Stability Convergence in Antibody Coformulations

Combined administration of antibody therapeutics has proven to be beneficial for patients with cancer or infectious diseases. As a result, there is a growing trend toward multiple antibodies premixed into a single product form and delivered to patients as a fixed-dose coformulation. However, combining antibodies into a single coformulation could be challenging as proteins have the potential to interact and alter their stability and degradation profiles in the mixture, compared to that in isolation. We show that in two specific antibody-antibody coformulations, the more stable antibody component increased the stability of the less stable component, which in return destabilized the more stable component, hence exhibiting an overall convergence of stability in the coformulation

Engineer flexible loops for improved enzyme thermostability

Enzyme thermostability is a critical factor for its wide applications in industrial fields. Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. The stereospecifically controlled carbon-carbon bond forming ability of Escherichia coli transketolase (TK) makes it very promising as a biocatalyst in industry. However, as a mesophilic enzyme, it suffers the limitation of low stability to elevated temperatures and extremes of pH, limiting its current use in industrial processes. In order to improve thermostability of TK, we have applied two parallel strategies to identify mutation candidates within the flexible loops. The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterized experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Furthermore, molecular dynamics (MD) simulations of variants confirmed a good inverse correlation between protein stability and local flexibility which was determined by the magnitude of fluctuations with respect to the average conformations. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzyme

Two strategies to engineer flexible loops for improved enzyme thermostability

The stereospecifically controlled carbon-carbon bond forming ability of Escherichia coli transketolase (TK) makes it very promising as a biocatalyst in industry. However, as a mesophilic enzyme the enzyme suffers the limitation of low stability to elevated temperatures and extremes of pH, limiting its current use in industrial processes. Flexible sites are potential targets for engineering the stability of enzymes. Nevertheless, the success rate of the rigidifying flexible sites (RFS) strategy is still low due to a limited understanding of how to determine the best mutation candidates. In this study, two parallel strategies were applied to identify mutation candidates within the flexible loops of TK. The first was a “back to consensus mutations” approach, and the second was computational design based on ΔΔG calculations in Rosetta. Forty-nine single variants were generated and characterised experimentally. From these, three single-variants I189H, A282P, D143K were found to be more thermostable than wild-type TK. The combination of A282P with H192P, a variant constructed previously, resulted in the best all-round variant with a 3-fold improved half-life at 60 °C, 5-fold increased specific activity at 65 °C, 1.3-fold improved kcat and a Tm increased by 5 °C above that of wild type. Based on a statistical analysis of the stability changes for all variants, the qualitative prediction accuracy of the Rosetta program reached 65.3%. Furthermore, molecular dynamics (MD) simulations of variants confirmed a good inverse correlation between protein stability and local flexibility which was determined by the magnitude of fluctuations with respect to the average conformations. Both of the two strategies investigated were useful in guiding mutation candidates to flexible loops, and had the potential to be used for other enzymes

Directed evolution strategies for improved enzymatic performance

The engineering of enzymes with altered activity, specificity and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. However, the general acceptance of these methods as a route to new biocatalysts for organic synthesis requires further improvement of the methods for both ease-of-use and also for obtaining more significant changes in enzyme properties than is currently possible. Recent advances in library design, and methods of random mutagenesis, combined with new screening and selection tools, continue to push forward the potential of directed evolution. For example, protein engineers are now beginning to apply the vast body of knowledge and understanding of protein structure and function, to the design of focussed directed evolution libraries, with striking results compared to the previously favoured random mutagenesis and recombination of entire genes. Significant progress in computational design techniques which mimic the experimental process of library screening is also now enabling searches of much greater regions of sequence-space for those catalytic reactions that are broadly understood and, therefore, possible to model. Biocatalysis for organic synthesis frequently makes use of whole-cells, in addition to isolated enzymes, either for a single reaction or for transformations via entire metabolic pathways. As many new whole-cell biocatalysts are being developed by metabolic engineering, the potential of directed evolution to improve these initial designs is also beginning to be realised

