In silico mediated development of orthogonally selective mAb downstream processes for the removal of process-related impurities.

Anion-exchange membrane adsorbers with silane-modulated biomimetic amination surface for efficient DNA separation

A new approach for downstream purification of rhamnolipid biosurfactants

Residual host cell protein impurities (HCPs) are a key component of biopharmaceutical process related impurities. These impurities need to be effectively cleared through chromatographic steps in the downstream purification process to produce safe and efficacious protein biopharmaceuticals. A variety of strategies to demonstrate robust host cell protein clearance using scale-down studies are highlighted and compared. A common strategy is the "spiking" approach, which is widely employed in clearance studies for well-defined impurities. For HCPs this approach involves spiking cell culture harvest, which is rich in host cell proteins, into the load material for all chromatographic steps to assess their clearance ability. However, for studying HCP clearance, this approach suffers from the significant disadvantage that the vast majority of host cell protein impurities in a cell culture harvest sample are not relevant for a chromatographic step that is downstream of the capture step in the process. Two alternative strategies are presented here to study HCP clearance such that relevance of those species for a given chromatographic step is taken into consideration. These include a "bypass" strategy, which assumes that some of the load material for a chromatographic step bypasses that step and makes it into the load for the subsequent step. The second is a "worst-case" strategy, which utilizes information obtained from process characterization studies. This involves operating steps at a combination of their operating parameters within operating ranges that yield the poorest clearance of HCPs over that step. The eluate from the worst case run is carried forward to the next chromatographic step to assess its ability to clear HCPs. Both the bypass and worst-case approaches offer significant advantages over the spiking approach with respect to process relevance of the HCP impurity species being studied. A combination of these small-scale validation approaches with large-scale HCP clearance data from clinical manufacturing and manufacturing consistency runs is used to demonstrate robust HCP clearance for the downstream purification process of an Fc fusion protein. The demonstration of robust HCP clearance through this comprehensive strategy can potentially be used to eliminate the need for routine analytical testing or for establishing acceptance criteria for these impurities as well as to demonstrate robust operation of the entire downstream purification process.

Biologics production using yeast or CHO with Yeastolate as cell culture additives often introduces β-glucan, which could potentially pose immunogenicity risk, if not adequately removed. Although a previous study has shown the effective clearance of Yeastolate-derived β-glucan by Protein A chromatography, the clearance pattern of yeast cell derived β-glucan remains unknown. In this study, we characterized the β-glucan clearance patterns during downstream processing of three monoclonal antibody (mAb) products, one mAb fragment from Pichia pastoris (mAb A) and two full mAbs from CHO expression system (mAb B and mAb C), by Glucatell assay. We demonstrated effective β-glucan clearance in both small (100 L) and large scale (5000 L) batches of mAb A as well as in one batch of mAb B. Protein A purification step removed an average of 97.74% (1.7 log10 reduction) of β-glucan detected in the two batches of mAb A microfiltration permeates (MFP) and 99.99% (3.9 log10 reduction) of β-glucan detected in mAb B clarified culture fluid harvest (HCCF). Residual β-glucans post Protein A purification in the two batches of mAb A were further removed by the two polishing chromatography steps (94.76% reduction on average). Residual β-glucan measured in the mAb A and mAb B drug substance ranges from 7.8 to 19 pg/mg, which is unlikely to alter physiological concentrations significantly in healthy adults when administered with typical intravenous doses. However, in mAb C, after almost complete removal (99.99%) by Protein A purification step, β-glucan level increased more than 20 fold in Viral Filtration (VF) product sample, indicating that it can be introduced from materials used in downstream process, such as cellulose-based filters and membranes. Our study results suggest that although β-glucan can be cleared by Protein A and other chromatographic steps such as AEX, monitoring β-glucan clearance during downstream process development remains very important to identify and avoid potential contaminations to the drug substance.

Emerging biomaterials for downstream manufacturing of therapeutic proteins

Preparation of liposome membrane adsorbers and testing for plasmid purification

Demonstrating comparability of secondary structure composition as part of higher order structure (HOS) in therapeutic proteins is a significant challenge. Previously, we showed that the variability of second derivative amide I Fourier transform infrared (FTIR) spectra were small enough that significant differences in secondary structures could be seen for a variety of model proteins. Those comparisons used spectral overlap and spectral correlation coefficients to quantify spectral differences. However, many of the excipients used in downstream purification process, drug substance, and drug product formulation, such as free amino acids and sugars, can interfere with the absorbance in the amide I region. In this study, analysis of amide II FTIR spectra is shown as an alternative to using spectral data from the amide I region to analyze protein secondary structure to assess their HOS. This research provided spectral overlap and spectral correlation coefficient mathematical approaches for analysis of amide II FTIR spectra to demonstrate comparability of protein secondary structure. Spectral overlap and spectral correlation coefficients results show strong correlations between changes in the second derivative of amide II and amide I FTIR spectra for various model proteins under different conditions, which demonstrate the applicability of using amide II FTIR spectra for the comparability of protein secondary structure. These results indicate that the analysis of the second derivative of amide II FTIR spectra may be used to monitor and demonstrate comparability of protein secondary structure during downstream process and formulation development of protein therapeutics.

