N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting.

In vitro-transcribed (IVT) mRNAs are modalities that can combat human disease, exemplified by their use as vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). IVT mRNAs are transfected into target cells, where they are translated into recombinant protein, and the biological activity or immunogenicity of the encoded protein exerts an intended therapeutic effect1,2. Modified ribonucleotides are commonly incorporated into therapeutic IVT mRNAs to decrease their innate immunogenicity3-5, but their effects on mRNA translation fidelity have not been fully explored. Here we demonstrate that incorporation of N1-methylpseudouridine into mRNA results in +1 ribosomal frameshifting in vitro and that cellular immunity in mice and humans to +1 frameshifted products from BNT162b2 vaccine mRNA translation occurs after vaccination. The +1 ribosome frameshifting observed is probably a consequence of N1-methylpseudouridine-induced ribosome stalling during IVT mRNA translation, with frameshifting occurring at ribosome slippery sequences. However, we demonstrate that synonymous targeting of such slippery sequences provides an effective strategy to reduce the production of frameshifted products. Overall, these data increase our understanding of how modified ribonucleotides affect the fidelity of mRNA translation, and although there are no adverse outcomes reported from mistranslation of mRNA-based SARS-CoV-2 vaccines in humans, these data highlight potential off-target effects for future mRNA-based therapeutics and demonstrate the requirement for sequence optimization.

Evaluating data integrity in ribosome footprinting datasets through modelled polysome profiles.

The assessment of transcriptome-wide ribosome binding to mRNAs is useful for studying the dynamic regulation of protein synthesis. Two methods frequently applied in eukaryotic cells that operate at different levels of resolution are polysome profiling, which reveals the distribution of ribosome loads across the transcriptome, and ribosome footprinting (also termed ribosome profiling or Ribo-Seq), which when combined with appropriate data on mRNA expression can reveal ribosome densities on individual transcripts. In this study we develop methods for relating the information content of these two methods to one another, by reconstructing theoretical polysome profiles from ribosome footprinting data. Our results validate both approaches as experimental tools. Although we show that both methods can yield highly consistent data, some published ribosome footprinting datasets give rise to reconstructed polysome profiles with non-physiological features. We trace these aberrant features to inconsistencies in RNA and Ribo-Seq data when compared to datasets yielding physiological polysome profiles, thereby demonstrating that modelled polysomes are useful for assessing global dataset properties such as its quality in a simple, visual approach. Aside from using polysome profile reconstructions on published datasets, we propose that this also provides a useful tool for validating new ribosome footprinting datasets in early stages of analyses.

Manipulation of mRNA translation elongation influences the fragmentation of a biotherapeutic Fc-fusion protein produced in CHO cells.

Mammalian cells, particularly Chinese hamster ovary cells, are the dominant system for the production of protein-based biotherapeutics, however, product degradation, particularly of Fc-fusion proteins, is sometimes observed that impacts the quality of the protein generated. Here, we identify the site of fragmentation of a model immunoglobulin G1 Fc-fusion protein, show that the observed clipping and aggregation are decreased by reduced temperature culturing, that the fragmentation/clipping is intracellular, and that reduced clipping at a lower temperature (<37°C) relates to mesenger RNA (mRNA) translation elongation. We subsequently show that reduced fragmentation can be achieved at 37°C by addition of chemical reagents that slow translation elongation. We then modified mRNA translation elongation speeds by designing different transcript sequences for the Fc-fusion protein based on alternative codon usage and improved the product yield at 37°C, and the ratio of intact to a fragmented product. Our data suggest that rapid elongation results in misfolding that decreases product fidelity, generating a region susceptible to degradation/proteolysis, whilst the slowing of mRNA translation improves the folding, reducing susceptibility to fragmentation. Manipulation of mRNA translation and/or the target Fc-fusion transcript is, therefore, an approach that can be applied to potentially reduce fragmentation of clipping-prone Fc-fusion proteins.

Constitutively active Rheb mutants [T23M] and [E40K] drive increased production and secretion of recombinant protein in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are high value agents used for disease therapy ("biologic drugs") or as diagnostic tools which are widely used in the healthcare sector. They are generally manufactured in mammalian cells, in particular Chinese hamster ovary (CHO) cells cultured in defined media, and are harvested from the medium. Rheb is a small GTPase which, when bound to GTP, activates mechanistic target of rapamycin complex 1, a protein kinase that drives anabolic processes including protein synthesis and ribosome biogenesis. Here, we show that certain constitutively active mutants of Rheb drive faster protein synthesis in CHO cells and increase the expression of proteins involved in the processing of secreted proteins in the endoplasmic reticulum, which expands in response to expression of Rheb mutants. Active Rheb mutants, in particular Rheb[T23M], drive increased cell number under serum-free conditions similar to those used in the biotechnology industry. Rheb[T23M] also enhances the expression of the reporter protein luciferase and, especially strongly, the secreted Gaussia luciferase. Moreover, Rheb[T23M] markedly (2-3 fold) enhances the amount of this luciferase and of a model immunoglobulin secreted into the medium. Our data clearly demonstrate that expressing Rheb[T23M] in CHO cells provides a simple approach to promoting their growth in defined medium and the production of secreted proteins of high commercial value.

