[Show abstract][Hide abstract] ABSTRACT: The convergent synthesis of a potent α1A-selective adrenoceptor antagonist is described. Salient features of the synthesis include the enzymatic resolution of a racemic dihydropyrimidinone and the use of a palladium coupling reaction in the synthesis of 2,4'-dipyridyl.Key words: dihydropyrimidinone, enzymatic resolution, palladium coupling.
Canadian Journal of Chemistry 02/2011; 80(6):646-652. DOI:10.1139/v02-079 · 1.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
[Show abstract][Hide abstract] ABSTRACT: Discovering and designing novel therapeutic monoclonal antibodies (mAb) is just the beginning. In order to support clinical evaluations and to reach the market place, rapid and cost effective production platforms are needed. Process development and production efficiency play a crucial role in this space since they influence the cost of good and ultimately wide access to these life-saving medications. Due to their therapeutic dosages and repeated uses, the yearly need for certain mAb, especially those used in the treatments of cancer and inflammation, amounts to several hundred of kilograms. Consequently, significant technological investments are needed to support these extraordinary large needs for such complex proteins, and the industry is constantly aiming at reducing production costs while maintaining product quality to high levels. This review discusses some of the critical scientific and engineering decisions, which span from the selection of cell-line expression platforms to choices of technologies, which influence mAbs cost of goods that need to be made along the development path of a therapeutic mAb.
Medecine sciences: M/S 12/2009; 25(12):1078-84. DOI:10.1051/medsci/200925121078 · 0.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The screening of 144 microbial strains yielded several suitable biocatalysts for the asymmetric bioreduction of an allylic ketosulfone to its corresponding (R)-alcohol. The production of the desired enantiomer with elevated optical purity (ee > 90%) was achieved by the yeast strain Candida magnoliae MY 1785. The enantiocomplementary chiral reduction, the production of the (5)-alcohol, was also achieved when employing other microbial strains. The production of preparative amounts (about 2.5 g) of the (R)-enantiomer with elevated optical purity (ee > 90%) was completed when employing the yeast strain Candida magnoliae.
Biocatalysis and Biotransformation 07/2009; 18(6):471-477. DOI:10.3109/10242420009015264 · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This article provides an overview of the upstream technologies used in the industrial production of therapeutic monoclonal antibodies (mAbs) based on the cultivation of mammalian cells. More specifically, in a first section, after a short discussion of relevant biochemical characteristics of antibodies, we review the cell lines currently employed in commercial production and the methods of constructing and isolating production clones. This is followed with a review of the most current methods of commercial scale production and their associated technologies. Selected references and short discussions pertaining to emerging and relevant technologies have been embedded throughout the text in order to give a sense of the overall direction the field is taking.
Current pharmaceutical biotechnology 01/2009; 9(6):447-67. DOI:10.2174/138920108786786367 · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We report the successful cultivation of cholesterol dependent NS0 cells in linear low-density polyethylene (LLDPE) Wave Bioreactors when employing a low ratio of cyclodextrin to cholesterol additive mixture. While cultivation of NS0 cells in Wave Bioreactors was successful when using a culture medium supplemented with fetal bovine serum (FBS), cultivation with the same culture medium supplemented with cholesterol-lipid concentrate (CLC), which contains lipids and synthetic cholesterol coupled with the carrier methyl-beta-cyclodextrin (mbetaCD), proved to be problematic. However, it was possible to cultivate NS0 cells in the medium supplemented with CLC when using conventional cultivation vessels such as disposable polycarbonate shake-flasks and glass bioreactors. A series of experiments investigating the effect of the physical conditions in Wave Bioreactors (e.g., rocking rate/angle, gas delivery mode) ruled out their likely influence, while the exposure of the cells to small squares of Wave Bioreactor film resulted in a lack of growth as in the Wave Bioreactor, suggesting an interaction between the cells, the CLC, and the LLDPE contact surface. Further experiments with both cholesterol-independent and cholesterol-dependent NS0 cells established that the concurrent presence of mbetaCD in the culture medium and the LLDPE film was sufficient to inhibit growth for both cell types. By reducing the excess mbetaCD added to the culture medium, it was possible to successfully cultivate cholesterol-dependent NS0 cells in Wave Bioreactors using a cholesterol-mbetaCD complex as the sole source of exogenous cholesterol. We propose that the mechanism of growth inhibition involves the extraction of cholesterol from cell membranes by the excess mbetaCD in the medium, followed with the irreversible adsorption or entrapment of the cholesterol-mbetaCD complexes to the LLDPE surface of the Wave Bioreactor. Controlling and mitigating these negative interactions enabled the routine utilization of disposable bioreactors for the cultivation of cholesterol-dependent NS0 cell lines in conjunction with an animal component-free cultivation medium.
