Purification Process Development Research Articles

Metallothermic reduction of Cerium Chloride in molten Salt using Li, Na, and Ca Metal

Options for reducing purified metal chlorides to metallic form were evaluated in support of the development of a metal purification process based on the conversion of impure metal via hydriding followed by chlorination. Metallothermic reduction of CeCl3 was tested using Li, Ca, and Na as reductant metals with and without a molten salt solvent in MgO and Y2O3-coated MgO crucibles. The effects of added reductant metal form, crucible material and dimensions, and mixture hold times above Ce melting temperature were studied. Direct contact of Li and CeCl3 was ineffective due to their immiscibility. A low-density alloy was formed between Li and Ce using Li in a LiCl solvent. Na was ineffective at reducing CeCl3 to any measurable degree. The highest percent recovery (71.4 %) and consolidation of Ce was achieved when using Ca in a CaCl2-NaCl solvent with 99.8 % removal of CeCl3 from the salt. Side reaction with MgO crucibles was found to be at least partially responsible for reduced Ce recovery.

Accelerated Development of AAV Purification Process Using a High-Throughput and Automated Crossflow System

Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr® Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF/DF process was further transferred to a larger scale to the Sartoflow® Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon® Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.

Challenges and solutions for the downstream purification of therapeutic proteins.

The innovation in recombinant protein technology has brought forth a host of challenges related to the purification of these therapeutic proteins. This article delves into the intricate landscape of developing purification processes for artificially designed therapeutic proteins. The key hurdles include controlling protein reduction, protein capture, ensuring stability, eliminating aggregates, removing host cell proteins and optimizing protein recovery. In this review, we outline the purification strategies in order to obtain products of high purity, highlighting the corresponding solutions to circumvent the unique challenges presented by recombinant therapeutic proteins, and exemplify the practical applications by case studies. Finally, a perspective towards future purification process development is provided.

Adaptation of an rVSV Ebola vaccine purification process for rapid development of a viral vaccine candidate for SARS-CoV-2.

During the COVID-19 pandemic, long development timelines typically associated with vaccines were challenged. The urgent need for a vaccine provided a strong driver to reevaluate existing vaccine development approaches. Innovative approaches to regulatory approval were realized, including the use of platform-based technology. In collaboration with the International AIDS Vaccine Initiative, Inc. (IAVI), Merck & Co., Inc., Rahway, NJ, USA rapidly advanced an investigational SARS-CoV-2 vaccine based on the recombinant vesicular stomatitis virus (rVSV) platform used for the Ebola vaccine ERVEBO (rVSV∆G-ZEBOV-GP). An rVSV∆G-SARS-CoV-2 vaccine candidate was generated using the SARS-CoV-2 spike protein to replace the VSV G protein. The purification process development for this vaccine candidate was detailed in this paper. Areas were highlighted where the ERVEBO platform process was successfully adopted and where additional measures were needed for the SARS-CoV-2 vaccine candidate. These included: (i) endonuclease addition directly into the bioreactor prior to harvest, (ii) inclusion of a core-shell chromatography step for improved purification, and (iii) incorporation of a terminal, sterile filtration step to eliminate the need for aseptic, closed processing. High infectious virus titers were achieved in Phase 3 clinical drug substance (>108 PFUmL-1 ), and process consistency was demonstrated across four large scale batches that were completed in 6 months from clone selection.

