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.

Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines.

The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.

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.

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling.

Despite many advances in the generation of high producing recombinant mammalian cell lines over the last few decades, cell line selection and development is often slowed by the inability to predict a cell line's phenotypic characteristics (e.g. growth or recombinant protein productivity) at larger scale (large volume bioreactors) using data from early cell line construction at small culture scale. Here we describe the development of an intact cell MALDI-ToF mass spectrometry fingerprinting method for mammalian cells early in the cell line construction process whereby the resulting mass spectrometry data are used to predict the phenotype of mammalian cell lines at larger culture scale using a Partial Least Squares Discriminant Analysis (PLS-DA) model. Using MALDI-ToF mass spectrometry, a library of mass spectrometry fingerprints was generated for individual cell lines at the 96 deep well plate stage of cell line development. The growth and productivity of these cell lines were evaluated in a 10L bioreactor model of Lonza's large-scale (up to 20,000L) fed-batch cell culture processes. Using the mass spectrometry information at the 96 deep well plate stage and phenotype information at the 10L bioreactor scale a PLS-DA model was developed to predict the productivity of unknown cell lines at the 10L scale based upon their MALDI-ToF fingerprint at the 96 deep well plate scale. This approach provides the basis for the very early prediction of cell lines' performance in cGMP manufacturing-scale bioreactors and the foundation for methods and models for predicting other mammalian cell phenotypes from rapid, intact-cell mass spectrometry based measurements.

¹H NMR spectroscopy profiling of metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture.

We report an NMR based approach to determine the metabolic reprogramming of Chinese hamster ovary cells upon a temperature shift during culture by investigating the extracellular cell culture media and intracellular metabolome of CHOK1 and CHO-S cells during culture and in response to cold-shock and subsequent recovery from hypothermic culturing. A total of 24 components were identified for CHOK1 and 29 components identified for CHO-S cell systems including the observation that CHO-S media contains 5.6 times the level of glucose of CHOK1 media at time zero. We confirm that an NMR metabolic approach provides quantitative analysis of components such as glucose and alanine with both cell lines responding in a similar manner and comparable to previously reported data. However, analysis of lactate confirms a differentiation between CHOK1 and CHO-S and that reprogramming of metabolism in response to temperature was cell line specific. The significance of our results is presented using principal component analysis (PCA) that confirms changes in metabolite profile in response to temperature and recovery. Ultimately, our approach demonstrates the capability of NMR providing real-time analysis to detect reprogramming of metabolism upon cellular perception of cold-shock/sub-physiological temperatures. This has the potential to allow manipulation of metabolites in culture supernatant to improve growth or productivity.

MALDI-ToF mass spectrometry coupled with multivariate pattern recognition analysis for the rapid biomarker profiling of Escherichia coli in different growth phases.

Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF MS) has been exploited extensively in the field of microbiology for the characterisation of bacterial species, the detection of biomarkers for early disease diagnosis and bacterial identification. Here, the multivariate data analysis technique of partial least squares-discriminant analysis (PLS-DA) was applied to 'intact cell' MALDI-ToF MS data obtained from Escherichia coli cell samples to determine if such an approach could be used to distinguish between, and characterise, different growth phases. PLS-DA is a technique that has the potential to extract systematic variation from large and noisy data sets by identifying a lower-dimensional subspace that contains latent information. The application of PLS-DA to the MALDI-ToF data obtained from cells at different stages of growth resulted in the successful classification of the samples according to the growth phase of the bacteria cultures. A further outcome of the analysis was that it was possible to identify the mass-to-charge (m/z) ratio peaks or ion signals that contributed to the classification of the samples. The Swiss-Prot/TrEMBL database and primary literature were then used to provisionally assign a small number of these m/z ion signals to proteins, and these tentative assignments revealed that the major contributors from the exponential phase were ribosomal proteins. Additional assignments were possible for the stationary phase and the decline phase cultures where the proteins identified were consistent with previously observed biological interpretation. In summary, the results show that MALDI-ToF MS, PLS-DA and a protein database search can be used in combination to discriminate between 'intact cell' E. coli cell samples in different growth phases and thus could potentially be used as a tool in process development in the bioprocessing industry to enhance cell growth and cell engineering strategies.

Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matrices.

Biopharmaceutical proteins are often formulated and freeze dried in agents that protect them from deleterious reactions that can compromise activity and authenticity. Although such approaches are widely used, a detailed understanding of the molecular mechanisms of protein stabilization in low water content amorphous glasses is lacking. Further, whilst deterioration chemistries are well described in dilute solution, relatively little is known about the extent and mechanisms by which protein integrity is compromised in the glassy state. Here we have investigated the relationship between protein modification and rate thereof, with variation of pH, carbohydrate excipient, temperature and the glass transition temperature using a model protein, lysozyme. Mass spectrometry analysis and peptide mapping confirm that protein modifications do occur in the glassy state in a time-, temperature-, and carbohydrate excipient-dependent manner. There were clear trends between the buffer pH and the primary modification detected (glycation). Most importantly, there were differences in the apparent reactivities of the lysine residues in the glass compared with those previously determined in solution, and therefore, the well-characterized solution reactivity of this reaction cannot be used to predict likely sites of modification in the glassy state. These findings have implications for (i) the selection and combinations of formulation components, particularly with regard to glycation in the glassy state, and (ii) the design of procedures and methodologies for the improvement of protein stability in the glassy state.

The effect of peptide glycation on local secondary structure.

Protein glycation is a non-enzymatic reaction between reducing sugars and amino groups that occurs in vivo and has been implicated in a number of disease states and pathologies including Alzheimer's and diabetes. Although glycation is thought to alter protein structure and function, there is currently little information on the structural consequences of this modification. We have used a model alpha-helix and a model beta-hairpin peptide, and NMR analysis, to investigate the effects of glycation upon secondary structure. Glycation of the dilysine motif within the alpha-helix peptide occurred preferentially at one lysine residue and resulted in severe disruption to the local secondary structure. The area immediately around the site of modification was extremely flexible and the peptide did not adopt a preferred conformation in this area of the helix in 30% TFE. Significant glycation of the beta-hairpin peptide was not detected and the structure was unchanged. These results show that glycation results in local secondary structure distortion of alpha-helices and that preferential glycation occurs in a sequence specific manner. The findings will allow us to interrogate the local environment in other peptides/proteins to predict the likelihood of glycation, and to model the potential effects such modification might have upon structure/function.

Comparison of the effects of 2,2,2-trifluoroethanol on peptide and protein structure and function.

The co-solvent 2,2,2-trifluoroethanol (TFE) has been often used to aid formation of secondary structure in solution peptides or alternately as a denaturant within protein folding studies. Hen egg white lysozyme (HEWL) and a synthetic model peptide defining HEWL helix-4 were used as comparative model systems to systematically investigate the effect of increasing TFE concentrations on the structure of proteins and peptides. HEWL was analyzed using NMR, far-UV CD and fluorescence spectroscopy; with correlation of these results towards changes in enzymatic activity and the helix-4 peptide was analysed using NMR. Data illustrates two conflicting modes of interaction: Low TFE concentrations stabilize tertiary structure, observed from an increase in the number of NMR NOE contacts. Higher TFE concentrations denatured HEWL with the loss of lysozyme tertiary structure. The effects of TFE upon secondary structural elements within HEWL are distinct from those observed for the helix-4 peptide. This illustrates a dissimilar interaction of TFE towards both protein and peptide at equivalent TFE concentrations. The concentration that TFE promotes stabilization over denaturation is likely to be protein dependent although the structural action can be extrapolated to other protein systems with implications for the use of TFE in structural stability studies.