Status and future developments for downstream processing of biological products: Perspectives from the Recovery XIX yield roundtable discussions.

Governments and biopharmaceutical organizations aggressively leveraged expeditious communication capabilities, decision models, and global strategies to make a COVID-19 vaccine happen within a period of 12 months. This was an unusual effort and cannot be transferred to normal times. However, this focus on a single vaccine has also led to other treatments and drug developments being sidelined. Society expects the pharmaceutical industry to provide an uninterrupted supply of medicines. However, it is often overlooked how complex the manufacture of these compounds is and what logistics are required, not to mention the time needed to develop new drugs. The overarching theme, therefore, is patient access and how we can help ensure access and extend it to low- and middle-income countries. Despite unceasing efforts to make medications available to all patient populations, this must never be done at the expense of patient safety. A major fraction of the costs in biopharmaceutical manufacturing are for drug discovery, process development, and clinical studies. Infrastructure costs are very difficult to quantify because they often depend on whether a greenfield facility or an existing, depreciated facility is used or adapted for a new product. To accelerate process development concepts of platform process and prior knowledge are increasingly leveraged. While more traditional protein therapeutics continue to dominate the field, we are also experiencing the exciting emergence and evolution of other therapeutic formats (bispecifics, tetravalent mAbs, antibody-drug conjugates, enzymes, peptides, etc.) that offer unique treatment options for patients. Protein modalities are still dominant, but new modalities are being developed that can be learned from including advanced therapeutics-like cell and gene therapies. The industry must develop a model-based strategy for process development and technologies such as continuous integrated biomanufacturing must be adopted. The overall conclusion is that the pandemic pace was unsustainable, focused on vaccine delivery at the expense of other modalities/disease targets, and had implications for professional and personal life (work-life balance). Routinely reducing development time from 10 years to 1 year is nearly impossible to achieve. Environmental aspects of sustainable downstream processing are also described.

Insights into product and process related challenges of lentiviral vector bioprocessing.

Lentiviral vectors (LVs) are used in advanced therapies to transduce recipient cells for long term gene expression for therapeutic benefit. The vector is commonly pseudotyped with alternative viral envelope proteins to improve tropism and is selected for enhanced functional titers. However, their impact on manufacturing and the success of individual bioprocessing unit operations is seldom demonstrated. To the best of our knowledge, this is the first study on the processability of different Lentiviral vector pseudotypes. In this work, we compared three envelope proteins commonly pseudotyped with LVs across manufacturing conditions such as temperature and pump flow and across steps common to downstream processing. We have shown impact of filter membrane chemistry on vector recoveries with differing envelopes during clarification and observed complete vector robustness in high shear manufacturing environments using ultra scale-down technologies. The impact of shear during membrane filtration in a tangential flow filtration-mimic showed the benefit of employing higher shear rates, than currently used in LV production, to increase vector recovery. Likewise, optimized anion exchange chromatography purification in monolith format was determined. The results contradict a common perception that lentiviral vectors are susceptible to shear or high salt concentration (up to 1.7 M). This highlights the prospects of improving LV recovery by evaluating manufacturing conditions that contribute to vector losses for specific production systems.

An ultra scale-down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes.

The availability of material for experimental studies is a key constraint in the development of full-scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale-down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modelling, and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only millilitre quantities before embarking on highly demanding full-scale studies. In this study the performance of a pilot-scale tangential flow filtration (TFF) system based on a membrane flat-sheet cassette using pumped flow was predicted by devising an USD device comprising a stirred cell using a rotating disc. The USD device operates with just 2.1 cm2 of membrane area and, for example, just 1.7 mL of feed for diafiltration studies. The novel features of the design involve optimisation of the disc location and the membrane configuration to yield an approximately uniform shear rate. This is characterised using computational fluid dynamics for a defined layer above the membrane surface. A pilot-scale TFF device operating at ~500-fold larger feed volume and membrane area was characterised in terms of the shear rate derived from flow rate-pressure drop relationships for the cassette. Good agreement was achieved between the USD and TFF devices for the flux and resistance values at equivalent average shear rates for a monoclonal antibody diafiltration stage.

Continuous manufacturing of lentiviral vectors using a stable producer cell line in a fixed-bed bioreactor.

Continuous manufacturing of lentiviral vectors (LVs) using stable producer cell lines could extend production periods, improve batch-to-batch reproducibility, and eliminate costly plasmid DNA and transfection reagents. A continuous process was established by expanding cells constitutively expressing third-generation LVs in the iCELLis Nano fixed-bed bioreactor. Fixed-bed bioreactors provide scalable expansion of adherent cells and enable a straightforward transition from traditional surface-based culture vessels. At 0.5 vessel volume per day (VVD), the short half-life of LVs resulted in a low total infectious titer at 1.36 × 104 TU cm-2. Higher perfusion rates increased titers, peaking at 7.87 × 104 TU cm-2 at 1.5 VVD. The supernatant at 0.5 VVD had a physical-to-infectious particle ratio of 659, whereas this was 166 ± 15 at 1, 1.5, and 2 VVD. Reducing the pH from 7.20 to 6.85 at 1.5 VVD improved the total infectious yield to 9.10 × 104 TU cm-2. Three independent runs at 1.5 VVD and a culture pH of 6.85 showed low batch-to-batch variability, with a coefficient of variation of 6.4% and 10.0% for total infectious and physical LV yield, respectively. This study demonstrated the manufacture of high-quality LV supernatant using a stable producer cell line that does not require induction.

