Designing an Artificial Golgi reactor to achieve targeted glycosylation of monoclonal antibodies

The therapeutic efficacy of monoclonal antibodies (mAbs) is dependent on their glycosylation patterns. As the largest group of currently approved biopharmaceuticals, the microheterogeneity in mAb oligosaccharide profiles deriving from mammalian cell production is a challenge to the biopharmaceutical industry. Disengaging the glycosylation process from the cell may offer significant enhancement of product quality and allow better control and reproducibility in line with the Quality‐by‐Design paradigm. Three potential designs of an Artificial Golgi reactor implementing targeted sequential glycosylation of mAbs are proposed including a (1) microcapillary film reactor, (2) packed bed reactor with nonporous pellets, and (3) packed bed reactor with porous pellets. Detailed mathematical models are developed to predict their performance for a range of design and operational parameters. While all three reactor designs can achieve desired conversion levels, the choice of a particular one depends on the required throughput and the associated cost of enzymes and co‐substrates. © 2016 The Authors AIChE Journal published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers AIChE J, 62: 2959–2973, 2016     

‘Closing the loop’ in biological systems modeling — From the in silico to the in vitro

This work presents a holistic ‘closed loop’ approach for the development of models of biological systems. The ever-increasing availability of experimental information necessitates the advancement of a systematic methodology to organise and utilise these data. Herein, we present a biological model building framework that maps the treatment of the information from the initial conception of the model, through its experimental validation and finally to its application in model-based optimisation studies. We highlight and discuss current issues associated with the development of mathematical models of biological systems and share our perspective towards a holistic ‘closed loop’ approach that will facilitate the control of the in vitro through the in silico.     

Mechanical Cues: Bidirectional Reciprocity in the Extracellular Matrix Drives Mechano-Signalling in Articular Cartilage

The composition and organisation of the extracellular matrix (ECM), particularly the pericellular matrix (PCM), in articular cartilage is critical to its biomechanical functionality; the presence of proteoglycans such as aggrecan, entrapped within a type II collagen fibrillar network, confers mechanical resilience underweight-bearing. Furthermore, components of the PCM including type VI collagen, perlecan, small leucine-rich proteoglycans—decorin and biglycan—and fibronectin facilitate the transduction of both biomechanical and biochemical signals to the residing chondrocytes, thereby regulating the process of mechanotransduction in cartilage. In this review, we summarise the literature reporting on the bidirectional reciprocity of the ECM in chondrocyte mechano-signalling and articular cartilage homeostasis. Specifically, we discuss studies that have characterised the response of articular cartilage to mechanical perturbations in the local tissue environment and how the magnitude or type of loading applied elicits cellular behaviours to effect change. In vivo, including transgenic approaches, and in vitro studies have illustrated how physiological loading maintains a homeostatic balance of anabolic and catabolic activities, involving the direct engagement of many PCM molecules in orchestrating this slow but consistent turnover of the cartilage matrix. Furthermore, we document studies characterising how abnormal, non-physiological loading including excessive loading or joint trauma negatively impacts matrix molecule biosynthesis and/or organisation, affecting PCM mechanical properties and reducing the tissue’s ability to withstand load. We present compelling evidence showing that reciprocal engagement of the cells with this altered ECM environment can thus impact tissue homeostasis and, if sustained, can result in cartilage degradation and onset of osteoarthritis pathology. Enhanced dysregulation of PCM/ECM turnover is partially driven by mechanically mediated proteolytic degradation of cartilage ECM components. This generates bioactive breakdown fragments such as fibronectin, biglycan and lumican fragments, which can subsequently activate or inhibit additional signalling pathways including those involved in inflammation. Finally, we discuss how bidirectionality within the ECM is critically important in enabling the chondrocytes to synthesise and release PCM/ECM molecules, growth factors, pro-inflammatory cytokines and proteolytic enzymes, under a specified load, to influence PCM/ECM composition and mechanical properties in cartilage health and disease.     

Immunoglobulin G N-glycan Biomarkers for Autoimmune Diseases: Current State and a Glycoinformatics Perspective

The effective treatment of autoimmune disorders can greatly benefit from disease-specific biomarkers that are functionally involved in immune system regulation and can be collected through minimally invasive procedures. In this regard, human serum IgG N-glycans are promising for uncovering disease predisposition and monitoring progression, and for the identification of specific molecular targets for advanced therapies. In particular, the IgG N-glycome in diseased tissues is considered to be disease-dependent; thus, specific glycan structures may be involved in the pathophysiology of autoimmune diseases. This study provides a critical overview of the literature on human IgG N-glycomics, with a focus on the identification of disease-specific glycan alterations. In order to expedite the establishment of clinically-relevant N-glycan biomarkers, the employment of advanced computational tools for the interpretation of clinical data and their relationship with the underlying molecular mechanisms may be critical. Glycoinformatics tools, including artificial intelligence and systems glycobiology approaches, are reviewed for their potential to provide insight into patient stratification and disease etiology. Challenges in the integration of such glycoinformatics approaches in N-glycan biomarker research are critically discussed.     

Osmolality Effects on CHO Cell Growth,Cell Volume,Antibody Productivity and Glycosylation

The addition of nutrients and accumulation of metabolites in a fed-batch culture of Chinese hamster ovary (CHO) cells leads to an increase in extracellular osmolality in late stage culture. Herein, we explore the effect of osmolality on CHO cell growth, specific monoclonal antibody (mAb) productivity and glycosylation achieved with the addition of NaCl or the supplementation of a commercial feed. Although both methods lead to an increase in specific antibody productivity, they have different effects on cell growth and antibody production. Osmolality modulation using NaCl up to 470 mOsm kg⁻¹ had a consistently positive effect on specific antibody productivity and titre. The addition of the commercial feed achieved variable results: specific mAb productivity was increased, yet cell growth rate was significantly compromised at high osmolality values. As a result, Feed C addition to 410 mOsm kg⁻¹ was the only condition that achieved a significantly higher mAb titre compared to the control. Additionally, Feed C supplementation resulted in a significant reduction in galactosylated antibody structures. Cell volume was found to be positively correlated to osmolality; however, osmolality alone could not account for observed changes in average cell diameter without considering cell cycle variations. These results help delineate the overall effect of osmolality on titre and highlight the potentially negative effect of overfeeding on cell growth.     

Which games are growing bacterial populations playing?

Microbial communities display complex population dynamics, both in frequency and absolute density. Evolutionary game theory provides a natural approach to analyse and model this complexity by studying the detailed interactions among players, including competition and conflict, cooperation and coexistence. Classic evolutionary game theory models typically assume constant population size, which often does not hold for microbial populations. Here, we explicitly take into account population growth with frequency-dependent growth parameters, as observed in our experimental system. We study the in vitro population dynamics of the two commensal bacteria (Curvibacter sp. (AEP1.3) and Duganella sp. (C1.2)) that synergistically protect the metazoan host Hydra vulgaris (AEP) from fungal infection. The frequency-dependent, nonlinear growth rates observed in our experiments indicate that the interactions among bacteria in co-culture are beyond the simple case of direct competition or, equivalently, pairwise games. This is in agreement with the synergistic effect of anti-fungal activity observed in vivo. Our analysis provides new insight into the minimal degree of complexity needed to appropriately understand and predict coexistence or extinction events in this kind of microbial community dynamics. Our approach extends the understanding of microbial communities and points to novel experiments.