Process simulation based decisional tool to evaluate strategies for continuous downstream bioprocess implementation - A CDMO perspective

To maintain a competitive space in the rapidly expanding and highly competitive market, many biopharmaceutical companies are outsourcing to contract development and manufacturing organizations (CDMOs) to accelerate research and development, shorten the time to market, alleviate internal capacity and technical constraints, and reduce risks associated with production [1]. To acquire new and maintain current clients, CDMOs must have strong, diverse technical offerings for development, manufacture, and testing of products with competitive pricing and timelines [2]. Adopting innovative technologies like continuous downstream processing can help debottleneck the process and reduce processing time, which is the most appealing to CDMOs as it translates to an increased number of batches per year. The majority of continuous processing assessments to date have focused on cost of goods and not on the time reduction potential [3-7]. End-to-end continuous downstream processing is not always practical as CDMOs must accommodate a wide range of molecules and processes. Hence, it is imperative to evaluate and customize continuous production based on client needs. Application of process simulation as a decisional tool to select an appropriate downstream processing strategy was evaluated. Two modelling programs were evaluated: BioSolve Process and SuperPro Designer®. Fully continuous and hybrid (continuous Protein A operation only) downstream processing were assessed for a 2000 L fed-batch bioreactor producing 1, 5, and 10 g/L of monoclonal antibody at 40 and 200 kg production demands. Hybrid and continuous processing decreased batch duration by 20% and 60%, respectively. Continuous processing was more favorable for higher titer processes (≥ 5 g/L). The largest cost reductions were observed for 5 and 10 g/L titer processes during 40 kg production. The results highlight the business case for continuous downstream bioprocessing especially at a CDMO. Selection of a processing method will be influenced by a range of factors and the impact can easily be assessed using process simulation. Therefore, it is recommended that CDMOs use process simulation to ensure the most favorable processing strategy is selected. [1] O. Gassmann, A. Schuhmacher, M. von Zedtwitz, G. Reepmeyer, The Make-or-Buy Challenge: How to In-and Outsource Innovation, Leading Pharmaceutical Innovation, Springer2018, pp. 79-110. [2] R. Hernandez, Contract Biomanufacturing Firms Become More Specialized, BioPharm International, 28 (2015) 22-27. [3] D. Pollard, M. Brower, Y. Abe, A.G. Lopes, Standardized Economic Cost Modeling for Next-Generation MAb Production, BioProcess Int, (2016). [4] A. Xenopoulos, A new, integrated, continuous purification process template for monoclonal antibodies: process modeling and cost of goods studies, Journal of biotechnology, 213 (2015) 42-53. [5] J. Hummel, M. Pagkaliwangan, X. Gjoka, T. Davidovits, R. Stock, T. Ransohoff, R. Gantier, M. Schofield, Modeling the Downstream Processing of Monoclonal Antibodies Reveals Cost Advantages for Continuous Methods for a Broad Range of Manufacturing Scales, Biotechnology journal, (2018) 1700665. [6] J. Pollock, J. Coffman, S.V. Ho, S.S. Farid, Integrated continuous bioprocessing: Economic, operational, and environmental feasibility for clinical and commercial antibody manufacture, Biotechnology progress, 33 (2017) 854-866. [7] S. Klutz, L. Holtmann, M. Lobedann, G. Schembecker, Cost evaluation of antibody production processes in different operation modes, Chemical Engineering Science, 141 (2016) 63-74

Design and optimization of membrane chromatography process for monoclonal antibody charge variant separation

Please click Additional Files below to see the full abstract

Characterizing Enterovirus 71 and Coxsackievirus A16 Virus-like Particles Production in Insect Cells

Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two viruses commonly responsible for hand, foot and mouth disease (HFMD) in children. The lack of prophylactic or therapeutic measures against HFMD is a major public health concern. Insect cell-based EV71 and CVA16 virus-like particles (VLPs) are promising vaccine candidates against HFMD and are currently under development. In this paper, the influence of insect cell line, incubation temperature, and serial passaging effect and stability of budded virus (BV) stocks on EV71 and CVA16 VLP production was investigated. Enhanced EV71 and CVA16 VLP production was observed in Sf9 cells compared to High Five (TM) cells. Lowering the incubation temperature from the standard 27 degrees C to 21 degrees C increased the production of both VLPs in Sf9 cells. Serial passaging of CVA16 BV stocks in cell culture had a detrimental effect on the productivity of the structural proteins and the effect was observed with only 5 passages of BV stocks. A 2.7x higher production yield was achieved with EV71 compared to CVA16. High-resolution asymmetric flow field-flow fractionation couple with multi-angle light scattering (AF4-MALS) was used for the first time to characterize EV71 and CVA16 VLPs, displaying an average root mean square radius of 15 +/- 1 nm and 15.3 +/- 5.8 nm respectively. This study highlights the need for different approaches in the design of production process to develop a bivalent EV71 and CVA16 vaccine. (C) 2015 Elsevier Inc. All rights reserved

Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

S-Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

A surface plasmon resonance assay to determine the effect of influenza neuraminidase mutations on its affinity with antiviral drugs.

The outbreak of pandemic influenza and its ability to spread rapidly makes it a severe threat to public health. Antiviral drugs such as oseltamivir (Roche’s Tamiflu™) and zanamivir (GlaxoSmithKline’s Relenza™) are neuraminidase (NA) inhibitors (NI), which bind more tightly to NA than its natural substrate, sialic acid. However, the virus can acquire resistance to antiviral drugs by developing single point mutations (such as H274Y) in the target protein. Thus in some cases the drugs may not be as effective as expected. The high level of inconsistency exhibited by fluorometric assays and the short half-life of the chemiluminescent assay for monitoring drug resistance lead to the need for a simple, label-free, reliable assay. To address this problem, this work focused on three main objectives: 1) to determine the binding affinities of two common anti-viral drugs (oseltamivir and zanamivir) against the influenza NA wild type and drug resistant mutants using bioinformatics software Schrodinger Suite™ 2010. 2) To develop a reliable label-free, real-time, surface plasmon resonance (SPR) assay to measure the binding affinity between influenza viral coat protein neuraminidase (wild type and mutant) and anti-viral drugs. 3) To develop an SPR inhibition assay to quantitatively compare the interactions of sialic acid, zanamivir and oseltamivir with the viral coat protein neuraminidase (wild type and mutant). The entire docking process was carried out using Schrödinger Suite™ 2010. The 2009 pandemic H1N1 neuraminidase (PDB: 3NSS) was used throughout the docking studies as the wild type structure. Five mutants (H274Y, N294S, H274N, A346N and I222V) and three ligands (sialic acid, oseltamivir and zanamivir) were built using the maestro module. The grid-based ligand docking with energetics (GLIDE) module and induced fit docking (IFD) module were used for docking studies. The binding affinities, Gibbs free energy change (∆G) and molecular mechanics-generalized born energy/ solvent accessible area (MM-GB/SA) values for wild-type NA interactions show that both the antiviral drugs studied interact strongly with the wild-type protein. The ∆G values for all antiviral interactions with mutant NA forms were reduced in magnitude, thereby indicating that they are less favourable than interactions with the wild-type protein. A similar trend was observed with MM-GB/SA results. Amongst all of the computed values, MM-GB/SA was the closest to the experimental data. In several cases, the interactions between the anti-viral drugs and NA mutants were markedly less favourable than those between sialic acid and the same mutants, indicating that these mutations could confer anti-viral resistance. Influenza NA wild-type and H274Y mutant were expressed in baculovirus expression system (BVES) in insect cells. The expressed proteins were partially purified using the standard purification techniques of anion exchange and size exclusion chromatography (SEC). A fluorometric activity assay was performed on the recombinant proteins. Both the wild type and the mutant showed similar level of activities. In addition, the recombinant NAs were used in an inhibition assay. Oseltamivir was found to be sensitive to wild type protein (IC50 = 0.59 nM) and resistant to the H274Y mutant protein (IC50 = 349.43 nM). On the other hand, zanamivir was sensitive to both wild type (IC50 = 0.26 nM) and the H274Y mutant (IC50 = 0.44 nM). This indicated that zanamivir was a more potent inhibitor than oseltamivir. These findings were in good agreement with the literature. An SPR assay for accurate monitoring of influenza antiviral drug resistance was developed. A spacer molecule (1, 6- hexanediamine) was site-specifically tethered to the inert 7-hydroxyl group of zanamivir. The tethered zanamivir was immobilized onto an SPR GLC chip to obtain a final immobilization response of 431 response units (RU). The reference subtracted binding responses obtained for NA wild-type and H274Y mutant were analysed using the ProteOn Manager™ Software tools. The SPR curves were fitted to a simple Langmuir 1:1 model with drift to obtain association rate constant (ka) and dissociation rate constants (kd). The relative binding values obtained from literature and the current SPR assay (1.9 and 1.7 respectively) suggested that the current SPR assay yielded similar results to the existing labelled enzymatic assay. In addition, an SPR inhibition assay was developed. The calculated IC50-spr values were compared and it was observed that oseltamivir was sensitive to wild type protein (IC50-spr = 7.7 nM) and resistant to the H274Y mutant protein (IC50-spr = 256 nM). On the other hand, zanamivir was sensitive to both wild type (IC50-spr = 2.16 nM) and the H274Y mutant (IC50-spr = 2.4 nM). Sialic acid was also found to be sensitive to both wild type (IC50-spr = 5.5 nM) and H274Y mutant (IC50-spr = 3.25 nM). In the cases studied, the viral proteins remained sensitive to sialic acid, consistent with retention of virulence of these mutant strains. It was concluded that zanamivir is a more potent inhibitor than oseltamivir for treating the H274Y mutant. Comparison of the SPR inhibition results with the docking results revealed a similar trend. The wild-type NA and H27Y mutant retained binding affinity for sialic acid and zanamivir. Oseltamivir showed a significant decrease in binding affinity for the H274Y mutant compared with the wild-type. This was because of the disruption of the salt bridge formation within NA that was vital for oseltamivir activity. To my knowledge, this is the first SPR biosensor assay developed to monitor influenza antiviral drug resistance. There is a tremendous scope to extend this study to more mutants and new antiviral drugs. This could pave the way for a reliable SPR biosensor assay to replace low consistency labelled enzymatic assays