(Invited) Synthesis of Ion-Endohedral Fullerenes by Plasma Implantation

Endohedral metallofullerenes (EMFs), which are the hybrid materials consisting of fullerene cage and encapsulated metal(s), are one of the well-studied nanocarbon materials during the past 3 decades.[1] As a result of significant progress in the development of synthetic procedures and purification processes, a variety of different types of EMFs, such as mono-metallofullerenes, di-metallofullerenes and metal-cluster-fullerenes with various carbon-cage sizes, are currently available. We previously reported development of an efficient method to access EMFs, so-called plasma implantation.[2] This approach allowed us to achieve scalable synthesis of lithium-ion-encapsulated [60]fullerene Li+@C60 X−, a stable ionic form of lithium encapsulated fullerene. The compound can be regarded as “ion-endohedral fullerene” consisting of encapsulated ion, neutral fullerene cage and external counter anion X−, and it should not be categorized into general metallofullerenes because of its ion-pair structure. Despite the unique properties arising from its ionic natures, no other ionic metallofullerenes have been well studied so far.[3] For systematic and deeper studies on ion-endohedral fullerenes, we focused on Li+-endohedral [70]fullerene (Li+@C70) because C70 is the second abundant and therefore widely studied fullerene next to C60. The Li+@C70 was synthesized as PF6 − and TFSI− (TFSI=bis(trifluoromethanesulfonyl)imide) salts using our improved plasma implantation method followed by oxidative purification process. 7Li NMR spectrum showed a sharp singlet signal at −32.81 ppm (vs LiCl/D2O standard) indicating that the Li+ was encapsulated in the C70 cage. 13C NMR spectrum showed 5 aromatic peaks, which evidenced the defectless C70 structure. In addition to the NMR signals originated from the Li+@C70 core, 19F and 31P NMR spectra showed clear signals assigned to each external counter anion. The structure of Li+@C70 was unambiguously confirmed by X-ray crystal structure analysis of the TFSI− salt. The crystal structure showed encapsulation of Li+ by C70 cage as well as the 1:1 ion-paired structure of the cationic Li+@C70 core and external TFSI− anion.Moreover, we also performed collision experiments using sodium plasma to synthesize sodium-ion-endohedral [60]fullerene. Laser desorption ionization (LDI) mass spectrum of the crude products showed signals at m/z= 720 and 743 assignable to the molecular ion of empty C60 and the Na+@C60, respectively. The target compound was isolated as TFSI− salt by oxidative purification process followed by electrolyte-added HPLC. There was only a mass peak at m/z= 743 after the purification, which evidenced the complete removal of empty C60. 23Na NMR spectrum showed a sharp singlet signal at −59.6 ppm (vs NaCl/D2O standard), evidenced the formation of the target Na+@C60.

Towards the effective distillation sequences for the purification of acetone-butanol-ethanol from the condensate of gas stripping-vapor permeation system

Gas stripping-vapor permeation (GSVP) is an attractive method for in situ acetone-butanol-ethanol (ABE) separation from fermentation broth to obtain renewable biological n-butanol. However, the downstream distillation systems for effective purification of ABE from the upstream GSVP process has not been developed. In this study, focusing on the generation of high purify A-B-E solvents as the final products, the downstream distillation systems for GSVP condensate purification were investigated. According to literature survey, four candidate distillation sequences were selected and optimized by steady-state simulation, followed by cross-over comparisons of energy requirements, techno-economics and environmental influences. The results indicate that the Scenario 3 that containing three individual columns and one decanter exhibited the lowest sensitivity to the key factors, lowest total annual cost (6.483 × 106 $/yr) and CO2 emission (3707.23 kg/h) in a scheme with 30 kt/yr n-butanol production among the four candidate distillation sequences. This study provides a valuable reference for the development of downstream ABE separation and purification processes in future works.

Aqueous biphasic systems comprising analogues of glycine-betaine ionic liquids: Toward greener separation platforms

The use of biobased and bioactive compounds has boosted the development of sustainable extraction and purification processes, in which ionic liquids have been playing a major role. Ionic-liquid-based aqueous biphasic systems (IL-based ABS) have been raising significant interest as advantageous extraction and purification platforms; however, imidazolium-based ILs have been the primary option as phase-forming agents. Aiming the development of more sustainable IL-based ABS, in this work, six novel analogues of glycine-betaine ILs (AGB-ILs) were investigated. These ILs were synthesized and characterized in terms of purity, physico-chemical properties, ecotoxicity and ability to form ABS with tri-potassium citrate. The investigated AGB-ILs are composed of tri(n-alkyl[2-ethoxy-2-oxoethyl]ammonium/phosphonium and tri(n-alkyl[4-ethoxy-4-oxobutyl]ammonium/phosphonium cations with ethyl, n-propyl and n-butyl substituents, and the tosylate ([Tos]-) anion. The synthesized ILs present degradation temperatures in the range from 220 to 265 °C, and are classified as relatively harmless regarding their ecotoxicity. The studied AGB-ILs display slightly lower thermal stability, but yet a lower ecotoxicity compared to the commercial IL 1-butyl-3-methylimidazolium tosylate. Their ability to form ABS was assessed, allowing to conclude that an expansion in the alkyl side chain length and spacer, as well as the presence of phosphonium instead of ammonium as central atom, increase the ability for phase separation in presence of tri-potassium phosphate aqueous solutions. The extraction performance of the studied AGB-ILs-based ABS was finally assessed with furfural, a platform chemical. In the majority of the studied systems, furfural preferentially migrates to the AGB-IL-rich phase, obtaining extraction efficiencies in a single step to the IL-rich phase up to 98%. These results reveal that novel ABS comprising AGB-ILs can be potentially used as separation platforms of bioactive compounds.

