An integrated modelling/experimental framework for protein-producing cell cutures

Imperial Users onl

Hybrid dynamic model of monoclonal antibody production using CHO cells

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A model-based approach towards accelerated process development: A case study on chromatography

Process development is typically associated with lengthy wet-lab experiments for the identification of good candidate setups and operating conditions. In this paper, we present the key features of a model-based approach for the identification and assessment of process design space (DSp), integrating the analysis of process performance and flexibility. The presented approach comprises three main steps: (1) model development & problem formulation, (2) DSp identification, and (3) DSp analysis. We demonstrate how such an approach can be used for the identification of acceptable operating spaces that enable the assessment of different operating points and quantification of process flexibility. The above steps are demonstrated on Protein A chromatographic purification of antibody-based therapeutics used in biopharmaceutical manufacturing.Comment: Pre-print paper under revie

NEXT-FLUX (Neural-net EXtracellular Trained Flux Constraints)

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Low-cost and user-friendly biosensor to test the integrity of mRNA molecules suitable for field applications

The use of mRNA in biotechnology has expanded with novel applications such as vaccines and therapeutic mRNA delivery recently demonstrated. For mRNA to be used in patients, quality control assays will need to be routinely established. Currently, there is a gap between the highly sophisticated RNA integrity tests available and broader application of mRNA-based products by non-specialist users, e.g. in mass vaccination campaigns. Therefore, the aim of this work was to develop a low-cost biosensor able to test the integrity of a mRNA molecule with low technological requirements and easy end-user application. The biosensor is based on a bi-functional fusion protein, composed by the λN peptide that recognizes its cognate aptamer encoded on the 5’ end of the RNA under study and β-lactamase, which is able to produce a colorimetric response through a simple test. We propose two different mechanisms for signal processing adapted to two levels of technological sophistication, one based on spectrophotometric measurements and other on visual inspection. We show that the proposed λN-βLac chimeric protein specifically targets its cognate RNA aptamer, boxB, using both gel shift and biolayer interferometry assays. More importantly, the results presented confirm the biosensor performs reliably, with a wide dynamic range and a proportional response at different percentages of full-length RNA, even when gene-sized mRNAs were used. Thus, the features of the proposed biosensor would allow to end-users of products such as mRNA vaccines to test the integrity of the product before its application in a low-cost fashion, enabling a more reliable application of these products

Controlling fab terminal sialylation of antibodies through culture conditions

Biologics are used for the treatment of a wide-range of diseases with specificity and minimal side effects. Safety and efficacy of the drugs has been linked to carbohydrate structures found on the antibody, termed N-linked glycans. These glycans are mainly found within the Fc-region of an antibody but 20% of all IgG antibodies also contain Fab glycans1. Glycans are composed of a range of sugars whose presence or absence affects the biological qualities of a drug. Sialic acid is one such sugar; its role is to “cap” the glycan chain, protecting the internal sugars, which when exposed are bound by receptors and cleared to host lysosomes2. The presence of sialic acid is linked to an increase in biologic half-life along with a reduced inflammatory response3. It has been established that bioprocess conditions such as cell culture temperature and pH directly impact glycan composition and site occupancy. A shift in the cell culture pH and its effect on the sialic acid content within the Fab region of an antibody product was examined. The product was produced in three CHO cell clones, each innately producing varying levels of sialic acid. An initial experiment utilized the Ambr 24® to run cultures at pH 7.1, 6.8 and with a shift from 7.1 to 6.8 on day 6. Data from this was used to establish a second experiment, utilizing an Ambr 48® system. This experiment looked at the supplementation of ManNAc, copper as well as the effect of pH and temperature shifts on sialylation. Glycan analysis was undertaken using a novel method for triple-quadrupole MS. A pH shift was found to produce overall more processed glycans. Although cell growth was negatively affected, antibody productivity and specific rate of sialylation were both increased at reduced culture pH (Figure 1). The extent of the effect differed between the clones and was correlated to how early the shift occurred. Due to the negative effects on growth, overall antibody yield was reduced with some clones having less than half that of the respective control. To determine the origin of the effect and the differences between the clones further analysis is being undertaken. The Ambr 48® experiment determined the effect of different supplements on sialylation, as well as the effect of a temperature shift and further understanding of the role of cell culture pH in increasing sialylation. Flux balance analysis and expression analysis of the enzymes involved in terminal glycosylation process is underway. Please click Additional Files below to see the full abstract

Immobilized enzyme cascade for targeted glycosylation

Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method. We reconstruct a reaction cascade mimicking a glycosylation pathway where promiscuity naturally exists to humanize a range of proteins derived from different cellular systems, yielding near-homogeneous glycoforms. Immobilized β-1,4-galactosyltransferase is used to enhance the galactosylation profile of three IgGs, yielding 80.2–96.3% terminal galactosylation. Enzyme recycling is demonstrated for a reaction time greater than 80 h. The platform is easy to implement, modular and reusable and can therefore produce homogeneous glycan structures derived from various hosts for functional and clinical evaluation.UKRI Engineering and Physical Sciences Research Council (EP/N509486/1)UKRI Engineering and Physical Sciences Research Council (EP/K038648/1, EP/H04986X/1 and EP/K038648/1)UK Research and Innovation ‘Global Challenges Research Fund’ BB/P02789X/1)UK Biotechnology and Biological Sciences Research Counci

In Situ Monitoring of Intracellular Glucose and Glutamine in CHO Cell Culture

The development of processes to produce biopharmaceuticals industrially is still largely empirical and relies on optimizing both medium formulation and cell line in a product-specific manner. Current small-scale (well plate-based) process development methods cannot provide sufficient sample volume for analysis, to obtain information on nutrient utilization which can be problematic when processes are scaled to industrial fermenters. We envision a platform where essential metabolites can be monitored non-invasively and in real time in an ultra-low volume assay in order to provide additional information on cellular metabolism in high throughput screens. Towards this end, we have developed a model system of Chinese Hamster Ovary cells stably expressing protein-based biosensors for glucose and glutamine. Herein, we demonstrate that these can accurately reflect changing intracellular metabolite concentrations in vivo during batch and fed-batch culture of CHO cells. The ability to monitor intracellular depletion of essential nutrients in high throughput will allow rapid development of improved bioprocesses