Quasi-perfusion studies for intensified lentiviral vector production using a continuous stable producer cell line.

Quasi-perfusion culture was employed to intensify lentiviral vector (LV) manufacturing using a continuous stable producer cell line in an 8-day process. Initial studies aimed to identify a scalable seeding density, with 3, 4, and 5 × 104 cells cm-2 providing similar specific productivities of infectious LV. Seeding at 3 × 104 cells cm-2 was selected, and the quasi-perfusion was modulated to minimize inhibitory metabolite accumulation and vector exposure at 37°C. Similar specific productivities of infectious LV and physical LV were achieved at 1, 2, and 3 vessel volumes per day (VVD), with 1 VVD selected to minimize downstream processing volumes. The optimized process was scaled 50-fold to 1,264 cm2 flasks, achieving similar LV titers. However, scaling up beyond this to a 6,320 cm2 multilayer flask reduced titers, possibly from suboptimal gas exchange. Across three independent processes in 25 cm2 to 6,320 cm2 flasks, reproducibility was high with a coefficient of variation of 7.7% ± 2.9% and 11.9% ± 3.0% for infectious and physical LV titers, respectively. The optimized flask process was successfully transferred to the iCELLis Nano (Cytiva) fixed-bed bioreactor, with quasi-perfusion at 1 VVD yielding 1.62 × 108 TU.

Production of indigo by recombinant bacteria.

Indigo is an economically important dye, especially for the textile industry and the dyeing of denim fabrics for jeans and garments. Around 80,000 tonnes of indigo are chemically produced each year with the use of non-renewable petrochemicals and the use and generation of toxic compounds. As many microorganisms and their enzymes are able to synthesise indigo after the expression of specific oxygenases and hydroxylases, microbial fermentation could offer a more sustainable and environmentally friendly manufacturing platform. Although multiple small-scale studies have been performed, several existing research gaps still hinder the effective translation of these biochemical approaches. No article has evaluated the feasibility and relevance of the current understanding and development of indigo biocatalysis for real-life industrial applications. There is no record of either established or practically tested large-scale bioprocess for the biosynthesis of indigo. To address this, upstream and downstream processing considerations were carried out for indigo biosynthesis. 5 classes of potential biocatalysts were identified, and 2 possible bioprocess flowsheets were designed that facilitate generating either a pre-reduced dye solution or a dry powder product. Furthermore, considering the publicly available data on the development of relevant technology and common bioprocess facilities, possible platform and process values were estimated, including titre, DSP yield, potential plant capacities, fermenter size and batch schedule. This allowed us to project the realistic annual output of a potential indigo biosynthesis platform as 540 tonnes. This was interpreted as an industrially relevant quantity, sufficient to provide an annual dye supply to a single industrial-size denim dyeing plant. The conducted sensitivity analysis showed that this anticipated output is most sensitive to changes in the reaction titer, which can bring a 27.8% increase or a 94.4% drop. Thus, although such a biological platform would require careful consideration, fine-tuning and optimization before real-life implementation, the recombinant indigo biosynthesis was found as already attractive for business exploitation for both, luxury segment customers and mass-producers of denim garments. Supplementary Information: The online version contains supplementary material available at 10.1186/s40643-023-00626-7.

Lentiviral Vector Bioprocessing.

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.

Evaluation of cell disruption effects on primary recovery of antibody fragments using microscale bioprocessing techniques.

Intracellular antibody Fab' fragments periplasmically expressed in Escherichia coli require the release of Fab' from the cells before initial product recovery. This work demonstrates the utility of microscale bioprocessing techniques to evaluate the influence of different cell disruption operations on subsequent solid-liquid separation and product recovery. Initially, the industrial method of Fab' release by thermochemical extraction was established experimentally at the microwell scale and was observed to yield Fab' release consistent with the larger scale process. The influence of two further cell disruption operations, homogenization and sonication, on subsequent Fab' recovery by microfiltration was also examined. The results showed that the heat-extracted cells give better dead-end microfiltration performance in terms of permeate flux and specific cake resistance. In contrast, the cell suspensions prepared by homogenization and sonication showed more efficient product release but with lower product purity and poorer microfiltration performance. Having established the various microscale methods the linked sequence was automated on the deck of a laboratory robotic platform and used to show how different conditions during thermochemical extraction impacted on the optimal performance of the linked unit operations. The results illustrate the power of microscale techniques to evaluate crucial unit operation interactions in a bioprocess sequence using only microliter volumes of feed.