Characterisation of the E. coli HMS174 and BLR host cell proteome to guide purification process development.

Mass-spectrometry-based proteomics is increasingly employed to monitor purification processes or to detect critical host cell proteins in the final drug substance. This approach is inherently unbiased and can be used to identify individual host cell proteins without prior knowledge. In process development for the purification of new biopharmaceuticals, such as protein subunit vaccines, a broader knowledge of the host cell proteome could promote a more rational process design. Proteomics can establish qualitative and quantitative information on the complete host cell proteome before purification (i.e., protein abundances and physicochemical properties). Such information allows for a more rational design of the purification strategy and accelerates purification process development. In this study, we present an extensive proteomic characterisation of two E.coli host cell strains widely employed in academia and industry to produce therapeutic proteins, BLR and HMS174. The established database contains the observed abundance of each identified protein, information relating to their hydrophobicity, the isoelectric point, molecular weight, and toxicity. These physicochemical properties were plotted on proteome property maps to showcase the selection of suitable purification strategies. Furthermore, sequence alignment allowed integration of subunit information and occurrences of post-translational modifications from the well-studied E.coli K12 strain.

Label-free shotgun proteomics: Exploiting a reliable and sensitive method to monitor residual host-cell proteins in monoclonal antibody products

The removal of host cell proteins (HCPs) remains a challenge in the downstream processing of monoclonal antibodies (mAbs). It is critical to monitor residual HCPs since they can have an impact on product stability and safety. The enzyme-linked immunosorbent assay (ELISA) only quantifies the total amount of HCPs and does not indicate the identity or quantity of any specific HCP. Mass spectrometry-based proteomics applications demonstrate an advantage in quantitatively profiling individual HCPs. However, technical reproducibility, dynamic range, and ensuring an acceptable statistical significance of scoring measurements are critical hurdles that ought to be overcome. In this paper, we describe a sensitive and reproducible shotgun proteomics approach that includes an efficient mAb depletion strategy and addresses the shortcomings of such workflows. Our methodology is potentially a valuable complement or alternative to ELISA. Additionally, it can facilitate the purification process development and the evaluation of ELISA kits upon process changes.

Universal protocol and standard-spiking strategy for profiling of host cell proteins in therapeutic growth hormone

Liquid chromatography coupled to mass spectrometry (LC-MS) is widely used for host cell proteins (HCP) identification in antibody drug development because of its sensitivity, selectivity, and adaptability. However, LC-MS based identification of HCP in biotherapeutics produced from the prokaryotic Escherichia coli-derived growth hormone (GH) has rarely been reported. Herein, we developed a universal and powerful workflow by combining optimized sample preparation with one-dimension ultra-high performance LC-MS based shotgun proteomics to support HCP profiling in GH samples from downstream pools and the final product, which would be beneficial to direct the purification process development and compare the difference of impurity of different products for guiding the development of the biosimilar. A standard-spiking strategy was also developed to increase the depth of HCP identification. Spiking with standards enables additional identification of HCP species, which is promising for trace-level HCP analysis. Our universal and standard-spiking protocols would open an avenue for profiling HCP in biotherapeutics derived from prokaryotic host cells.

Development of Oil Purification Process in Metal Sheet Products

Today, the use of metal sheet is quite high in order to provide the required strength and endurance values in the automotive industry. Metal sheets take form by going through different operations. Special oils are used in order to create the geometric forms they have easily and to prevent the sheet metal from being damaged. These used oils come into contact with metals during the geometric forming and machining processes and most of them remain on the metals. These oils cause difficulties in operations such as welding, coating and painting after the forming process. For this reason, a clean surface is considered as the most important step in the preliminary preparation of the next processes. There is an increase in the stickiness of the oil on the metal, which reaches a certain temperature by applying the welding process. This situation causes problems such as non-adhesion to the surface and peeling of the coating in the powder paint and zinc coating applied to increase the corrosion resistance of metals and the life of the product. In addition, when the oil residues on the metal are not cleaned, undesirable boiling occurs in the coating pool and causes an ash problem. 
 In this study, it is aimed to develop a metal-specific degreasing process to clean the oils of different viscosities used in different processes while forming the geometric form of the metal. Thus, the surface quality will increase and it will be ensured that the errors that occur in the next operations that require surface cleaning will be prevented.