Journal of Bioscience and Bioengineering 02/2007; 103(1):50-9. DOI:10.1263/jbb.103.50 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The need for large quantities of purified plasmid DNA has increased as the applications of DNA vaccines continue to expand. This chapter describes a simple, scaleable procedure based on the fed-batch cultivation of various Escherichia coli clones, which can be easily implemented and scaled-up to large bioreactors. Although some clones may require minor modifications to the feeding strategy, in general, this procedure, implemented as described, is likely to support the production of milligram to gram quantities of plasmid DNA.
Methods in molecular medicine 02/2006; 127:295-309. DOI:10.1385/1-59745-168-1:295
[Show abstract][Hide abstract] ABSTRACT: The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introduction of genes into mammalian cells for use in both gene therapy and vaccine applications. “Naked DNA vaccines” allow the foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immune responses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially less consideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, for both clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typical small-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigations towards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the final product of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliance with existing regulatory guidelines. The impact of the nature of the plasmid vector on the choice of fermentation protocols is presented, along with the effect of varying cultivation conditions on final plasmid content. Practical considerations for the large-scale purification of plasmid DNA are also discussed.
[Show abstract][Hide abstract] ABSTRACT: Selected examples of asymmetric bioreductions of pharmaceutically relevant prochiral ketones are reviewed. These data show that microbial screens lead to the identification of appropriate biocatalysts, and that the use of miniaturized and semi-automated technology can greatly reduce both labor and lead times. The same data also highlight the need to evaluate a relatively large and/or diverse microbial population (highlighting biodiversity). We also found that in many instances the luxury of producing either enantiomers with high optical purity, enantiocomplementarity, can be achieved when employing different microbial strains. Process development studies reviewed here demonstrate that it is possible in some cases to understand and control the production of an unwanted enantiomer or by-product. Finally, a specific example, the asymmetric bioreduction of a ketone by Candida sorbophila, shows that process development studies which optimized, the bioreduction environmental conditions (pH, temperature…), the addition of ketone, and the implementation of a nutrient feeding strategy in conjunction with the use of a defined cultivation medium were key in achieving increased bioreduction rates and product titers. When scaled-up in pilot plant bioreactors, the bioreduction process supported the production of several kilograms of (R)-alcohol (enantiomeric excess (e.e.)>98%), with an isolated product yield of about 80%.
Journal of Molecular Catalysis B Enzymatic 01/2001; 11(4-11):503-512. DOI:10.1016/S1381-1177(00)00170-3 · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The feasibility of five potential biocatalytic routes were investigated for the chiral synthesis of key intermediates of an experimental endothelin receptor antagonist. Two asymmetric bioreductions of a ketoester and a chlorinated ketone to their corresponding chiral alcohol yielded very encouraging leads. Pichia delftensis (strain MY 1569) and Rhodotorula piliminae (ATCC 32762) were found to respectively bioreduce the esterified ketone and chlorinated substrate to their corresponding (S) alcohol with enantiomeric excesses > 98% and > 99% respectively. When scaled up in laboratory bioreactors (23-liter scale), both processes produced the desired (S) alcohol intermediate with elevated yield, about 88% and 97% for the ketoester and chloroketone respectively. Investigative chemical syntheses employing the (S) ester alcohol showed that unfavorable racemization occurred during the subsequent synthetic steps. However, the use of the (S) chloroalcohol as chiral synthon for the production of the endothelium receptor antagonist was successfully demonstrated at a preparative scale.
