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Synthesis of Important Chiral Building Blocks for Pharmaceuticals Using Lactobacillus and Rhodococcus Alcohol Dehydrogenases
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Marion Rauter, Simon Krebs, Gotthard Kunze
(S)-Duloxetine building blocks were synthesized by the reduction of the corresponding ketone with an ADH variant from L. kefir generated by directed evolution methods. The engineered enzyme converted the substrate at a rate 15 times higher than that of the reference ADH. In a 100 ml reaction the engineered enzyme gave a yield of 99% with an ee of >99% after one day under vacuum for acetone removal from the isopropanol-coupled NADPH regenerating process. Substrate concentrations were high with up to 150 g L−1. These improvements allowed the effective bioproduction of this drug.
Insights into the IgG heavy chain engineering patent landscape as applied to IgG4 antibody development
Published in mAbs, 2019
Christophe Dumet, Jérémy Pottier, Valérie Gouilleux-Gruart, Hervé Watier
Pharmacological differences between IgG1 and IgG4 subclasses result from structural differences between the γ1 and γ4 heavy chains. However, depending on allotypes (genetic variants), comparison between these two isotypes can be misleading. Here, we use γ1 and γ4 to refer to the chains encoded by the IGHG1*01 and IGHG4*01 alleles, respectively. The differences are mainly located in and around the hinge (Figure 1(a,b)) and can also affect the downstream processing during bioproduction. For example, the purification process can contribute to aggregation, but to a different extent according to the subclass. Indeed, IgG4 have been showed to be less stable than IgG1s at low pH conditions.19,20 In particular, the serine at position 228 is specific to γ4 and is one of the key amino-acids playing a role in the stability of IgG4s. A study has shown that S228P substitution resulted in a homogenous IgG4 when analyzed by SDS-PAGE.21 It was later demonstrated that this substitution could block the half-IgG exchange phenomenon usually known as Fab-arm exchange (Figure 2).22,23
Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes
Published in Journal of Extracellular Vesicles, 2018
Dionysios C. Watson, Bryant C. Yung, Cristina Bergamaschi, Bhabadeb Chowdhury, Jenifer Bear, Dimitris Stellas, Aizea Morales-Kastresana, Jennifer C. Jones, Barbara K. Felber, Xiaoyuan Chen, George N. Pavlakis
Using the scalable production and purification workflow described in this study, we obtained large amounts of highly purified multivalent EV incorporating bioactive, fully human hetIL-15/lactadherin complexes. Future studies will test these EV as delivery vehicles of immunotherapy in preclinical models. The described methods are based on well-established technologies, which can be readily applied to generate purified preparations of engineered EV for research and development. Cost for the consumables required (TFF-devices, chromatography columns) is proportional to the volume of the starting material, making it an appealing workflow for various application scales. Confirmation of the utility of the method proposed in this study in industrial-scale production of EV remains to be demonstrated in an appropriate setting. Given the established use of TFF and SEC in industrial bioproduction, and their compatibility with cGMP settings, the proposed methodology comprises a promising candidate for production and purification of EV for clinical use.
Computational re-design of protein structures to improve solubility
Published in Expert Opinion on Drug Discovery, 2019
Susanna Navarro, Salvador Ventura
In most cases, the size and complexity of therapeutic proteins hinder their chemical synthesis and they should be produced in biofactories [4–6]. The inherent instability of proteins and the associated propensity to degradation makes the bioproduction and purification of these molecules in their native and active conformations challenging [7]. Therefore, strategies to improve the production yield, specificity, purity or half-life of the product are usually required [8–10]. In addition, the need to administrate therapeutic proteins at high concentrations (≥ 100 mg/mL), exceeding in several orders of magnitude their natural abundances, unavoidably compromises their solubility in pharmaceutical formulations, often leading to the formation of protein aggregates during long-term storage.