Demonstration of continuous gradient elution functionality with automated liquid handling systems for high-throughput purification process development

Various high-throughput systems and strategies are employed by the biopharmaceutical industry for early to late-stage process development for biologics manufacturing. The associated increases to experiment productivity and reduction in material consumption makes high throughput tools integral for bioprocess development. While these high-throughput systems have been successfully leveraged to generate high quality data representative of manufacturing scale processes, their data interpretation often requires complex data transformation and time-intensive system characterization. With respect to high throughput purification development, RoboColumns by Repligen operated on Tecan automated liquid handling systems offer superior performance scalability, but lack an optimized liquid delivery system that is representative of preparative chromatography. Particularly, stock Tecan liquid handling systems lack the capability to provide high-capacity continuous liquid flow and ideal linear gradient chromatography conditions. These limitations impact protein chromatography performance and hinder the application of high-throughput gradient elution experiments. In this work, we describe a Tecan Freedom EVO high-throughput purification tool that provides more continuous liquid delivery enabling continuous gradient elution capability for RoboColumn experiments as demonstrated by generation of highly linear conductivity gradients. Results demonstrate that the tool can provide RoboColumn performance and product quality data that is in agreement with larger, bench scale chromatography formats for two model purification methods. The described gradient purification method also provides more consistent performance between RoboColumns and larger column formats compared to step elution methods using the same optimized Tecan system. Lastly, new insights into the impact of discontinuous flow on RoboColumn elution performance are introduced, which may help further improve application of these data towards bioprocess development.

Identification and Quantification of a Problematic Host Cell Protein to Support Therapeutic Protein Development

Monitoring of residual host cell proteins (HCPs) in therapeutic protein is essential to ensure product quality, safety and efficacy. Despite the development of advanced mass spectrometry techniques and optimized workflows, identifying and quantifying all problematic HCPs present at low levels remain challenging. Here, we developed a practical, effective strategy for the identification and quantification of low abundance HCPs, which facilitates the improvement of downstream purification process to eliminate potentially problematic HCPs. A case study of using this strategy to investigate a problematic HCP is presented. Initially, a commonly used native digestion approach coupled with UPLC-MS/MS was applied for HCP profiling, wherein several lipases and proteases were identified in a monoclonal antibody named mAb1 in early stages of purification process development. A highly active lipase, liver carboxylesterase (CES), was found to be responsible for polysorbate 80 degradation. To facilitate process improvement, after the identification of CES, we developed a highly sensitive LC-MS/MS-MRM assay with a lower limit of quantification of 0.05 ppm for routine monitoring of the CES in mAb1 produced through the different processes. This workflow was applied in low-level lipase identification and absolute quantification, which facilitated the investigation of polysorbate degradation and downstream purification improvement to further remove the problematic HCP. The current MRM method increased the sensitivity of HCP quantification by over 10-fold that in previously published studies, thus meeting the needs for quantification of problematic HCPs at sub-ppm to ppb levels during drug development. This workflow could be readily adapted to the detection and quantification of other problematic HCPs present at extremely low levels in therapeutic protein drug candidates.

Development of versatile affinity-based system for one step purification process: Case of Group A Streptococcus vaccine.

Affinity capture is one of the most attractive strategies for simplifying downstream processing. Although it is a key mainstream approach for antibody purification, the same is not true for other biologics such as vaccines, mainly due to the lack of suitable affinity material. In this study, a novel custom affinity system is introduced permitting widespread adoption of affinity capture for the purification of biologics beyond antibodies. This is illustrated here by the development of a one-step purification process of a mutant form of streptolysin O (SLO), a vaccine candidate against Streptococcus pyogenes infection. The system consists of the association of custom ligands based on the Nanofitin protein scaffold, with Eshmuno® industry-grade chromatography medium. The Nanofitins were selected for their specificity to the target product. The newly developed affinity medium was used at different column sizes to monitor scalability from process development (1 ml) and robustness verification (5 ml) to pilot (133 ml) and technical (469 ml) runs. The single-step affinity purification consistently delivered high purity product (above > 90%) and improved performances compared with the current three-step process: reduced process time and footprint (3 to 1 step) and increased product yields (0.31 g vs. 0.04 g of SLO per kg of harvest broth). The custom affinity system herein described can potentially be applied to any biologic for which a specific Nanofitin is identified, thus establishing a platform with a strong impact on the manufacturing of vaccines and other biological targets.

Evaluation of lentiviral vector stability and development of ion exchange purification processes.

