From R&D to Manufacturing of Drugs and Biotechnology Products

A pre-clinical drug discovery company and a manufacturer of antibodies wanted to discuss with us on options to use the new information and statistics from proteomics and genomics to plan R&D and avoid discrepancies in the scaling-up phase.

Our conversations started with the need to apply multidisciplinary approaches. The integration of Bioinformatics with Crystallography and Metallo-proteomics was our initial suggestion. To illustrate this, we used as models two types of protein: enzyme Methionine Aminopeptidase (MetAP) as a cancer marker, and human IgG antibodies. 

MetAP as a cancer marker

The MetAP enzyme was extensively studied for over twenty years as a cancer marker. However, still, the results are controversial due to the lack of enough information on the enzyme structure-activity correlations. We collected the data available for crystal structures of MetAPs from different organisms, their primary sequences and the most prominent articles published and available in PubMed.

Sequence and structure comparisons of MetAPs: Figure A. Domain organization of MetAP from E. coli, M. tuberculosis, human MetAPI, human mitochondrial MetAP, rat MetAPII and human MetAPII. K1, K2 and D boxes at the N-termini of MetAPII signify basic and acidic motifs, respectively; the metal-binding residues are shown in pink, the highly conserved residues (cyan), Cys (blue), based on the sequence alignment presented in Figure B. Divalent metal coordination in human MetAPI and MetAPII. Figure C. Ribbon structure of human MetAPI, the divalent metal (pink spheres) binding site and the NXV motif (boxed), PDB 2B3K. Figure D. Ribbon structure of human MetAPII, the divalent metal (pink spheres) binding site and the disulphide bond (boxed), PDB 1BN5. Figure E. The NXV motif in human MetAPI. Figure F. The disulfide bond in human MetAPII. The colours of the residues in the structures are the same as in Figure A: His and Met are coloured in firebrick and forest, respectively. PyMOL was used for the ribbon drawings. For more details, please refer to our article.

A new model for the enzyme mechanism of action was just proposed and published in a scientific journal. This new finding means that a different approach to planning the experiments is required. Moreover, we identified a substantial structural difference between the MetAP iso-forms derived from bacterial and higher organisms types. This finding is another new evidence that will help to understand MetAPs contribution to cancers in research better. The data explained the controversial results from the past that cost companies billion dollars to bring new anticancer drugs to the market.

Human IgG Antibodies

Monoclonal antibodies are the best example of heterogeneity in purified proteins. Partial modifications may come from cellular type, strains and stages of growth, media and buffer composition, primary and quaternary structure, temperature, etc. We collected data from the crystal structures, sequences and literature of the most studied IgG2 and IgG4 subclasses antibodies.

Human IgG antibodies. Figure A. Schematic representation of IgG2 with disulfide bonding, originally proposed (left) and observed rearrangements (right, dotted red lines), Figure B. Schematic representation of IgG4 with disulfide bonding, originally proposed (left) and observed rearrangements (right, dotted red lines). Figure C. Structure of intact monoclonal IgG2ak antibody (PDB 1IGT) with the two heavy chains (red and blue) and the two light chains (pink and light blue) such as colored in the scheme. Figure D. The hinge region of IgG2ak with the disulfide bonds (yellow) formed between the light and the heavy chains (CL214-CH128), and between the two heavy chains: CH237-CH237, CH240-CH240 and CH242-CH242. Data used from Ref. [70-72] (please refer to our article) and PyMOL was used for drawing the figures.

The existence of heterogeneity in terms of variations of post-translational modifications and oxidation were documented. This data illustrates the need for more careful preliminary research from literature and databases to plan future experiments for purification strategies and protein stability. Otherwise, abnormalities, such as aggregations and toxicity, may compromise the R&D project with substantial costs associated.

The two examples above indicated that the intricate nature of proteins requires complex case-by-case approaches when a research team endeavours to study a particular drug target. With the rapid development of modern biotechnology, the proper bio-application requires a thorough understanding of protein nature and data interpretation.  The collective efforts of scientists working in diverse closely-related fields make it possible to understand better how proteins function in cells. And this is the recommended approach by the regulatory authorities that mitigate risk and minimize costs, waste and time to bring high-quality products to the market.

With early research from databases and literature and the application of appropriate analytical tools, a reduction in subsequent unwanted processes is achievable. In the article, we did a review of the topic to illustrate the need for extensive preliminary research, multidisciplinary approaches, statistical DoE, and integration of classic with advanced analytical techniques. This organization provides an excellent environment to collect an enormous amount of data to describe the drug target better. For more details, please refer to our article.