Progression of continuous downstream processing of monoclonal antibodies: current trends and challenges

Rapid advances in intensifying upstream processes for biologics production have left downstream processing as a bottleneck in the manufacturing scheme. Biomanufacturers are pursuing continuous downstream process development to increase efficiency and flexibility, reduce footprint and cost of goods and improve product consistency and quality. Even after successful laboratory trials, the implementation of a continuous process at manufacturing scale is not easy to achieve. This paper reviews specific challenges in converting each downstream unit operation to a continuous mode. Key elements of developing practical strategies for overcoming these challenges are detailed. These include equipment valve complexity, favourable column aspect ratio, Protein-A resin selection, quantitative assessment of chromatogram peak size and shape, holistic process characterization approach and a customized process economic evaluation. Overall, this article provides a comprehensive review of current trends and the path forward for implementing continuous downstream processing at the manufacturing scale. This article is protected by copyright. All rights reserved

Intensified downstream processing of monoclonal antibodies using membrane technology

The need to intensify downstream processing of monoclonal antibodies to complement the advances in upstream productivity has led to increased attention towards implementing membrane technologies. With the industry moving towards continuous operations and single use processes, membrane technologies show promise in fulfilling the industry needs due to their operational flexibility and ease of implementation. Recently, the applicability of membrane-based unit operations in integrating the downstream process has been explored. In this article, we review the major developments in the application of membrane-based technologies in the bioprocessing of monoclonal antibodies. We focus on the recent progress towards developing intensified end-to-end bioprocesses and the critical role membrane technology will play in achieving this goal. This article is protected by copyright. All rights reserved

Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which Detchanamurthy, S. et al. 7 modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies

Cloning and expression of S-Adenosyl Methionine Synthetase gene in recombinant E. coli strain for large scale production of SAMe

Adenosyl Methionine (SAMe) Synthetase is an enzyme which catalyses the synthesis of S-Adenosyl Methionine using methionine and ATP. It is also known as AdoMet which is well known methyl donor, which modifies DNA, RNA, histones and other proteins, dictating replicational, transcriptional and translational fidelity, mismatch repair, chromatin modeling, epigenetic modifications and imprinting. The objective of the present work is to clone the SAMe Synthetase gene in recombinant E. coli strain in order to express, characterize and purify it for further synthesis of SAMe in a large scale fermentation. Expression was induced by 1 mM IPTG and expressed protein was characterized by SDS-PAGE. The recombinant E. coli cells were used for the production of SAMe through batch and fed batch fermentation operations. The produced SAMe was purified through paper chromatography in order to use it in our future studies