Engineered transient and stable overexpression of translation factors eIF3i and eIF3c in CHOK1 and HEK293 cells gives enhanced cell growth associated with increased c-Myc expression and increased recombinant protein synthesis.

There is a desire to engineer mammalian host cell lines to improve cell growth/biomass accumulation and recombinant biopharmaceutical protein production in industrially relevant cell lines such as the CHOK1 and HEK293 cell lines. The over-expression of individual subunits of the eukaryotic translation factor eIF3 in mammalian cells has previously been shown to result in oncogenic properties being imparted on cells, including increased cell proliferation and growth and enhanced global protein synthesis rates. Here we report on the engineering of CHOK1 and HEK cells to over-express the eIF3i and eIF3c subunits of the eIF3 complex and the resultant impact on cell growth and a reporter of exogenous recombinant protein production. Transient over-expression of eIF3i in HEK293 and CHOK1 cells resulted in a modest increase in total eIF3i amounts (maximum 40% increase above control) and an approximate 10% increase in global protein synthesis rates in CHOK1 cells. Stable over-expression of eIF3i in CHOK1 cells was not achievable, most likely due to the already high levels of eIF3i in CHO cells compared to HEK293 cells, but was achieved in HEK293 cells. HEK293 cells engineered to over-express eIF3i had faster growth that was associated with increased c-Myc expression, achieved higher cell biomass and gave enhanced yields of a reporter of recombinant protein production. Whilst CHOK1 cells could not be engineered to over-express eIF3i directly, they could be engineered to over-express eIF3c, which resulted in a subsequent increase in eIF3i amounts and c-Myc expression. The CHOK1 eIF3c engineered cells grew to higher cell numbers and had enhanced cap- and IRES-dependent recombinant protein synthesis. Collectively these data show that engineering of subunits of the eIF3 complex can enhance cell growth and recombinant protein synthesis in mammalian cells in a cell specific manner that has implications for the engineering or selection of fast growing or high producing cells for production of recombinant proteins.

Engineering of Chinese hamster ovary cell lipid metabolism results in an expanded ER and enhanced recombinant biotherapeutic protein production.

Chinese hamster ovary (CHO) cell expression systems have been exquisitely developed for the production of recombinant biotherapeutics (e.g. standard monoclonal antibodies, mAbs) and are able to generate efficacious, multi-domain proteins with human-like post translational modifications at high concentration with appropriate product quality attributes. However, there remains a need for development of new CHO cell expression systems able to produce more challenging secretory recombinant biotherapeutics at higher yield with improved product quality attributes. Amazingly, the engineering of lipid metabolism to enhance such properties has not been investigated even though the biosynthesis of recombinant proteins is at least partially controlled by cellular processes that are highly dependent on lipid metabolism. Here we show that the global transcriptional activator of genes involved in lipid biosynthesis, sterol regulatory element binding factor 1 (SREBF1), and stearoyl CoA desaturase 1 (SCD1), an enzyme which catalyzes the conversion of saturated fatty acids into monounsaturated fatty acids, can be overexpressed in CHO cells to different degrees. The amount of overexpression obtained of each of these lipid metabolism modifying (LMM) genes was related to the subsequent phenotypes observed. Expression of a number of model secretory biopharmaceuticals was enhanced between 1.5-9 fold in either SREBF1 or SCD1 engineered CHO host cells as assessed under batch and fed-batch culture. The SCD1 overexpressing polyclonal pool consistently showed increased concentration of a range of products. For the SREBF1 engineered cells, the level of SREBF1 expression that gave the greatest enhancement in yield was dependent upon the model protein tested. Overexpression of both SCD1 and SREBF1 modified the lipid profile of CHO cells and the cellular structure. Mechanistically, overexpression of SCD1 and SREBF1 resulted in an expanded endoplasmic reticulum (ER) that was dependent upon the level of LMM overexpression. We conclude that manipulation of lipid metabolism in CHO cells via genetic engineering is an exciting new approach to enhance the ability of CHO cells to produce a range of different types of secretory recombinant protein products via modulation of the cellular lipid profile and expansion of the ER.