Biocatalysis and Biotransformation 01/2001; 19(4):267-279. DOI:10.3109/10242420109003644 · 0.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tools of metabolic and enzyme engineering have been well developed in academic laboratories and are now being applied for the optimization of biocatalysts used in the production of a wide range of pharmaceutically important molecules. Engineered microorganisms with a diverse set of modified or non-native enzyme activities are being used both to generate novel products and to provide improved processes for the manufacture of established products, such as in the production of precursors, intermediates, and complete compounds of importance to the pharmaceutical industry, including polyketides, nonribosomal peptides, steroids, vitamins, and unnatural amino acids. The use of directed evolution has rapidly emerged to be the method of choice for the development and selection of mutated enzymes with improved properties. A variety of such methods have been used to alter the activity, stability and availability of an array of enzymes. The industrial practice of these technologies at large scale is, however, in its infancy and stands as an exciting challenge for process scientists today.
Current Opinion in Biotechnology 05/2000; 11(2):209-14. DOI:10.1016/S0958-1669(00)00081-1 · 7.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In reviewing how biocatalysis can be applied to improve chiral synthesis for pharmaceuticals it becomes clear that there will be many opportunities using a simple enzyme system but that many of the more useful applications will require the whole cell because of the requirement for cofactors. An assessment is made of the opportunities to apply metabolic engineering to construct de novo metabolic pathways for the biosynthesis of useful advanced intermediates and a conceptual example is provided for the biosynthesis of cis-aminoindanol. We predict that in the future novel pathways will be assembled for a one-step biosynthesis of many semisynthetic natural products.
[Show abstract][Hide abstract] ABSTRACT: A rapid and efficient microbial screening procedure was developed utilizing a 24-well plate format in conjunction with an automated liquid handling system and an HPLC. For the evaluation of this miniaturized and automated screening system, we selected the bioreduction of 6-bromo-β-tetralone to 6-bromo-β-tetralol. This procedure employed both yeast and rhodococci libraries, representing a culture collection comprised of several hundred strains, from which to screen for desirable bioconversion activity. Most of these strains had demonstrated bioreducing activity during previous screens to insure a “hit rate” as high as possible. The cultivation of microbes in the plate format was facile, time saving, and efficient compared to the standard method of screening utilizing larger volumes, such as test tubes or shake flasks. This improved method of screening for bioconversion activity, employing pre-selected microbial libraries based on microtiter plates and a fully roboticized analytical system, proved to rapidly yield valuable leads which compared advantageously with a more classical approach. A total of 192 yeast strains and 48 rhodococci strains were screened using this procedure. Analytical data revealed that 78% of the strains tested bioconverted the tetralone to the desired alcohol.
Journal of Bioscience and Bioengineering 02/2000; 89(4-89):367-371. DOI:10.1016/S1389-1723(00)88960-4 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The screening of 310 microbial strains yielded eight as suitable biocatalysts for the asymmetric bioreduction of a highly hindered bisaryl ketone to its corresponding alcohols. The production of both enantiomers with elevated optical purity (ee>96%) was achieved by different microorganisms. When scaling up the asymmetric bioreduction process in laboratory bioreactors (23 l scale), the production of preparative amounts (1.5 g) of the (S) enantiomer with elevated optically purity (ee>96%) was achieved when employing the yeast Rhodotorula pilimanae ATCC 32762. Achieving this asymmetric bioreduction with enantiocomplementarity in employing such a hindered substrate is remarkable and highlights the potential of such biological approach.
