Peter Salmon

Metabolic engineering and directed evolution for the production of pharmaceuticals.

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.

High-level protein expression in scalable CHO transient transfection

Chinese hamster ovary cells (CHO) have been extensively utilized as the production platform for therapeutic proteins including monoclonal antibodies in pharmaceutical industry. For early development, it would be advantageous to rapidly produce large amounts of protein in the same cell line; therefore, development of a CHO transient transfection platform with high protein expression level is highly desirable. Here, we describe the development of such a platform in CHO cells. Polyethylenimine (PEI) was used as the transfection reagent. Different media were screened for the best transfection and expression performance, and UltraCHO was chosen as the best performer. DMSO and lithium acetate (LiAc) were discovered to improve CHO transient transfection expression levels significantly. A 14‐day fed‐batch process was successfully developed to further increase production yield. With an optimized transient transfection process, we were able to express monoclonal antibody (Mab) in CHO cells at a high level, averaging 80 mg/L. The process was successfully scaled up to 10 L working volume in a 20 L wave bioreactor. As expected, the Mabs had similar glycosylation patterns in comparison to the Mabs produced from a stably transfected CHO cell line, while in contrast Mabs expressed transiently from HEK293EBNA cells differed. Biotechnol. Bioeng. 2009;103: 542–551.

Purification and characterization of a novel bioconverting lipase from Pseudomonas aeruginosa MB 5001

Abstract The purification and characterization of a lipase produced by Pseudomonas aeruginosa strain MB 5001 that was selected for its unique bioconversion properties is described herein. The purified lipase bioconverts dimethyl 5-(3-(2-(7-chloroquinolin-2-yl)-ethyl)phenyl)4, 6-dithianonanedioate (diester) to its (S)-ester acid, an intermediate in the synthesis of Verlukast, a leukotriene receptor antagonist. In its native form, the enzyme exists as high-molecular-weight aggregates that are dissociated with Triton X-100. The purified enzyme has a molecular weight of 29,000 daltons, a pH optimum of 8.0, a temperature optimum of 55°C, and is stable for 1 h at 40°C. This lipase is strongly inhibited by 1 m m ZnSo 4 (94% inhibition) but is stimulated by the addition of 10 m m CaCl 2 (1.24-fold) and 200 m m taurocholic acid (1.6-fold). It is more active on short-chain versus long-chain triglycerides, and hydrolyzes C18-unsaturated fatty acid esters more efficiently than it hydrolyzes C18-saturated fatty acid esters. In light of its catalytic and physicochemical properties, this enzyme is regarded as a novel lipase .

Rapid protein production using CHO stable transfection pools

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post‐transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence‐activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three‐fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin‐opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines.

Development of a highly productive and scalable plasmid DNA production platform.

With the applications of DNA vaccines extending from infectious diseases to cancer, achieving the most efficient, reproducible, robust, scalable, and economical production of clinical grade plasmid DNA is paramount to the medical and commercial success of this novel vaccination paradigm. A first generation production process based on the cultivation of Escherichia coli in a chemically defined medium, employing a fed‐batch strategy, delivered reasonable volumetric productivities (500–750 mg/L) and proved to perform very well across a wide range of E. coli constructs upon scale‐up at industrial scale. However, the presence of monosodium glutamate (MSG) in the formulation of the cultivation and feed solution was found to be a potential cause of process variability. The development of a second generation process, based on a defined cultivation medium and feed solution excluding MSG, was undertaken. Optimization studies, employing a plasmid coding for the HIV gag protein, resulted in cultivation conditions that supported volumetric plasmid titers in excess of 1.2 g/L, while achieving specific yields ranging from 25 to 32 μg plasmid DNA/mg of dry cell weight. When used for the production of clinical supplies, this novel process demonstrated applicability to two other constructs upon scale‐up in 2,000‐L bioreactors. This second generation process proved to be scalable, robust, and highly productive.

Development of transfection and high-producer screening protocols for the CHOK1SV cell system

To date, the FDA has approved 18 monoclonal antibody (MAb) therapeutic drugs with targets ranging from asthma and rheumatoid arthritis to leukemia. Many of these approved products are produced in Chinese hamster ovary cells (CHO) making CHO a significant and relevant host system. We studied the applicability of CHOK1SV cells as a potential host cell line for MAb production in terms of timelines, achievable titers, transfectant stability, and reproducibility. CHOK1SV, developed by Lonza Biologics, is a suspension, protein-free-adapted CHOK1-derivative utilizing the glutamine synthetase (GS) gene expression system. CHOK1SV expresses the GS enzyme endogenously; thus, positive transfectants were obtained under the dual selection of methionine sulfoximine (MSX) and glutamine-free media. We examined outgrowth efficiencies, specific productivities, and achievable batch titers of three different IgG MAbs transfected into CHOK1SV. Reducing the MSX concentration in the initial selection medium resulted in a decreased incubation time required for transfectant colonies to appear. Specific productivities of “high-producers” ranged between 11 and 49 pg/c/d with batch titers ranging from 105 to 519 mg/L. Transfectant stability and the effects of MSX also were investigated, which indicated that the addition of MSX was necessary to maintain stable MAb production. Cell growth was stable regardless of MSX concentration.

