Identifying opportunities in cell engineering for the production of ‘difficult to express’ recombinant proteins

There is a growing demand for production of recombinant proteins of many structural varieties in mammalian expression systems, either as therapeutics or for protein characterisation. However, certain recombinant proteins are “difficult to express” in mammalian expression systems requiring extensive cell line and process optimisation which, as a result, can have significant consequences for drug development processes. The Tissue Inhibitors of Metalloproteinase (TIMP) protein family, TIMP-2, -3 and -4, are naturally secreted proteins that share significant structural homology (~50% identity and ~70% similarity in amino acid sequence), but show profound differences in secretion in mammalian expression systems. Computational sequence analysis of the TIMPs shows areas of significant amino acid difference mainly locating to flexible loop regions. This study has investigated the molecular mechanisms that selectively restrict expression of recombinant proteins of extensive sequence similarity. The loci of the molecular steps that limit successful expression have been defined by quantitative real-time polymerase chain reaction, proteomic analyses, cellular fractionation and immunofluorescence microscopy. All three TIMPs were readily detectable at mRNA and protein level within the cell but only TIMP-2 was secreted effectively into the culture medium. Analysis of protein localisation showed intracellular protein for all three TIMPs, mainly co-localised in the organellar and cytoskeleton fractions. In addition, immunofluorescence microscopy showed all three TIMPs to be detectable within the endoplasmic reticulum. TIMP-3, which was not secreted, was detected within the cell in both expected glycosylated and non-glycosylated forms. Treatment of intracellular TIMP-3 with glycosidases suggests the presence of an immature high mannose glycoform. Knockout of the TIMP-3 glycan site did not result in secretion. These data suggest that the post-translational processing of poorly expressed TIMPs limits transit through the secretory pathway. To overcome this challenge, cell engineering of limiting secretory pathway components could enhance production of these “difficult to express” recombinant proteins

Use of a ‘molecular tug’ to overcome limitations in the production of ‘difficult to express’ recombinant proteins

In recent years there has been an increased drive towards the production of recombinant proteins in large amounts using rapid cell culture processes. Mammalian expression systems such as Chinese Hamster Ovary (CHO) cells have remained the preferred choice for large-scale recombinant protein production (Walsh, 2014). However, in mammalian cells certain recombinant targets can prove to be ‘difficult to express’ and require extensive upstream process optimisation which can have a negative impact on industrial processes. This study has investigated the molecular mechanisms that are responsible for poor recombinant protein production. Model proteins from the Tissue Inhibitors of Metalloproteinase (TIMP) family, TIMP-2, TIMP-3 and TIMP-4, were subject to detailed study to characterise the molecular mechanisms that limit production of recombinant proteins with high sequence homology (Hussain et al., 2017). TIMP-2, -3 and -4, share significant sequence/structural homology (Douglas et al., 1997, Garcia et al., 2012), but show differences when produced in a transient CHO expression system (Hussain et al., 2017) . A systematic screen of the protein expression pathway showed all three TIMPs were detectable at the mRNA and protein level within the cell but only TIMP-2 was secreted in significant amounts into the culture medium. Analysis of the intracellular protein suggested the post-translational processing of poorly expressed TIMPs was limiting. A protein engineering approach was employed to overcome challenges in the production of these ‘difficult to express’ TIMP proteins. This approach involved the attachment of a furin-cleavable pro-sequence from a secretory growth factor to recombinant targets. The pro-sequence was predicted to act as a ‘molecular tug’ to aid transit through the protein expression pathway and/or promote correct post-translational processing. Initially, the furin-cleavable pro-sequence was added to TIMP-3 (non-secreted), which resulted in secretion of TIMP-3, however incomplete processing of the pro-sequence was observed. The protein engineering approach was optimised further and applied in combination with cell engineering (furin overexpression) to TIMP-4 (poorly secreted), which was also successfully detected in significantly higher amounts in the culture medium (Hussain et al., 2017). Together, the described protein engineering approach presents a novel strategy to increase the production of ‘difficult-to-express’ recombinant targets. References: Douglas, D. A., Shi, Y. E. & Sang, Q. X. A. 1997. Computational sequence analysis of the tissue inhibitor of metalloproteinase family. Journal of Protein Chemistry, 16, 237-255. Garcia, M. P. S., Suarez-Penaranda, J. M., Gayoso-Diz, P., Barros-Angueira, F., Gandara-Rey, J. M. & Garcia-Garcia, A. 2012. Tissue inhibitor of metalloproteinases in oral squamous cell carcinomas - A therapeutic target? Cancer Letters, 323, 11-19. Hussain, H., Fisher, D. I., Abbott, W. M., Roth, R. G. & Dickson, A. J. 2017. Use of a protein engineering strategy to overcome limitations in the production of Difficult to Express recombinant proteins. Biotechnology and Bioengineering, 114, 2348-2359. Walsh, G. 2014. Biopharmaceutical benchmarks 2014. Nature Biotechnology, 32, 992-1000