Journal of Molecular Catalysis B Enzymatic 02/2000; 8(4):285-288. DOI:10.1016/S1381-1177(99)00103-4 · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An attractive method for producing cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene (toluene cis glycol) was developed employing a cis dihydrodiol dehydrogenase "deficient" strain of Rhodococcus (MA 7249). The toluene cis glycol produced was found to have optical rotations of [alpha]D25 = +25.8 (c 0.45, CH3OH) and +72.8 (c 0.42, CHCl3) which indicated an absolute configuration of (1S,2R) when compared with previously published values. When cultivated in laboratory fermentor in the presence of toluene vapors, MA 7249 reached a toluene cis glycol concentration up to 18 g/l in 110 h. Culture MA 7249 also accumulated cis (1S,2R) dihydrodiols from dihydronaphthalene, biphenyl, chlorobenzene, and styrene.
Journal of Bioscience and Bioengineering 02/2000; 90(3):321-7. DOI:10.1263/jbb.90.321 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A chemoenzymatic synthesis of β3 agonist 1 suitable for large scale preparation is described. The key chiral 3-pyridylethanolamine intermediate (R)-7 was prepared via an improved Neber rearrangement and a yeast-mediated asymmetric reduction. The tetrazolone fragment of the molecule was constructed via a dipolar cycloaddition between 1-(cyclopentyl)-3-propylazide and p-chlorosulfonyl phenylisocyanate. Sulfonamide coupling of these two intermediates under Shotten-Baumann conditions, followed by a borane reduction of the amide afforded 1 in 20–32% overall yield from 3-acetylpyridine.
[Show abstract][Hide abstract] ABSTRACT: A microbial screen identified the yeast Candida sorbophila MY 1833 as a suitable biocatalyst for the asymmetric bioreduction of a ketone (2-(4-nitro-phenyl)-N-(2-oxo-2-pyridin-3-yl-ethyl)-acetamide) to its corresponding (R) alcohol [(R)-N-(2-hydroxy-2-pyridin-3-yl-ethyl)-2-(4-nitro-phenyl)-acetamide]. Studies yielded the formulation of a chemically defined cultivation medium that supported excellent growth and bioconversion activity. Process development showed that the optimization of the bioreduction environmental conditions (pH, temperature), the timing of ketone addition, and the implementation of a nutrient feeding strategy were key factors in achieving increased bioreduction rates. The optimized process achieved a 10-fold bioreduction rate improvement over the original process, while reaching final product concentrations of up to 60 g/l. When scaled up in Pilot Plant bioreactors (280 l), the bioreduction process supported the production of several kilograms of highly optically pure (R) alcohol (enantiomeric excess > 98%).
[Show abstract][Hide abstract] ABSTRACT: Cis (1S,2R) indanediol is a potential precursor to (-)-cis (1S,2R)-1-aminoindan-2-ol, a key chiral synthon for a leading HIV protease inhibitor, Crixivan (Indinavir). A potential route to the biosynthesis of this important precursor, the microbial asymmetric direduction of 1,2-indanedione to its corresponding diol, cis (1S,2R) indanediol, was investigated. The screening of 32 yeast strains yielded Trichosporon cutaneum MY 1506 as a suitable biocatalyst. At the 2-l shake-flask scale, 1,2-indanedione (charged at 1.0 g/l) was bioconverted to cis (1S,2R) indanediol at a final bioconversion yield of 99.1% and an enantiomeric excess of >99%. When scaled up in a 23-l bioreactor, T. cutaneum produced 8.4 g of pure cis (1S,2R) indanediol, and the isolated yield of cis (1S,2R) indanediol was 52%. Purification of the scale-up also yielded 0.9 g of the more polar trans (1S,2R) indanediol diastereomer, a minor bioreduction product. Supercritical fluid chromatography analyses of the purified cis (1S,2R) and trans (1S,2S) indanediol demonstrated that the enantiomeric excesses during this bioconversion scale-up were 99% and 26%, respectively.
Journal of Bioscience and Bioengineering 02/1999; 88(5):495-9. DOI:10.1016/S1389-1723(00)87665-3 · 1.88 Impact Factor