An ex-ante Life Cycle Assessment was conducted to assess the cradle-to-factory gate environmental impact of polyethylenefuranoate (PEF). The two monomers used to synthesize a 100% bio-based PEF, namely 2,5- furan dicarboxylic acid (2,5-FDCA) and mono ethylene glycol (MEG), are synthesized simultaneously from a novel electrochemical reactor using bio-based raw materials. The technology is currently at a low Technological Readiness Level (TRL 2–3), and was scaled up to a theoretical TRL4 using process design. The purposes of this study are two folds: 1) to identify the significant environmental issues at an early development stage and 2) to gain insights into and experience of ex-ante assessment for a low-TRL bio-based innovation. The electrochemical technology investigated offers the opportunity of electrification of the chemical sector in the future. Ex-ante LCA was applied based on recently suggested TRL-frameworks. Primary data from the foreground system, covering the electrochemical reactor and the downstream purification processes, were obtained from lab-scale experiments and conceptual design. Five environmental indicators were assessed: namely, climate change, non-renewable energy use (NREU), acidification, eutrophication and land use. The results show that the electricity demand from the electrochemical reactor is the most important contributor of the environmental impacts, yet downstream processes contribute significantly as well. Future scenarios show that a carbon neutral electricity in 2050 could help to significantly reduce the climate change impact (by up to 60%). As a proof-of-concept, the assessed electrochemical reactor shows its important potential of the electrification of the chemical sector for monomer and polymer production, provided that a zero emission electricity in the future can be achieved. • Ex-ante LCA of a bio-based PEF based on an early stage electrochemical innovation. • Detailed mass balance flowsheet for the electrochemical conversion to FDCA and MEG. • Downstream processes are complex and mainly based on fossil-based heat. • Carbon neutral electricity could reduce climate change impact by up to 60%. • TERRA technology offers opportunity for future electrification of the chemical sector.

Aqueous Methods for Extraction/Recovery of Macromolecules From Microorganisms of Atypical Environments: A Focus on Three Phase Partitioning

Polyhydroxyalkanoates (PHAs) represent a category of microbial polyesters that offer both biodegradability and biocompatibility, if produced in sufficient quantities, they could serve as an alternative to many conventional plastics in use today. However, these microbial polymers are intracellularly stored, necessitating a more complex downstream extraction and purification process. Downstream processes often constitute the most financially burdensome stage in biomolecule production. One significant drawback of many existing extraction processes is their reliance on harsh organic solvents, such as chloroform, and high temperatures. This study presents and compares two novel downstream processes for the extraction and purification of poly(3-hydroxybutyrate) (PHB), a type of short-chain-length PHA, utilizing bio-based green solvents and natural deep eutectic solvents (NADES), respectively. The soil bacterium Pseudomonas putida, engineered to produce PHB from sugars, was adopted as a model for testing these extraction procedures. Initially, biomass was disrupted using a hypotonic buffer containing lysozyme to enhance the extraction efficiency in the downstream process. After extensive screening, the bio-based solvent ethyl acetate was selected for PHB extraction from P. putida biomass, yielding ∼ 95 wt% of the homo-polymer with a purity of ∼ 97 wt%, results comparable to those achieved with the traditional benchmark solvent, chloroform. Furthermore, a hydrophobic natural deep eutectic solvent (hydrophobic NADES) was synthesized, comprising L-menthol and acetic acid in a 1:3 M ratio, and employed as the extraction solvent in combination with methanol as the anti-solvent. The optimized extraction process resulted in a homo-polymer yield of ∼ 66 wt% with a high purity of ∼ 85 wt%. These results are promising considering the benefits associated with the use of NADES, they are less toxic and much easier to handle than ethyl acetate and have the potential to be recycled. Therefore, it represents a promising avenue for a more sustainable PHB extraction process, devoid of harmful organic solvents.

A rapid and sensitive ELISA to quantify an HBsAg specific monoclonal antibody and a plant-derived antibody during their downstream purification process

Efficient host cell protein clearance: A study of membrane adsorbers and resins in biopharmaceutical processes.

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) increases the need for a rapid development of efficient vaccines. Among other vaccines in clinical trials, a recombinant VSV-∆G-spike vaccine was developed by the Israel Institute for Biological Research (IIBR) and is being evaluated. The development of an efficient downstream purification process (DSP) enables the vaccine to be advanced to clinical trials. The DSP must eliminate impurities, either process- or product-related, to yield a sufficient product with high purity, potency and quality. To acquire critical information on process restrictions and qualities, the application of in-line monitoring is vital and should significantly impact the process yield, product quality and economy of the entire process. Here, we describe an in-line monitoring technique that was applied in the DSP of the VSV-∆G-spike vaccine. The technique is based on determining the concentrations of metabolites, nutrients and a host cell protein using the automatic chemistry analyzer, Cobas Integra 400 Plus. The analysis revealed critical information on process parameters and significantly impacted purification processes. The technique is rapid, easy and efficient. Adopting this technique during the purification process improves the process yield and the product quality and enhances the economy of the entire downstream process for biotechnology and bio pharmaceutical products.

Development of Vero Cell-Derived Inactivated Japanese Encephalitis Vaccine

A novel approach based on solvent displacement crystallisation for iron removal and copper recovery from solutions of semi-pilot scale bioleaching of WPCBs