Pro-Environmental Behaviors and Ecological Responsibilities: An Evaluation of Pakistani University Students’ Behavioral Intentions Towards Climate Change

Rapid and devastating changes in climate and environment have affected individuals from all around the globe to indulge in climate change mitigation and adaptation. The aim of this research is to evaluate the role of students in climate change mitigation and adaptation by analyzing their behavioral intentions. We incorporated the theory of planned behavior in evaluating the environmental knowledge among university students in Pakistan. The results implicated that the attitudes, societal norms and perceived behavioral control contributes significantly in an individual’s pro-environmental behavioral intentions. Additionally, education was found to be contributing merely in building attitudes towards pro-environmental behaviors. However the societal norms and individual’s perceived behavioral control were found to be less influenced by the educational background of individuals.This research provides strategic policy suggestions in face of formulating encouraging engagement in deliberative discussions, interaction with scientists, and formulation of community projects to induce education, resources and opportunities which can be beneficial in changing behaviors of individuals on a larger scale in society

Where energy flows, passion grows: testing a moderated mediation model of work passion through a cross-cultural lens

This study examines how and when passionate leaders can instigate work passion in their followers. We propose relational energy as a social interaction mediator that can facilitate the crossover of work passion from leader to followers. Additionally, we introduce a moderator of culture (Anglo culture, e.g., Canada vs. Confucian Asian culture, e.g., China) as it plays a vital role in the dynamics of interpersonal relations within a leader-follower dyad. We collected two-wave data from MBA students of two Confucian Asian countries (China and Singapore, n = 120) and two Anglo countries (Canada and Australia, n = 265) to test our moderated mediation model. The results show that interactions with passionate leaders can generate relational energy in followers and subsequently lead to followers’ passion for work. Furthermore, the findings shed light on the moderating effect of culture, such that the leader-follower work passion relationship via follower relational energy was stronger for followers from Anglo culture than the followers from Confucian Asian culture. Limitations of the study and directions for future research are discussed

Predictive approaches to guide the expression of recombinant vaccine targets in Escherichia coli: a case study presentation utilising Absynth Biologics Ltd. proprietary Clostridium difficile vaccine antigens

From Springer Nature via Jisc Publications RouterHistory: received 2021-04-28, rev-recd 2021-06-02, accepted 2021-06-08, registration 2021-06-11, pub-electronic 2021-06-28, online 2021-06-28, pub-print 2021-07Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; doi: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/P004237/1Abstract: Bacterial expression systems remain a widely used host for recombinant protein production. However, overexpression of recombinant target proteins in bacterial systems such as Escherichia coli can result in poor solubility and the formation of insoluble aggregates. As a consequence, numerous strategies or alternative engineering approaches have been employed to increase recombinant protein production. In this case study, we present the strategies used to increase the recombinant production and solubility of ‘difficult-to-express’ bacterial antigens, termed Ant2 and Ant3, from Absynth Biologics Ltd.’s Clostridium difficile vaccine programme. Single recombinant antigens (Ant2 and Ant3) and fusion proteins (Ant2-3 and Ant3-2) formed insoluble aggregates (inclusion bodies) when overexpressed in bacterial cells. Further, proteolytic cleavage of Ant2-3 was observed. Optimisation of culture conditions and changes to the construct design to include N-terminal solubility tags did not improve antigen solubility. However, screening of different buffer/additives showed that the addition of 1–15 mM dithiothreitol alone decreased the formation of insoluble aggregates and improved the stability of both Ant2 and Ant3. Structural models were generated for Ant2 and Ant3, and solubility-based prediction tools were employed to determine the role of hydrophobicity and charge on protein production. The results showed that a large non-polar region (containing hydrophobic amino acids) was detected on the surface of Ant2 structures, whereas positively charged regions (containing lysine and arginine amino acids) were observed for Ant3, both of which were associated with poor protein solubility. We present a guide of strategies and predictive approaches that aim to guide the construct design, prior to expression studies, to define and engineer sequences/structures that could lead to increased expression and stability of single and potentially multi-domain (or fusion) antigens in bacterial expression systems