Development of a defined medium fermentation process for physostigmine production by Streptomyces griseofuscus

Physostigmine is a plant alkaloid of great interest as a therapeutic candidate for the treatment of Alzheimers disease. Fortunately, this compound is also produced by Streptomyces griseofuscus NRRL 5324 during submerged cultivation. A fermentation process that used chemically defined medium was therefore developed for its production. By means of statistical experimentation, the physostigmine titer was quickly increased from 20 mg/l to 520 mg/l with a culture growth of 19 gl dry cell weight on the shake-flask scale. Further medium optimization resulted in a yield of 790 mg/l in a 23-l bioreactor using a batch process. A titer of 880 mg/l was attained during scale-up in a 800-l fermentor by employing a nutrient-feeding strategy. This production represents a 44-fold increase over the yield from the initial process in shake-flasks. The defined-medium fermentation broth was very amenable to downstream processing.

Effects of amino acid and trace element supplementation on pneumocandin production by Glarea lozoyensis: impact on titer, analogue levels, and the identification of new analogues of pneumocandin B0

Addition of the amino acids threonine, serine, proline, and arginine to fermentations of the fungus Glarea lozoyensis influenced both the pneumocandin titer and the spectrum of analogues produced. Addition of threonine or serine altered the levels of the “serine analogues” of pneumocandins B0 and B5 and allowed for their isolation and identification. Proline supplementation resulted in a dose-dependent increase in the levels of pneumocandins B0 and E0, whereas pneumocandins C0 and D0 decreased as a function of proline level. Moreover, proline supplementation resulted in an overall increase in the synthesis of both trans-3- and trans-4-hydroxyproline while maintaining a low trans-4-hydroxyproline to trans-3-hydroxyproline ratio compared to the unsupplemented culture. Pneumocandin production and the synthesis of hydroxyprolines was also affected by addition of the proline-related amino acid arginine but not by the addition of glutamine or ornithine. Zinc, cobalt, copper, and nickel, trace elements that are known to inhibit α-ketoglutarate-dependent dioxygenases, affected the pneumocandin B0 titer and altered the levels of pneumocandins B1, B2, B5, B6, and E0, analogues that possess altered proline, ornithine, and tyrosine hydroxylation patterns. Journal of Industrial Microbiology & Biotechnology (2001) 26, 216–221.

Conversion of indene to cis-(1S),(2R)-indandiol by mutants of Pseudomonas putida F1

Two mutation and selection methods were used to isolate mutants of Pseudomonas putida F1 which convert indene to cis-(1S),(2R)-indandiol in a toluene-independent fashion. Using soybean or silicone oil as a second phase to deliver indene to the culture, cis-(1S),(2R)-indandiol, cis-(1R),(2S)-indandiol, 1,2-indenediol (or the keto-hydroxy indan tautomer), and the monooxygenation products 1-indenol and 1-indanone were produced from indene as a function of time. Similarly the enantiomeric excess of the cis-(1S),(2R)-indandiol produced also increased with increasing time. In addition, mutants were isolated which produced cis-(1S),(2R)-indandiol of lower optical purity which corresponded to reduced levels of 1,2-indenediol. These data suggest this toluene dioxygenase produces cis-(1S),(2R)-indandiol of low optical purity and that cis-glycol dehydrogenase plays a role in resolving the two cis-1,2-indandiol enantiomers.

Enhancement of Lipase Production during Fed-Batch Cultivation of Pseudomonas aeruginosa MB 5001

Abstract Lipase production by Pseudomonas aeruginosa (strain MB 5001) was enhanced 6.6-fold by developing a fed-batch fermentation process. In batch culturing, lipase synthesis occurred during decelerated cell growth. Building on this information, lipase production was increased by feeding a balanced solution of glucose and ammonium chloride (carbon to nitrogen ratio of 4.13). Best performance was achieved when feeding was initiated during mid growth phase, prior to lipase biosynthesis. This lipase fermentation process was found to be unusually sensitive to dissolved oxygen tension, requiring cultivation of the microorganisms initially under dissolved oxygen limiting conditions followed by non-limiting dissolved oxygen conditions (lipase production phase). These process improvements yielded rapidly to scale up from laboratory bioreactors (23-l) to pilot plant bioreactors (1,900-l).