In this manuscript, we employ parallel batch stability and chromatographic screens in concert with linear and step gradient experiments to develop a high yield, HCP clearance anion exchange capture process for lentiviral vector (LVV) purification. An initial broad resin screen is carried out to determine anion exchange-based resins that exhibit high recovery of LVV. LVV stability is then evaluated and conditions are established where the vector exhibits good stability, namely phosphate buffer at pH 6.5-7.5, with low to moderate salt concentrations. A subsequent high-throughput batch screen is then carried out with a subset of resins selected from the first screen under stable conditions to identify optimal wash and elution steps to further improve product yield and protein clearance. Linear gradient experiments are also conducted in mini-column format to refine the operating conditions and final step gradient processes are established that exhibit greater than 70% yield of infectious LVV while also achieving up to 2.89 log reduction values (LRV) of HCPs during the process. The large set of stability and chromatographic data provided in this work represent an important contribution to knowledge in the field about the chromatographic efficacy of a wide range of resins for LVV bioprocessing under stable conditions.

Manufacturing considerations underpinning viral and non-viral platform selection

With an ever-increasing range of viral and non-viral technologies available to advanced biotherapeutics developers, manufacturing considerations must play a key role in the decision-making process behind platform selection. These considerations include the current level of innovation in the bioprocessing toolkit and its corresponding capability to address the specific challenges facing individual technology areas.In this article a panel of experts spanning the lentiviral vector (LVV), exosome, and oncolytic virus fields discuss the impact of manufacturing considerations on their respective platform selection and ongoing product/process development strategies, comparing the state of the art in enabling technology in each application area, and discussing related challenges, needs, and opportunities.Allison Hagerman, P.Eng., PMP, MBTVice President, Product Development, Oncolytics BiotechA Professional Engineer focused on biotechnology, Ms. Hagerman joined Oncolytics in 2010 and has been integral to the progress of its product development program ever since. Prior to being appointed as Vice President of Product Development, Ms. Hagerman was the Director, Manufacturing and Engineering from 2013-2017 and Project Manager from 2010-2013, during which time she led the process performance qualification for pelareorep drug substance. Ms. Hagerman is a Professional Engineer (P.Eng., APEGA) and Project Management Professional (PMP, PMI). She holds a Master of Biomedical Technology (MBT) degree from the University of Calgary, and B.Sc. degrees in both Chemical Engineering and Biological Sciences. She is an accomplished equestrian and spends her spare time on horseback.Aaron NoyesVice President of Development, Codiak BiosciencesAaron is a Vice President of Integrated Drug Substance Development at Codiak BioSciences where he leads a team focused on developing scalable production processes for exosomes and robust technology to load varied payloads into exosomes. At the start of his industrial career, Aaron worked at Millipore before joining Wyeth Biopharma/Pfizer Biotech for 12 years where he developed purification processes and focused on scale-up of biologics, including mAbs, recombinant proteins, ADCs, vaccines, cell therapies, and viral vectors. Aaron received a B.S in Biochemistry from the University of Massachusetts at Amherst, a M.E in Biotechnology Engineering from Tufts University and an Engineering Doctorate in Biochemical Engineering at University College London.Laurens SierkstraSenior Director, Business Leader, Purification Thermo Fisher ScientificLaurens received his PhD in biotechnology in 1994 from the University of Utrecht after studying biology at the University of Leiden. He then joined Unilever as Project Manager and Unit Leader. In 2005, after the spinout of BAC BV from Unilever, he became CEO of BAC BV and set up the business in using single- domain antibodies for affinity purification, called CaptureSelect, which was sold in 2013 to Life Technologies. Since the acquisition by Thermo Fisher Scientific, he has been the business leader for the affinity purification business within the Bioproduction Division.Dr. Vladimir SlepushkinGlobal Head of Manufacturing, MedTherapy BiotechnologyDr. Vladimir Slepushkin is Global Head of Manufacturing at MedTherapy Biotechnology. He is leading all functions associated with manufacturing of CAR-T cells and viral vectors. Previously he was Executive Director of Vector Technology at Autolus Therapeutics, leading process development for manufacturing of lentivirus vectors in suspension cell culture, guiding assay development to support process de¬velopment for lentiviral vectors, managing CMO for GMP vector production and T-cell processing. Before that, Dr. Slepushkin was directing research vector core, and providing lentiviral, retroviral and AAV vectors for Kite Pharma. Vladimir proved successful in developing novel high-quality products by managing diverse technical groups and cross-functional teams, developing first-in-class clinical product from scratch, including facilities, equipment, manufacturing process, quality systems, regulatory CMC submissions and clinical trials design. He has proven expertise in technically understanding and leading the development and improvement of cell culture and purification processes, and operations and analytical methods, adhering to customer, regulatory, safety and environmental requirements and guidelines. Vladi¬mir is experienced in identifying and resolving regulatory and manufacturing technical problems, as well as intellectual property assessment and licensing. He has authored 61 scientific papers in peer-reviewed journals and he’s an author on 14 patents and patent applications.