Cooling-induced SUMOylation of EXOSC10 down-regulates ribosome biogenesis.

The RNA exosome is essential for 3' processing of functional RNA species and degradation of aberrant RNAs in eukaryotic cells. Recent reports have defined the substrates of the exosome catalytic domains and solved the multimeric structure of the exosome complex. However, regulation of exosome activity remains poorly characterized, especially in response to physiological stress. Following the observation that cooling of mammalian cells results in a reduction in 40S:60S ribosomal subunit ratio, we uncover regulation of the nuclear exosome as a result of reduced temperature. Using human cells and an in vivo model system allowing whole-body cooling, we observe reduced EXOSC10 (hRrp6, Pm/Scl-100) expression in the cold. In parallel, both models of cooling increase global SUMOylation, leading to the identification of specific conjugation of SUMO1 to EXOSC10, a process that is increased by cooling. Furthermore, we define the major SUMOylation sites in EXOSC10 by mutagenesis and show that overexpression of SUMO1 alone is sufficient to suppress EXOSC10 abundance. Reducing EXOSC10 expression by RNAi in human cells correlates with the 3' preribosomal RNA processing defects seen in the cold as well as reducing the 40S:60S ratio, a previously uncharacterized consequence of EXOSC10 suppression. Together, this work illustrates that EXOSC10 can be modified by SUMOylation and identifies a physiological stress where this regulation is prevalent both in vitro and in vivo.

The effect of formulation variables on protein stability and integrity of a model IgG4 monoclonal antibody and translation to formulation of a model ScFv.

OBJECTIVES: There are a number of blockbuster monoclonal antibodies on the market used for the treatment of a variety of diseases. Although the formulation of many antibodies is achieved in 'platform' formulations, some are so difficult to formulate that it can result in an inability to develop a finished drug product. Further, a large number of antibody-inspired or-based molecules are now being developed and assessed for biotherapeutic purposes and less is understood around the required active protein drug concentrations, excipients and additives required in final product formulations. RESULTS: We investigated the effect of formulation variables (pH, buffer composition, glycine and NaCl concentration, time and temperature of accelerated stability studies) on antibody solubility/aggregation and activity using a Plackett-Burman Experimental Design approach. We then used the findings from this study and applied these to the formulation of a single chain variable fragment (ScFv) molecule. Our data shows that prediction of ScFc stability from a model monoclonal antibody could be achieved although further formulation optimization was required. Mass spectrometry analysis confirmed changes to the mass and hence authenticity of both the model antibody and ScFv under formulation conditions that did not provide appropriate conditions for protection of the molecules. CONCLUSIONS: The role of the different formulation conditions on maintaining protein integrity is described and using mass spectrometry shows that protein integrity is compromised under particular conditions. The implications for predicting successful formulations for protein molecules is discussed and how antibody formulations could be used to predict formulation components for novel antibody based molecules.

mTORC1 signalling and eIF4E/4E-BP1 translation initiation factor stoichiometry influence recombinant protein productivity from GS-CHOK1 cells.

Many protein-based biotherapeutics are produced in cultured Chinese hamster ovary (CHO) cell lines. Recent reports have demonstrated that translation of recombinant mRNAs and global control of the translation machinery via mammalian target of rapamycin (mTOR) signalling are important determinants of the amount and quality of recombinant protein such cells can produce. mTOR complex 1 (mTORC1) is a master regulator of cell growth/division, ribosome biogenesis and protein synthesis, but the relationship between mTORC1 signalling, cell growth and proliferation and recombinant protein yields from mammalian cells, and whether this master regulating signalling pathway can be manipulated to enhance cell biomass and recombinant protein production (rPP) are not well explored. We have investigated mTORC1 signalling and activity throughout batch culture of a panel of sister recombinant glutamine synthetase-CHO cell lines expressing different amounts of a model monoclonal IgG4, to evaluate the links between mTORC1 signalling and cell proliferation, autophagy, recombinant protein expression, global protein synthesis and mRNA translation initiation. We find that the expression of the mTORC1 substrate 4E-binding protein 1 (4E-BP1) fluctuates throughout the course of cell culture and, as expected, that the 4E-BP1 phosphorylation profiles change across the culture. Importantly, we find that the eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity. Furthermore, eIF4E amounts appear to be co-regulated with 4E-BP1 amounts. This may reflect a sensing of either change at the mRNA level as opposed to the protein level or the fact that the phosphorylation status, as well as the amount of 4E-BP1 present, is important in the co-regulation of eIF4E and 4E-BP1.

