Systematic downstream process development for purification of baccatin III with key performance indicators

Winkelnkemper, T.; Schuldt, S.; Schembecker, Gerhard

doi:10.1016/j.seppur.2011.01.004

Cited by 15 publications

(17 citation statements)

References 28 publications

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“…Despite differences in polarity of the triterpenoids betulin and betulinic acid, assessed from 1‐octanol/water partition coefficients (betulin: 9.01, betulinic acid: 8.94) estimated with ACD/Chemsketch freeware, none of the screened extraction solvents was suited to selectively extract the acid. Equally, an additional purification step of adsorption/desorption with both ethyl acetate and acetone extracts using different adsorber materials as described in Winkelnkemper () turned out to be nonselective and gave low yields (data not shown). As the solubility of betulinic acid and other hydrophobic triterpenoids is pH‐dependent and decreases at low pH (Jäger, Winkler, Pfuller, & Scheffler, ), we evaluated polar acetone‐miscible antisolvents enabling precipitation for product recovery.…”

Section: Resultsmentioning

confidence: 99%

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Czarnotta

Dianat

Korf

et al. 2017

Biotech & Bioengineering

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Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L, and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering.

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Section: Resultsmentioning

confidence: 99%

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Czarnotta

Dianat

Korf

et al. 2017

Biotech & Bioengineering

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“…q resin;jÀ1 (19) Therein, V product,j-1 describes the size of the product pool collected in step j-1. In case of a flow-through, in which no fractionation takes place, the product mass balance simplifies to: c prod;in;j ¼ Y jÀ1 c prod;in;jÀ1 (20) The resulting rate-dependent specific costs as well as the additionally required flow velocities and product pool sizes are listed in Tab.…”

Section: Purification Stepsmentioning

confidence: 99%

“…These three key performance indicators are successfully applied by Winkelnkemper et al on literature examples [17,18] as well as on the example of purifying Baccatin III from a Taxus culture [19]. The resulting three-step purification sequence for Baccatin III consists of adsorption on Amberlite XAD-7, washing with water, and desorption with toluene.…”

Section: Introductionmentioning

confidence: 99%

Production Rate‐Dependent Key Performance Indicators for a Systematic Design of Biochemical Downstream Processes

Brandt

Schembecker

2016

Chem Eng & Technol

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Key performance indicators have become a common tool in bioprocess development, as they allow an objective and independent rating and ranking of process alternatives screened. A disadvantage of all key performance indicators developed so far is their strong focus on costs, which makes them neglect the influence of the production rate on the profit of the process. But, since both production costs and production rate have an equally strong impact, both should be considered when decisions have to be made. Therefore, two new key performance indicators are introduced in this work, one for batch and one for continuous processing. They combine product yield, purity performance, variable manufacturing costs, and production rate to one objective. Thereby, misguided trade-offs between these four individual measures can be replaced by one key performance indicator. This ensures a systematic development of biochemical downstream processes.

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“…Such methods are often used to optimize conditions for mAb polishing steps involving product variant removal, and several groups have also used these techniques to design non‐mAb purification processes (Bhambure, Gupta, & Rathore, ; Kateja, Kumar, Godara, Kumar, & Rathore, ). To effectively utilize the large amount of data collected in HTS experiments and to span a wider breadth of design space, in silico techniques have been used to further inform a knowledge‐based synthesis of new downstream processes (Asenjo & Andews, ; Asenjo, Herrera, & Byrne, ; Eriksson, Sandahl, Forslund, & Österlund, ; Petrides, ; Swanson, Xu, Nettleson, & Glatz, ; Watanabe, Tsoka, & Asenjo, ; Winkelnkemper & Schembecker, ; Winkelnkemper & Schembecker, ). Reported strategies, however, often rely on time‐consuming proteomic analysis of host cell proteins (HCPs), extensive characterization of the biophysical or biochemical attributes of the proteins, or tracking of individual impurities (Hanke & Ottens, ; Hanke et al, ).…”

Section: Introductionmentioning

confidence: 99%

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product‐related impurity removal

Vecchiarello

Timmick

Goodwine

et al. 2019

Biotech & Bioengineering

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Integrated designs of chromatographic processes for purification of biopharmaceuticals provides potential gains in operational efficiency and reductions of costs and material requirements. We describe a combined method using screening and in silico algorithms for ranking chromatographic steps to rapidly design orthogonally selective integrated processes for purifying protein therapeutics from both process-and product-related impurities. IFN-α2b produced in Pichia pastoris containing a significant product variant challenge was used as a case study. The product and product-related variants were screened on a set of 14 multimodal, ion exchange, and hydrophobic charge induction chromatography resins under various pH and salt linear gradient conditions. Data generated from reversed-phase chromatography of the fractions collected were used to generate a retention database for IFN-α2b and its variants. These data, in combination with a previously constructed process-related impurity database for P. pastoris, were input into an in silico process development tool that generated and ranked all possible integrated chromatographic sequences for their ability to remove both process and product-related impurities. Top-ranking outputs guided the experimental refinement of two successful three step purification processes, one comprising all bind-elute steps and the other having two bind-elute steps and a flowthrough operation. This approach suggests a new platform-like approach for rapidly designing purification processes for a range of proteins where separations of both process-and product-related impurities are needed. K E Y W O R D Shigh throughput process development, integrated downstream processing, mixed mode chromatography, process development tool, protein chromatography, straight-through processing

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Systematic downstream process development for purification of baccatin III with key performance indicators

Cited by 15 publications

References 28 publications

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Production Rate‐Dependent Key Performance Indicators for a Systematic Design of Biochemical Downstream Processes

A combined screening and in silico strategy for the rapid design of integrated downstream processes for process and product‐related impurity removal

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