacturing of biopharmaceuticals.

Accelerated and intensified manufacturing of an adenovirus-vectored vaccine to enable rapid outbreak response.

The Coalition for Epidemic Preparedness Innovations' "100-day moonshot" aspires to launch a new vaccine within 100 days of pathogen identification, followed by large-scale vaccine availability within the "second hundred days." Here, we describe work to optimize adenoviral vector manufacturing for rapid response, by minimizing time to clinical trial and first large-scale supply, and maximizing output from the available manufacturing footprint. We describe a rapid virus seed expansion workflow that allows vaccine release to clinical trials within 60 days of antigen sequence identification, followed by vaccine release from globally distributed sites within a further 40 days. We also describe a perfusion-based upstream production process, designed to maximize output while retaining simplicity and suitability for existing manufacturing facilities. This improves upstream volumetric productivity of ChAdOx1 nCoV-19 by approximately fourfold and remains compatible with the existing downstream process, yielding drug substance sufficient for 10,000 doses from each liter of bioreactor capacity. This accelerated manufacturing process, along with other advantages such as thermal stability, supports the ongoing value of adenovirus-vectored vaccines as a rapidly adaptable and deployable platform for emergency response.

Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs.

Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing" approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.

Improved virus purification processes for vaccines and gene therapy.

The downstream processing of virus particles for vaccination or gene therapy is becoming a critical bottleneck as upstream titers keep improving. Moreover, the growing pressure to develop cost-efficient processes has brought forward new downstream trains. This review aims at analyzing the state-of-the-art in viral downstream purification processes, encompassing the classical unit operations and their recent developments. Emphasis is given to novel strategies for process intensification, such as continuous or semi-continuous systems based on multicolumn technology, opening up process efficiency. Process understanding in the light of the pharmaceutical quality by design (QbD) initiative is also discussed. Finally, an outlook of the upcoming breakthrough technologies is presented.

Rational development of two flowthrough purification strategies for adenovirus type 5 and retro virus-like particles.

We report on the rational design and implementation of flowthrough (FT) platforms for purification of virus vectors (VVs) and virus-like particles (VLPs), combining anion-exchange polyallylamine membranes (Sartobind STIC) and core-shell octylamine resins (CaptoCore 700). In one configuration, the VV bulk is concentrated and conditioned with appropriate buffer in a ultra/diafiltration (UF/DF) unit prior to injection into the STIC chromatography membrane. The FT pool and an intermediate cut of the elution pool of the STIC membrane are admixed and directed to a second UF/DF. Finally, the retentate is injected into a CC700 packed bed adsorber where the purified VVs are collected in the FT pool, whereas the residual amount of DNA and host cell protein (HCP) are discarded in the eluate. The experimental recovery achieved with this downstream processing (DSP) platform is close to 100%, the DNA clearance is roughly a 4-log reduction, and the HCP level is reduced by 5 logs. The platform developed for VLP purification is simpler than the previous one, as the STIC membrane adsorber and CC700 bed are connected in series with no UF/DF unit in between. Experimentally, the FT scheme for VLP purification gave a recovery yield of 45% in the chromatography train; the experimental log reduction of DNA and HCP were 2.0 and 3.5, respectively. These results are in line with other purification strategies in the specific field of enveloped VLPs. Both DSP platforms were successfully developed from an initial design space of the binding of the major contaminant (DNA) to the two ligands, determined by surface plasmon resonance, which was subsequently scaled up and confirmed experimentally.

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.

Evaluation of novel large cut-off ultrafiltration membranes for adenovirus serotype 5 (Ad5) concentration.

The purification of virus particles and viral vectors for vaccine and gene therapy applications is gaining increasing importance in order to deliver a fast, efficient, and reliable production process. Ultrafiltration (UF) is a widely employed unit operation in bioprocessing and its use is present in several steps of the downstream purification train of biopharmaceuticals. However, to date few studies have thoroughly investigated the performance of several membrane materials and cut-offs for virus concentration/diafiltration. The present study aimed at developing a novel class of UF cassettes for virus concentration/diafiltration. A detailed study was conducted to evaluate the effects of (i) membrane materials, namely polyethersulfone (PES), regenerated cellulose (RC), and highly cross-linked RC (xRC), (ii) nominal cut-off, and (iii) UF device geometry at different production scales. The results indicate that the xRC cassettes with a cut-off of approximately 500 kDa are able to achieve a 10-fold concentration factor with 100% recovery of particles with a process time twice as fast as that of a commercially available hollow fiber. DNA and host cell protein clearances, as well as hydraulic permeability and fouling behavior, were also assessed.

Impact of grafting on the design of new membrane adsorbers for adenovirus purification.

The impacts of quaternary amine ligand density and matrix structure, namely hydrogel grafted and directly grafted, on state-of-the-art chromatographic membranes operated in bind-and-elute mode were evaluated for the purification of adenovirus serotype 5. The experiments were performed on a 96-well plate membrane holder, which is a convenient high-throughput screening tool for obtaining the best operating conditions for a process yield optimization. The results show that the hydrogel-grafted membranes are more suitable for virus purification than the directly grafted ones. By reducing the number of grafted ligands to low (1.7µmol/cm(2)) or medium (2.4µmol/cm(2)) density, it is possible to increase the recovery of purified virus by 60% compared to a highly charged membrane (3.3µmol/cm(2)) that yielded a recovery rate lower than 30%. In the reported experiments, Sartobind(®) Q, chosen as benchmark comparison, provides a better compromise between high recovery and large dynamic binding capacity. Overall, this work contributes to the understanding and development of new membrane adsorbers specifically designed for virus purification.

Adenovirus purification by two-column, size-exclusion, simulated countercurrent chromatography.

Adenovirus serotype 5 (Ad5) was successfully separated by size-exclusion chromatography (SEC) using a simple, yet efficient, two-column, quasi-continuous, simulated moving-bed process operated in an open-loop configuration. The operating cycle is divided into two identical half-cycles, each of them consisting of the following sequence of sub-steps: (i) elution of the upstream column and direction of the effluent of the downstream column to waste; (ii) elution of the upstream column and redirection of its effluent to waste while the downstream column is fed with the clarified bioreaction bulk and its effluent collected as purified product; (iii) operation of the system as in step (i) but collecting the effluent of the downstream column as product; (iv) elution of the upstream column and direction of its effluent to waste while the flow through the downstream column is temporarily halted. Clearance of impurities, namely DNA and host cell protein (HCP), were experimentally assessed. The pilot-scale run yielded a virus recovery of 86%, and a clearance of 90% and 89% for DNA and HCP, respectively, without any fine tunning of the predetermined operating parameters. These figures compare very favorably against single-column batch chromatography for the same volume of size-exclusion resin. However, and most importantly, the virus yield was increased from 57% for the batch system to 86% for the two-column SEC process because of internal recycling of the mixed fractions of contaminated Ad5, even though the two-column process was operated strictly in an open-loop configuration. And last, but not least, the productivity was increased by 6-fold with the two-column process. In conclusion, the main drawbacks of size-exclusion chromatography, namely low productivity and low product titer, were overcome to a considerable extent by an innovative two-column configuration that keeps the mixed fractions inside the system at all times.