Investigations into, and development of, a lyophilized and formulated recombinant human factor IX produced from CHO cells.

OBJECTIVES: To develop a recombinant human factor IX (rFIX) formulation equivalent to commercially available products in terms of cake appearance, residual moisture, proportion of soluble aggregates and activity maintenance for 3 months at 4-8 °C. RESULTS: NaCl and low bulking agent/cryoprotectant mass ratio had a negative impact on cake quality upon lyophilisation for a wide range of formulations tested. Particular devised formulations maintained rFIX activity after lyophilization with a similar performance when compared with the rFIX formulated using the excipients reported for a commercially available FIX formulation (Benefix). rFIX remained active after 3 months when stored at 4 °C, though this was not the case with samples stored at 40 °C. Interestingly, particular formulations had an increase in residual moisture after 3 months storage, but not above a 3% threshold. All four formulations tested were equivalent to the Benefix formulation in terms of particle size distribution and cake appearance. CONCLUSIONS: Three specific formulations, consisting of surfactant polysorbate-80, sucrose or trehalose as cryoprotectant, mannitol or glycine as bulking agent, L-histidine as buffering agent, and NaCl added in the reconstitution liquid at 0.234% (w/v) were suitable for use with a CHO cell-derived recombinant FIX.

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity.

p58IPK is an inhibitor of the eIF2α kinase GCN2 and its localization and expression underpin protein synthesis and ER processing capacity.

One of the key cellular responses to stress is the attenuation of mRNA translation and protein synthesis via the phosphorylation of eIF2α (eukaryotic translation initiation factor 2α). This is mediated by four eIF2α kinases and it has been suggested that each kinase is specific to the cellular stress imposed. In the present study, we show that both PERK (PKR-like endoplasmic reticulum kinase/eIF2α kinase 3) and GCN2 (general control non-derepressible 2/eIF2α kinase 4) are required for the stress responses associated with conditions encountered by cells overexpressing secreted recombinant protein. Importantly, whereas GCN2 is the kinase that is activated following cold-shock/hypothermic culturing of mammalian cells, PERK and GCN2 have overlapping functions since knockdown of one of these at the mRNA level is compensated for by the cell by up-regulating levels of the other. The protein p58IPK {also known as DnaJ3C [DnaJ heat-shock protein (hsp) 40 homologue, subfamily C, member 3]} is known to inhibit the eIF2α kinases PKR (dsRNA-dependent protein kinase/eIF2α kinase 2) and PERK and hence prevent or delay eIF2α phosphorylation and consequent inhibition of translation. However, we show that p58IPK is a general inhibitor of the eIF2α kinases in that it also interacts with GCN2. Thus forced overexpression of cytoplasmic p58 delays eIF2α phosphorylation, suppresses GCN2 phosphorylation and prolongs protein synthesis under endoplasmic reticulum (ER), hypothermic and prolonged culture stress conditions. Taken together, our data suggest that there is considerable cross talk between the eIF2α kinases to ensure that protein synthesis is tightly regulated. Their activation is controlled by p58 and the expression levels and localization of this protein are crucial in the capacity the cells to respond to cellular stress via control of protein synthesis rates and subsequent folding in the ER.

Eukaryotic elongation factor 2 kinase regulates the cold stress response by slowing translation elongation.

Cells respond to external stress conditions by controlling gene expression, a process which occurs rapidly via post-transcriptional regulation at the level of protein synthesis. Global control of translation is mediated by modification of translation factors to allow reprogramming of the translatome and synthesis of specific proteins that are required for stress protection or initiation of apoptosis. In the present study, we have investigated how global protein synthesis rates are regulated upon mild cooling. We demonstrate that although there are changes to the factors that control initiation, including phosphorylation of eukaryotic translation initiation factor 2 (eIF2) on the α-subunit, the reduction in the global translation rate is mediated by regulation of elongation via phosphorylation of eukaryotic elongation factor 2 (eEF2) by its specific kinase, eEF2K (eukaryotic elongation factor 2 kinase). The AMP/ATP ratio increases following cooling, consistent with a reduction in metabolic rates, giving rise to activation of AMPK (5'-AMP-activated protein kinase), which is upstream of eEF2K. However, our data show that the major trigger for activation of eEF2K upon mild cooling is the release of Ca2+ ions from the endoplasmic reticulum (ER) and, importantly, that it is possible to restore protein synthesis rates in cooled cells by inhibition of this pathway at multiple points. As cooling has both therapeutic and industrial applications, our data provide important new insights into how the cellular responses to this stress are regulated, opening up new possibilities to modulate these responses for medical or industrial use at physiological or cooler temperatures.