Carbon nanotubes incorporated Z-scheme assembly of AgBr/TiO2 for photocatalytic hydrogen production under visible light irradiations

Photocatalytic H2 production is a promising strategy toward green energy and alternative to carbon-based fuels which are the root cause of global warming and pollution. In this study, carbon nanotubes (CNTs) incorporated Z-scheme assembly of AgBr/TiO2 was developed for photocatalytic H2 production under visible light irradiations. Synthesized photocatalysts were characterized through transmission electron microscope (TEM), X-ray photoelectron spectra (XPS), X-ray diffractometer (XRD), Fourier transform infrared (FTIR), photoluminescence spectra (PL), Brunauer Emmet-Teller(BET), and UV-vis spectroscopy analysis techniques. The composite photocatalysts exhibited a H2 production of 477 ppm which was three-folds higher than that produced by TiO2. The good performance was attributed to the strong interaction of three components and the reduced charge recombination, which was 89 and 56.3 times lower than the TiO2 and AgBr/TiO2. Furthermore, the role of surface acidic and basic groups was assessed and the photocatalytic results demonstrated the importance of surface functional groups. In addition, the composites exhibited stability and reusability for five consecutive cycles of reaction. Thus, improved performance of the photocatalyst was credited to the CNTs as an electron mediator, surface functional groups, higher surface area, enhanced charge separation and extended visible light absorption edge. This work provides new development of Z-scheme photocatalysts for sustainable H2 production

A comparative analysis of recombinant Fab and full‐length antibody production in Chinese hamster ovary cells

Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen‐binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full‐length antibodies are mammalian cell‐based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small‐ and large‐scale Fab production. Using a panel of full‐length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full‐length antibody format resulted in the recovery of fewer mini‐pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody‐producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full‐length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats

A comparative analysis of recombinant Fab and full‐length antibody production in Chinese hamster ovary cells

From Wiley via Jisc Publications RouterHistory: received 2021-06-18, rev-recd 2021-08-31, accepted 2021-09-12, pub-electronic 2021-10-06Article version: VoRPublication status: PublishedFunder: UCB UK; Id: http://dx.doi.org/10.13039/100011111Funder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268; Grant(s): BB/R001731/1, BB/R002096/1Abstract: Monoclonal antibodies are the leading class of biopharmaceuticals in terms of numbers approved for therapeutic purposes. Antigen‐binding fragments (Fab) are also used as biotherapeutics and used widely in research applications. The dominant expression systems for full‐length antibodies are mammalian cell‐based, whereas for Fab molecules the preference has been an expression in bacterial systems. However, advances in CHO and downstream technologies make mammalian systems an equally viable option for small‐ and large‐scale Fab production. Using a panel of full‐length IgG antibodies and their corresponding Fab pair with different antigen specificities, we investigated the impact of the IgG and Fab molecule format on production from Chinese hamster ovary (CHO) cells and assessed the cellular capability to process and produce these formats. The full‐length antibody format resulted in the recovery of fewer mini‐pools posttransfection when compared to the corresponding Fab fragment format that could be interpreted as indicative of a greater overall burden on cells. Antibody‐producing cell pools that did recover were subsequently able to achieve higher volumetric protein yields (mg/L) and specific productivity than the corresponding Fab pools. Importantly, when the actual molecules produced per cell of a given format was considered (as opposed to mass), CHO cells produced a greater number of Fab molecules per cell than obtained with the corresponding IgG, suggesting that cells were more efficient at making the smaller Fab molecule. Analysis of cell pools showed that gene copy number was not correlated to the subsequent protein production. The amount of mRNA correlated with secreted Fab production but not IgG, whereby posttranscriptional processes act to limit antibody production. In summary, we provide the first comparative description of how full‐length IgG and Fab antibody formats impact on the outcomes of a cell line construction process and identify potential limitations in their production that could be targeted for engineering increases in the efficiency in the manufacture of these recombinant antibody formats