Design-for-Six-Sigma for Development of a Bioprocess Quality-by-Design Framework.

An initial quality-by-design (QbD) framework was assembled for biopharmaceutical product, process, and analytical development using the design-for-six-sigma (DFSS) methodology. This technique was both streamlined and efficient, which permitted development of a QbD framework with minimized team leader and member resources. DFSS also highly emphasized voice-of-the-customer, information considered crucial to development and implementation of a bioprocess QbD framework appropriate for current development needs of the organization and its regulatory environment. The bioprocess QbD final design and implementation plan was comprised of seven teams, constructed from six QbD elements plus a communication/training team. Each element's detailed design was evaluated against internal and external established best practices, the QbD charter, and design inputs. Gaps were identified and risks mitigated to assure robustness of the proposed framework. Aggregated resources and timing were estimated to obtain vital implementation sponsorship. Where possible, existing governance and information technology efforts were leveraged to minimize additional bioprocess resources required. Finally, metrics were selected to track success of pilots and eventual implementation. LAY ABSTRACT: An initial quality-by-design (QbD) framework was assembled to guide biopharmaceutical product, process, and analytical development. QbD starts by defining the patient requirements which then are translated into required quality attributes for the product. The production process then is designed to consistently meet these quality requirements by identifying and understanding those parameters which influence them. A control strategy is developed that specifically relates each point of control to a desired quality measure. Overall, this approach results in a robust process, capable of reliably producing quality product. The bioprocess QbD framework was developed to guide implementation of the desired QbD strategy. It was comprised of seven teams, constructed from six QbD elements plus a communication/training team. Each element's detailed design was evaluated against internal and external established best practices, the charter, and design inputs. Gaps were identified and risks mitigated to assure robustness of the proposed framework. Aggregated resources and timing were estimated to obtain vital implementation sponsorship. Where possible, existing governance and information technology efforts were leveraged to minimize additional bioprocess resources required. Finally, metrics were selected to track success of pilots and eventual implementation.

[Producing several hundred of kilograms of monoclonal antibodies for therapy: a constant challenge]. / Du milligramme à la tonne d'anticorps monoclonaux : outils et perspectives de production.

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.

Induction studies with Escherichia coli expressing recombinant interleukin-13 using multi-parameter flow cytometry.

The expression of interleukin-13 (IL13) following induction with IPTG in Escherichia coli results in metabolic changes as indicated by multi-parameter flow cytometry and traditional methods of fermentation profiling (O2 uptake rate, CO2 evolution rate and optical density measurements). Induction early in the rapid growth phase was optimal although this led to lower overall biomass concentrations and lower maximum specific growth rates. In contrast, induction in the mid-rapid growth phase was the most detrimental to cell quality as measured by cytoplamsic membrane depolarisation.

Development and production of commercial therapeutic monoclonal antibodies in Mammalian cell expression systems: an overview of the current upstream technologies.

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.

Cholesterol delivery to NS0 cells: challenges and solutions in disposable linear low-density polyethylene-based bioreactors.

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.

A simple method for the production of plasmid DNA in bioreactors.

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.

An Asymmetric Synthesis of L-694,458, a Human Leukocyte Elastase Inhibitor, via Novel Enzyme Resolution of beta-Lactam Esters.

A convergent synthesis of [S-(R,S)]-2-[4-[(4-methylpiperazin-1-yl)carbonyl]phenoxy]-3,3-diethyl-N-[1-[3,4-(methylenedioxy)phenyl]butyl]-4-oxo-1-azetidinecarboxamide (L-694,458, 1), a potent human leukocyte elastase inhibitor, was achieved via chiral synthesis of key intermediates: (S)-3,3-diethyl-4-[4'-[(N-methylpiperazin-1-yl)carbonylphenoxy]-2-azetidinone (2) and (R)-alpha-propylpiperonyl isocyanate (3). Synthesis of beta-lactam 2 was achieved by a novel enantioselective lipase hydrolysis of ester 5 to produce (S)-3,3-diethyl-4-(4'-carboxyphenoxy)-2-azetidinone (6) (60% yield, three cycles, 93% ee) with isolation, epimerization, and recycling of the undesired (R)-ester 5. Isocyanate 3 was prepared by chiral addition of Zn(n-Pr)(2) to piperonal (98% yield, 99.2% ee), azide displacement and reduction to (R)-alpha-propylpiperonylamine (11) (58% yield, 85% ee), crystallization as the D-pyroglutamic acid salt (92% yield, 98.2% ee), and isocyanate formation (98% yield) with phosgene.