Engineering 2’O-mRNA methyltransferases for industrial biocatalysis

Eukaryotic messenger RNA (mRNA) are universally modified at their 5’ end into a cap 0 structure consisting of an N7-methylguanosine and an inverted 5’-5’ triphosphate bridge linking the penultimate nucleoside. Multicellular eukaryotes possess the capacity to further modify this cap by 2’O-methylating the ribose of the penultimate nucleotide producing a so-called cap 1 structure1. This methylation seems to be a molecular signature for the discrimination between self and non-self mRNA2. In order to escape the innate immune system of the infected cell, some viruses have also evolved the ability to methylate their cap structures1. By analogy, therapeutic mRNAs must be non-immunogenic in order to restore or supplement the function of altered genes by mRNA-based therapy3. In this context, we propose to exploit the capacity of Vaccinia virus to produce non-immunogenic mRNAs. More specifically, VP39 is a 39 kDa-enzyme directly involved in the mRNAs’ post-transcriptional modifications. It catalyses the 2’O-methylation in the 5’ cap structure producing the cap 1 mRNA and acts by heterodimerisation as a processivity factor with the poly(A) RNA polymerase4. However, the low expression level of VP39 in Escherichia coli (E. coli) as well as its low in vitro catalytic efficiency have so far limited its use for industrial biocatalysis. Here, the two above-mentioned limitations are tackled by complementary approaches: i) we use a Split-GFP5 strategy coupled with ultrahigh throughput screening to select for higher soluble expression in E. coli and ii) we design smart libraries seeking to directly improve the catalytic turnover of the enzyme. 1. Leung, D. W. & Amarasinghe, G. K. When your cap matters: structural insights into self vs non-self recognition of 5’ RNA by immunomodulatory host proteins. Curr. Opin. Struct. Biol. 36, 133–141 (2016). 2. Zust, R. et al. Ribose 2’-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5. Nat Immunol 12, 137–143 (2011). 3. Sahin, U., Kariko, K. & Tureci, O. mRNA-based therapeutics - developing a new class of drugs. Nat. Rev. Drug Discov. 13, 759–780 (2014). 4. Hodel, A. E., Gershon, P. D., Shi, X. & Quiocho, F. A. The 1.85 A structure of vaccinia protein VP39: A bifunctional enzyme that participates in the modification of both mRNA ends. Cell 85, 247–256 (1996). 5. Cabantous, S. & Waldo, G. S. In vivo and in vitro protein solubility assays using split GFP. Nat. Methods 3, 845–854 (2006)

Advancements in the co-formulation of biologic therapeutics

Biologic therapeutics are the medicines of the future and are destined to transform the approaches by which the causes and symptoms of diseases are cured and alleviated. These approaches will be accelerated through the development of novel strategies that target multiple pharmacologically active sites using a combination of different biologics, or mixtures of biologics and small molecule therapeutics. However, for this potential to be realised, advancements in co-formulation strategies for biologic therapeutics must be established. This review describes the current and emerging developments within this field and highlights the challenges and potential solutions, that will pave-the-way towards their clinical translation

Column-free optical deconvolution of intrinsic fluorescence for a monoclonal antibody and its product-related impurities

The quantification of monoclonal antibody (mAb) aggregates and fragments using high pressure liquid chromatography-size exclusion chromatography (HPLC-SEC) typically requires off-line measurements that are time-consuming and therefore not compatible with real-time monitoring. However, it has been crucial to manufacturing and process development, and remains the industrial standard in the assessment of product-related impurities. Here we demonstrate that our previously established intrinsic time-resolved fluorescence (TRF) approach can be used to quantify the bioprocess critical quality attribute (CQA) of antibody product purity at various stages of a typical downstream process, with the potential to be developed for in-line bioprocess monitoring. This was directly benchmarked against industry-standard HPLC-SEC. Strong linear correlations were observed between outputs from TRF spectroscopy and HPLC-SEC, for the monomer and aggregate-fragment content, with R2 coefficients of 0.99 and 0.69, respectively. At total protein concentrations above 1.41 mg/mL, HPLC-SEC UV-Vis chromatograms displayed signs of detector saturation which reduced the accuracy of protein quantification, thus requiring additional sample dilution steps. By contrast, TRF spectroscopy increased in accuracy at these concentrations due to higher signal-to-noise ratios. Our approach opens the potential for reducing the time and labour required for validating aggregate content in mAb bioprocess stages from the several hours required for HPLC-SEC to a few minutes per sample

Lyophilization to enable distribution of ChAdOx1 and ChAdOx2 adenovirus-vectored vaccines without refrigeration

