Despite the advent of many new therapies, therapeutic monoclonal antibodies remain a prominent biologics product, with a market value of billions of dollars annually. A variety of downstream processing technological advances have led to a paradigm shift in how therapeutic antibodies are developed and manufactured. A key driver of change has been the increased adoption of single-use technologies for process development and manufacturing. An early-stage developability assessment of potential lead antibodies, using both in silico and high-throughput experimental approaches, is critical to de-risk development and identify molecules amenable to manufacturing. Both statistical and mechanistic modelling approaches are being increasingly applied to downstream process development, allowing for deeper process understanding of chromatographic unit operations. Given the greater adoption of perfusion processes for antibody production, continuous and semi-continuous downstream processes are being increasingly explored as alternatives to batch processes. As part of the Quality by Design (QbD) paradigm, ever more sophisticated process analytical technologies play a key role in understanding antibody product quality in real-time. We should expect that computational prediction and modelling approaches will continue to be advanced and exploited, given the increasing sophistication and robustness of predictive methods compared to the costs, time, and resources required for experimental studies.

Antibody therapeutic development often involves significant demands for purified protein samples, from initial assessments of numerous constructs from early stage screening campaigns through to lead identification and then for process development and pilot scale runs. Efforts to reduce timelines and cost per sample are common to both platform purification and for process development. In the earliest stages, high-throughput purification platforms that utilize liquid handlers or other small volume approaches can be suitable, as the quantity requirements for assays are minimal. However, as the number of candidate molecules diminishes, the scope of assays can quickly expand and include a variety of cell-based and in vivo experiments which can require tens or hundreds of milligrams of products of defined purity and with low endotoxin levels. Purification of these samples in a high-throughput, parallelized manner represents a significant challenge with relatively few available off-the-shelf solutions. Process development requirements are also amendable to high-throughput purification strategies combined with statistical approaches in order to optimize the design space and narrow initial process operation parameters suitable for a given purification unit operation. While less often utilized, non-chromatographic purification methods may also be amenable to automation and parallelization at the initial stages of purification development.

Abstract One of the most promising of the next generation of biologic-based cancer therapeutics builds on the molecular targeting abilities of antibodies by combining them with drugs to generate highly specific antibody-drug conjugates (ADCs). However, the development of ADCs requires time-consuming selection of the antibody for every target and cancer type. High-throughput screening technologies based on the use of conjugated secondary antibodies provide a fast and efficient surrogate assay from which to identify which antibodies are best internalized and suitable for immunoconjugate development into ADCs. As part of its integrated antibody development initiative, NRC has isolated and characterized anti- mouse Fc and anti-human Fc monoclonal antibodies to serve as very selective detective reagents for various IgG isotypes. We have shown that these secondary antibodies are species specific, selective and of high affinity. When conjugated to pH sensitive fluorophores, we have used them to specifically identify internalizing antibodies against tumor targets, which were later validated as ADCs. Furthermore, these secondary conjugates exhibit high specific potency and low background toxicity once conjugated to linkered drugs. This approach allow us to optimize the selection of an antibody for a particular target, tumor type, linker and drug for ADC development. NRC will present results of a screen of 285 mouse antibodies against 20 different targets in 7 different cancer cell lines, using either MCC-DM1 or vc-MMAE-conjugated secondary antibodies. The NRC ADC discovery platform is combining this methodology with our proprietary mRNA and DNA expression database for the selection of appropriate ADC targets. NRC Biologics and Biomanufacturing program is in the process of screening thousands of NRC antibodies generated against a variety of cancer-associated cell surface targets to deliver a steady pipeline of ADCS as part of its drug discovery efforts. This functional screening platform further promotes the integration and advancement of NRC’s capabilities and strengths in the area of biologic-based therapeutics lead candidate selection, including quality attributes and characterization and biomanufacturing. The combined expertise in cell biology, high throughput screening, antibody generation and selection, bioinformatics and expression analysis forms the foundation by which NRC can establish strategic collaborations with other Canadian or international partners to develop antibodies into novel ADC biologics. Citation Format: Maria Luz Jaramillo, Luc Meury, Patrice Bouchard, Allan Matte, Anne Marcil, Mauro Acchione, Jennifer Hill, Francois Fauteux. Screening platform for development of antibody-drug conjugates against novel targets at the National Research Council of Canada [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3669. doi:10.1158/1538-7445.AM2017-3669

Within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mAbs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we describe specific applications and associated workflows of the Protein Maker liquid handling system utilized in both of these contexts. To meet the requirements for various in vitro assays, for the identification and validation of new therapeutic targets, small quantities of large numbers of purified antibodies or antibody fragments are often required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various wash buffers is an example of how parallelized protein purification can be leveraged to improve a process development outcome. Stability testing under various conditions of in-process intermediates, as an example, the mAb product from a clarified harvest, requires parallelized protein purification to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid scale platform purification or for process development applications to generate the necessary purified protein samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a significant reduction in time and cost per sample compared to serial purification using a traditional FPLC system. By combining the Protein Maker purification system with a TECAN Freedom EVO liquid handler for automated buffer exchange we have created a new, integrated platform for a variety of protein purification and process development applications.