Excellent removal of knob-into-hole bispecific antibody byproducts and impurities in a single-capture chromatography

Bispecific antibodies (bsAbs) are therapeutically promising due to their ability to bind to two different antigens. However, the bsAb byproducts and impurities, including mispaired homodimers, half-antibodies, light chain mispairings, antibody fragments and high levels of high molecular weight (HMW) species, all pose unique challenges to their downstream processing. Here, using two knob-into-hole (KiH) constructs of bsAbs as model molecules, we demonstrate the excellent removal of bsAb byproducts and impurities in a single Protein A chromatography under optimized conditions, including hole–hole homodimer mispaired products which are physicochemically very similar to the target bsAbs and still present even with the use of the KiH format, though at reduced levels. The removal occurs through the incorporation of an intermediate low-pH wash step and optimal elution conditions, achieving ~ 60% monomeric purity increase in a single Protein A step, without the introduction of sequence-specific bsAb modifications to specifically induce differential Protein A binding. Our results also suggest that the higher aggregation propensity of bsAbs may cause aggregation during the column process, hence an optimization of the appropriate loading amount, which may be lower than that of monoclonal antibodies (mAbs), is required. With the use of loading at 50% of 10% breakthrough (QB10) at 6-min residence time, we show that an overall high monomer purity of 92.1–93.2% can be achieved with good recovery of 78.4–90.6% within one capture step, which is a significant improvement from a monomer purity of ~ 30% in the cell culture supernatant (CCS). The results presented here would be an insightful guidance to all researchers working on the purification process development to produce bispecific antibodies, especially for knob-into-hole bispecific antibodies.Graphical

Recent advances to accelerate purification process development: A review with a focus on vaccines

The safety requirements for vaccines are extremely high since they are administered to healthy people. For that reason, vaccine development is time-consuming and very expensive. Reducing time-to-market is key for pharmaceutical companies, saving lives and money. Therefore the need is raised for systematic, general and efficient process development strategies to shorten development times and enhance process understanding. High throughput technologies tremendously increased the volume of process-related data available and, combined with statistical and mechanistic modeling, new high throughput process development (HTPD) approaches evolved. The introduction of model-based HTPD enabled faster and broader screening of conditions, and furthermore increased knowledge. Model-based HTPD has particularly been important for chromatography, which is a crucial separation technique to attain high purities. This review provides an overview of downstream process development strategies and tools used within the (bio)pharmaceutical industry, focusing attention on (protein subunit) vaccine purification processes. Subsequently high throughput process development and other combinatorial approaches are discussed and compared according to their experimental effort and understanding. Within a growing sea of information, novel modeling tools and artificial intelligence (AI) gain importance for finding patterns behind the data and thereby acquiring a deeper process understanding.

All-in-one biofabrication and loading of recombinant vaults in human cells

One of the most promising approaches in the drug delivery field is the use of naturally occurring self-assembling protein nanoparticles, such as virus-like particles, bacterial microcompartments or vault ribonucleoprotein particles as drug delivery systems (DDSs). Among them, eukaryotic vaults show a promising future due to their structural features, in vitro stability and non-immunogenicity. Recombinant vaults are routinely produced in insect cells and purified through several ultracentrifugations, both tedious and time-consuming processes. As an alternative, this work proposes a new approach and protocols for the production of recombinant vaults in human cells by transient gene expression of a His-tagged version of the major vault protein (MVP-H6), the development of new affinity-based purification processes for such recombinant vaults, and the all-in-one biofabrication and encapsulation of a cargo recombinant protein within such vaults by their co-expression in human cells. Protocols proposed here allow the easy and straightforward biofabrication and purification of engineered vaults loaded with virtually any INT-tagged cargo protein, in very short times, paving the way to faster and easier engineering and production of better and more efficient DDS.

Development of an Industry-Applicable Distillation Purification Process to Produce High-Purity Furfural

Development of an Industry-Applicable Distillation Purification Process to Produce High-Purity Furfural