Biotherapeutic protein formulation variables influence protein integrity and can promote post-translational modifications as shown using chicken egg white lysozyme as a model system.

OBJECTIVES: The effect of different formulations variables on protein integrity were investigated using lysozyme as a model protein for the development of biotherapeutic protein formulations for use in the clinic. RESULTS: Buffer composition/concentration was the key variable of formulation reagents investigated in determining lysozyme stability and authenticity independent of protein concentration whilst the storage temperature and time, not surprisingly, were also key variables. Tryptic peptide mapping of the protein showed that the modifications occurred when formulated under specific conditions but not others. A model peptide system was developed that reflected the same behavior under formulation conditions as intact lysozyme. CONCLUSIONS: Peptide models may mirror the stability of proteins, or regions of proteins, in the same formulations and be used to help develop a rapid screen of formulations for stabilisation of biotherapeutic proteins.

The future of host cell protein (HCP) identification during process development and manufacturing linked to a risk-based management for their control.

The use of biological systems to synthesize complex therapeutic products has been a remarkable success. However, during product development, great attention must be devoted to defining acceptable levels of impurities that derive from that biological system, heading this list are host cell proteins (HCPs). Recent advances in proteomic analytics have shown how diverse this class of impurities is; as such knowledge and capability grows inevitable questions have arisen about how thorough current approaches to measuring HCPs are. The fundamental issue is how to adequately measure (and in turn monitor and control) such a large number of protein species (potentially thousands of components) to ensure safe and efficacious products. A rather elegant solution is to use an immunoassay (enzyme-linked immunosorbent assay [ELISA]) based on polyclonal antibodies raised to the host cell (biological system) used to synthesize a particular therapeutic product. However, the measurement is entirely dependent on the antibody serum used, which dictates the sensitivity of the assay and the degree of coverage of the HCP spectrum. It provides one summed analog value for HCP amount; a positive if all HCP components can be considered equal, a negative in the more likely event one associates greater risk with certain components of the HCP proteome. In a thorough risk-based approach, one would wish to be able to account for this. These issues have led to the investigation of orthogonal analytical methods; most prominently mass spectrometry. These techniques can potentially both identify and quantify HCPs. The ability to measure and monitor thousands of proteins proportionally increases the amount of data acquired. Significant benefits exist if the information can be used to determine critical HCPs and thereby create an improved basis for risk management. We describe a nascent approach to risk assessment of HCPs based upon such data, drawing attention to timeliness in relation to biosimilar initiatives. The development of such an approach requires databases based on cumulative knowledge of multiple risk factors that would require national and international regulators, standards authorities (e.g., NIST and NIBSC), industry and academia to all be involved in shaping what is the best approach to the adoption of the latest bioanalytical technology to this area, which is vital to delivering safe efficacious biological medicines of all types.

The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h.

eIF3 (eukaryotic initiation factor 3) is the largest and most complex eukaryotic mRNA translation factor in terms of the number of protein components or subunits. In mammals, eIF3 is composed of 13 different polypeptide subunits, of which five, i.e. a, b, c, g and i, are conserved and essential in vivo from yeasts to mammals. In the present study, we show that the eukaryotic cytosolic chaperonin CCT [chaperonin containing TCP-1 (tailless complex polypeptide 1)] binds to newly synthesized eIF3b and promotes the correct folding of eIF3h and eIF3i. Interestingly, overexpression of these last two subunits is associated with enhanced translation of specific mRNAs over and above the general enhancement of global translation. In agreement with this, our data show that, as CCT is required for the correct folding of eIF3h and eIF3i subunits, it indirectly influences gene expression with eIF3i overexpression enhancing both cap- and IRES (internal ribosome entry segment)-dependent translation initiation, whereas eIF3h overexpression selectively increases IRES-dependent translation initiation. Importantly, these studies demonstrate the requirement of the chaperonin machinery for the correct folding of essential components of the translational machinery and provide further evidence of the close interplay between the cell environment, cell signalling, cell proliferation, the chaperone machinery and translational apparatus.

Control and regulation of mRNA translation.

Translational control is central to the gene expression pathway and was the focus of the 2013 annual Translation UK meeting held at the University of Kent. The meeting brought together scientists at all career stages to present and discuss research in the mRNA translation field, with an emphasis on the presentations on the research of early career scientists. The diverse nature of this field was represented by the broad range of papers presented at the meeting. The complexity of mRNA translation and its control is emphasized by the interdisciplinary research approaches required to address this area with speakers highlighting emerging systems biology techniques and their application to understanding mRNA translation and the network of pathways controlling it.