Many hexose sugars in bacteria undergo a variety of modifications, including oxidation/reduction, amination and acetylation, as part of biosynthesis into their final biologically active forms. Enzymes that catalyse these reactions normally utilize nucleotide-linked sugar substrates, utilizing the nucleotide as an 'ancient handle' to bind and orient the sugar within the enzymes' active site. We and others have focused efforts on elucidating structure function relationships for a subset of such biosynthetic enzymes, those associated with the synthesis of trideoxy-diacetamidohexoses, and the nonulsonate sugars subsequently derived from them. With structural information combined with site-directed mutagenesis, enzymatic analysis and molecular modelling, these studies have been essential to understanding the chemistry of how these enzymes bind their substrates and effect catalysis. Enzymes having different folds, such as N-acetyltransferases, can utilize different scaffolds to attain the same 4-acetamido-sugar product. In the case of dehydratases/epimerases and aminotransferases, the enzymes have a conserved structure, but utilize subtle differences within their active site to confer substrate binding and the nature of the final product. These studies depict the structural relationships between these enzymes, while at the same time high-lighting important differences that are beginning to reveal their function at the molecular level.

The Escherichia coli dihydroxyacetone (Dha) kinase is an unusual kinase because (i) it uses the phosphoenolpyruvate carbohydrate: phosphotransferase system (PTS) as the source of high-energy phosphate, (ii) the active site is formed by two subunits, and (iii) the substrate is covalently bound to His218K* of the DhaK subunit. The PTS transfers phosphate to DhaM, which in turn phosphorylates the permanently bound ADP coenzyme of DhaL. This phosphoryl group is subsequently transferred to the Dha substrate bound to DhaK. Here we report the crystal structure of the E. coli Dha kinase complex, DhaK–DhaL. The structure of the complex reveals that DhaK undergoes significant conformational changes to accommodate binding of DhaL. Combined mutagenesis and enzymatic activity studies of kinase mutants allow us to propose a catalytic mechanism for covalent Dha binding, phosphorylation, and release of the Dha-phosphate product. Our results show that His56K is involved in formation of the covalent hemiaminal bond with Dha. The structure of H56NK with noncovalently bound substrate reveals a somewhat different positioning of Dha in the binding pocket as compared to covalently bound Dha, showing that the covalent attachment to His218K orients the substrate optimally for phosphoryl transfer. Asp109K is critical for activity, likely acting as a general base activating the γ-OH of Dha. Our results provide a comprehensive picture of the roles of the highly conserved active site residues of dihydroxyacetone kinases.

Abstract Adenosine 5′‐triphosphate (ATP) binds to a great number of proteins to elicit a wide variety of effects, including energy production and molecular signalling. Proteins have evolved different strategies to specifically recognize ATP, utilizing different ways of binding the phosphoryl moieties as well as the adenine base. The most common, conserved sequence and structural motif for binding ATP is the Walker‐A motif, or P‐loop, found in many different protein structural families. Greater variation in the sequence of the P‐loop is being recognized, as more ATP‐binding proteins are being structurally and functionally characterized. In contrast to the P‐loop, recognition of the adenine base often makes use of conserved structural motifs of main‐chain atoms via hydrogen‐bonding interactions, or side‐chains in stacking interactions, without a definitive amino acid sequence pattern. Key concepts: A major class of ATP‐binding proteins are those that contain a P‐loop or Walker‐A motif. P‐loops or glycine‐rich loops function by binding the phosphoryl groups of ATP. Several sequence variations on the Walker‐A motif are now known and have been functionally characterized. The Walker‐B motif contains a conserved acidic residue (Glu/Asp) that functions to bind directly or indirectly a metal ion important in catalysis. Adenine‐binding does not occur through specific sequence motifs, but rather uses a conserved pattern of polar and nonpolar interactions within a structural motif. Both main‐chain hydrogen bonding and aromatic residue stacking contribute to adenine‐binding by proteins.

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters.

The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a β-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the β-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.