Distribution of vaccines which require refrigerated or frozen storage can be challenging and expensive. The adenovirus vector platform has been widely used for COVID-19 vaccines while several further candidate vaccines using the platform are in clinical development. In current liquid formulations, adenoviruses require distribution at 2-8 °C. The development of formulations suitable for ambient temperature distribution would be advantageous. Previous peer-reviewed reports of adenovirus lyophilization are relatively limited. Here, we report the development of a formulation and process for lyophilization of simian adenovirus-vectored vaccines based on the ChAdOx1 platform. We describe the iterative selection of excipients using a design of experiments approach, and iterative cycle improvement to achieve both preservation of potency and satisfactory cake appearance. The resulting method achieved in-process infectivity titre loss of around 50%. After drying, there was negligible further loss over a month at 30 °C. Around 30% of the predrying infectivity remained after a month at 45 °C. This performance is likely to be suitable for 'last leg' distribution at ambient temperature. This work may also facilitate the development of other product presentations using dried simian adenovirus-vectored vaccines

Directed evolution for soluble and active periplasmic expression of bovine enterokinase in Escherichia coli

Bovine enterokinase light chain (EKL) is an industrially useful protease for accurate removal of affinity-purification tags from high-value biopharmaceuticals. However, recombinant expression in Escherichia coli produces insoluble inclusion bodies, requiring solubilisation, refolding, and autocatalytic activation to recover functional enzyme. Error-prone PCR and DNA shuffling of the EKL gene, T7 promoter, lac operon, ribosome binding site, and pelB leader sequence, yielded 321 unique variants after screening ~ 6500 colonies. The best variants had > 11,000-fold increased total activity in lysates, producing soluble enzyme that no longer needed refolding. Further characterisation identified the factors that improved total activity from an inactive and insoluble starting point. Stability was a major factor, whereby melting temperatures > 48.4 °C enabled good expression at 37 °C. Variants generally did not alter catalytic efficiency as measured by kcat/Km, which improved for only one variant. Codon optimisation improved the total activity in lysates produced at 37 °C. However, non-optimised codons and expression at 30 °C gave the highest activity through improved protein quality, with increased kcat and Tm values. The 321 variants were statistically analysed and mapped to protein structure. Mutations detrimental to total activity and stability clustered around the active site. By contrast, variants with increased total activity tended to combine stabilising mutations that did not disrupt the active site

Crystal structures and molecular dynamics simulations of a humanised antibody fragment at acidic to basic pH

Antibody-fragment (Fab) therapy development has the potential to be accelerated by computational modelling and simulations that predict their target binding, stability, formulation, manufacturability, and the impact of further protein engineering. Such approaches are currently predicated on starting with good crystal structures that closely represent those found under the solution conditions to be simulated. A33 Fab, is an undeveloped immunotherapeutic antibody candidate that was targeted to the human A33 antigen homogeneously expressed in 95% cases of primary and metastatic colorectal cancers. It is now used as a very well characterised testing ground for developing analytics, formulation and protein engineering strategies, and to gain a deeper understanding of mechanisms of destabilisation, representative of the wider therapeutic Fab platform. In this article, we report the structure of A33 Fab in two different crystal forms obtained at acidic and basic pH. The structures overlapped with RMSD of 1.33 Å overall, yet only 0.5 Å and 0.76 Å for the variable- and constant regions alone. While most of the differences were within experimental error, the switch linker between the variable and the constant regions showed some small differences between the two pHs. The two structures then enabled a direct evaluation of the impact of initial crystal structure selection on the outcomes of molecular dynamics simulations under different conditions, and their subsequent use for determining best fit solution structures using previously obtained small-angle x-ray scattering (SAXS) data. The differences in the two structures did not have a major impact on MD simulations regardless of the pH, other than a slight persistence of structure affecting the solvent accessibility of one of the predicted APR regions of A33 Fab. Interestingly, despite being obtained at pH 4 and pH 9, the two crystal structures were more similar to the SAXS solution structures obtained at pH 7, than to those at pH 4 or pH 9. Furthermore, the P65 crystal structure from pH 4 was also a better representation of the solution structures at any other pH, than was the P1 structure obtained at pH 9. Thus, while obtained at different pH, the two crystal structures may represent highly (P65) and lesser (P1) populated species that both exist at pH 7 in solution. These results now lay the foundation for confident MD simulations of A33 Fab that rationalise or predict behaviours in a range of conditions