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Cambridge, MA, United States

Mcelearney K.,Cell Culture Development | Ali A.,Cell Culture Development | Gilbert A.,Cell Culture Development | Kshirsagar R.,Cell Culture Development | Zang L.,Analytical Development
Biotechnology Progress | Year: 2015

Chemically defined media have been widely used in the biopharmaceutical industry to enhance cell culture productivities and ensure process robustness. These media, which are quite complex, often contain a mixture of many components such as vitamins, amino acids, metals and other chemicals. Some of these components are known to be sensitive to various stress factors including photodegradation. Previous work has shown that small changes in impurity concentrations induced by these potential stresses can have a large impact on the cell culture process including growth and product quality attributes. Furthermore, it has been shown to be difficult to detect these modifications analytically due to the complexity of the cell culture media and the trace level of the degradant products. Here, we describe work performed to identify the specific chemical(s) in photodegraded medium that affect cell culture performance. First, we developed a model system capable of detecting changes in cell culture performance. Second, we used these data and applied an LC-MS analytical technique to characterize the cell culture media and identify degradant products which affect cell culture performance. Riboflavin limitation and N-formylkynurenine (NFK), a tryptophan oxidation catabolite, were identified as chemicals which results in a reduction in cell culture performance. © 2015 American Institute of Chemical Engineers. Source

Hart M.,Analytical Development | Acott S.,Sanofi S.A.
ecancermedicalscience | Year: 2010

A one-vial formulation of the chemotherapy agent Taxotere® (docetaxel) is likely to be commercially available in some countries in 2010. Through control of two significant potential risk factors for crystallization - intervention into the infusion bag (by using a one-step technique) and temperature (by use of refrigerated storage) - it was postulated that the risk of precipitation would be reduced and the storage time of Taxotere® increased using the one-vial formulation versus the two-vial formulation. Furthermore, improved convenience and flexibility were anticipated as a result of the easier and quicker preparation associated with the one-vial formulation versus the twovial formulation. The results of the physicochemical stability study presented here indicate that the one-vial formulation is indeed associated with reduced risk of precipitation of docetaxel. Moreover, refrigerated storage has been shown to extend the physical and chemical stability for up to seven days, versus 4 h for the currently approved two-vial formulation and 6 h for the one-vial formulation stored at room temperature. However, only physical and chemical stability have been assessed in this study. Copyright: © the authors; licensee ecancermedicalscience. Source

Wright C.,Cell Culture Development | Groot J.,Computational Biology and Genomics | Swahn S.,Cell Culture Development | McLaughlin H.,Computational Biology and Genomics | And 5 more authors.
Biotechnology Progress | Year: 2016

A central goal for most biopharmaceutical companies is to reduce the development timeline to reach clinical proof of concept. This objective requires the development of tools that ensure the quality of biotherapeutic material destined for the clinic. Recent advances in high throughput protein analytics provide confidence in our ability to assess productivity and product quality attributes at early stages of cell line development. However, one quality attribute has, until recently, been absent from the standard battery of analytical tests facilitating informed choices early in cell line selection: genetic sequence confirmation. Techniques historically used for mutation analysis, such as detailed mass spectrometry, have limitations on the sample number and turnaround times making it less attractive at early stages. Thus, we explored the utility of Next-Generation Sequencing (NGS) as a solution to address these limitations. Amplicon sequencing is one such NGS technique that is robust, rapid, sensitive, and amenable to multiplexing, all of which are essential attributes for our purposes. Here we report a NGS method based upon amplicon sequencing that has been successfully incorporated into our cell line development workflow alongside other high-throughput protein analytical assays. The NGS method has demonstrated its value by identifying at least one Chinese hamster ovary (CHO) clone expressing a variant form of the biotherapeutic in each of the four clinical programs in which it has been utilized. We believe this sequence confirmation method is essential to safely accelerating the time to clinical proof of concept of biotherapeutics, and guard against delays related to sequence mutations. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:813–817, 2016. © 2016 American Institute of Chemical Engineers Source

Szekrenyes A.,Debrecen University | Park S.S.,Amgen | Santos M.,SCIEX | Lew C.,SCIEX | And 28 more authors.
mAbs | Year: 2016

An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene- 1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra highperformance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established. © 2016, Ákos Szekrényes…. Source

Coughlin J.,Analytical Development | Masci A.,Analytical Development | Gronke R.S.,Process Biochemistry | Bergelson S.,Analytical Development | Co C.,Analytical Development
Analytical Biochemistry | Year: 2016

Immobilized protein receptors and enzymes are tools for isolating or enriching ligands and substrates based on affinity. For example, glutathione S-transferase (GST) is fused to proteins as a tag for binding to its substrate glutathione (GSH) linked to solid supports. One issue with this approach is that high-affinity interactions between receptors and ligands require harsh elution conditions such as low pH, which can result in leached receptor. Another issue is the inherent nonspecific chemical conjugation of reactive groups such as N-hydroxysuccinimide (NHS) that couple lysines to solid supports; the nonspecificity of NHS may result in residue modifications near the binding site(s) of the receptor that can affect ligand specificity. In this study, a simple conjugation procedure is presented that overcomes these limitations and results in immobilized GST fusion proteins that are functional and specific. Here, the affinity of GST for GSH was used to generate an enzyme–substrate site-specific cross-linking reaction; GSH–Sepharose was preactivated with 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) and then incubated Fc gamma receptor IIIa (FcγRIIIa)–GST. The immobilized FcγRIIIa–GST more specifically bound glycosylated immunoglobulin G1s (IgG1s) and was used to enrich nonfucosylated IgG1s from weaker binding species. This technique can be used when modifications of amino acids lead to changes in activity. © 2016 Elsevier Inc. Source

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