CE-MS for the analysis of intact proteins

Haselberg, Rob; Somsen, Govert W.

doi:10.1002/9783527693801.ch7

Cited by 14 publications

(15 citation statements)

References 141 publications

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“…surfactants) from the samples, or to concentrate the samples [12,13]. Intact proteins in biopharmaceutical products are commonly analyzed by spectrometry [14], electrophoresis [15], liquid chromatography [16,17], or mass spectrometry [14,[18][19][20]. More specifically, viral proteins are typically analyzed by SDS-PAGE [10,21,22], capillary gel electrophoresis (CGE) [23][24][25] or RP-HPLC [12,13,26].…”

Section: Introductionmentioning

confidence: 99%

Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography

Tricht

Raadt

Verwilligen

et al. 2018

Journal of Chromatography A

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a b s t r a c tA method for the quantitative determination of the protein composition of adenovirus-vector based vaccines was developed. The final method used RP-UPLC with UV absorbance detection, a C4 column (300 Å, 1.7 m, 2.1 × 150 mm), and a water-acetonitrile (ACN) gradient containing trifluoroacetic acid (TFA) as ion-pairing agent. The chromatographic resolution between the various adenovirus proteins was optimized by studying the effect of the TFA concentration and the column temperature, applying a full factorial design of experiments. A reproducible baseline separation of all relevant adenovirus proteins could be achieved within 17 min run time. Samples containing adenovirus particles could be directly injected into the UPLC system without sample pretreatment. The viruses reproducibly dissociate into proteins in the UPLC system upon contact with the mobile phase containing ACN. The new RP-UPLC method was successfully validated for protein profiling and relative quantification of proteins in vaccine products based on adenovirus vector types 26 and 35. The intermediate precision of the relative peak areas of all proteins was between 1% and 14% RSD, except for the peak assigned to protein V (26% RSD). The method proved to be stability indicating with respect to thermal and oxidation stress of the adenovirusvector based vaccine and was successfully implemented for the characterization of adenovirus-based products.

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Section: Introductionmentioning

confidence: 99%

Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography

Tricht

Raadt

Verwilligen

et al. 2018

Journal of Chromatography A

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“…[12][13][14] Mass spectrometry techniques are often coupled with chromatographic separation techniques producing methods that have very high analyte resolution which are often the best means of characterizing complex biological samples in detail. Again, these combined MS/chromatography technologies are widely adopted and are the subject of much scientific literature (e.g., HPLC-MS, 15,16 UHPLC-MS, 17,18 CE-MS 19,20 ). Chromatographic and MS-based techniques are well understood, very effective, and widely used; they do, however, have several significant drawbacks.…”

Section: Analytical Technologies In the Biopharmaceutical Industrymentioning

confidence: 99%

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

2017

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The production of active pharmaceutical ingredients (APIs) is currently undergoing its biggest transformation in a century. The changes are based on the rapid and dramatic introduction of protein-and macromolecule-based drugs (collectively known as biopharmaceuticals) and can be traced back to the huge investment in biomedical science (in particular in genomics and proteomics) that has been ongoing since the 1970s. Biopharmaceuticals (or biologics) are manufactured using biological-expression systems (such as mammalian, bacterial, insect cells, etc.) and have spawned a large (>E35 billion sales annually in Europe) and growing biopharmaceutical industry (BioPharma). The structural and chemical complexity of biologics, combined with the intricacy of cell-based manufacturing, imposes a huge analytical burden to correctly characterize and quantify both processes (upstream) and products (downstream). In small molecule manufacturing, advances in analytical and computational methods have been extensively exploited to generate process analytical technologies (PAT) that are now used for routine process control, leading to more efficient processes and safer medicines. In the analytical domain, biologic manufacturing is considerably behind and there is both a huge scope and need to produce relevant PAT tools with which to better control processes, and better characterize product macromolecules. Raman spectroscopy, a vibrational spectroscopy with a number of useful properties (nondestructive, non-contact, robustness) has significant potential advantages in BioPharma. Key among them are intrinsically high molecular specificity, the ability to measure in water, the requirement for minimal (or no) sample pre-treatment, the flexibility of sampling configurations, and suitability for automation. Here, we review and discuss a representative selection of the more important Raman applications in BioPharma (with particular emphasis on mammalian cell culture). The review shows that the properties of Raman have been successfully exploited to deliver unique and useful analytical solutions, particularly for online process monitoring. However, it also shows that its inherent susceptibility to fluorescence interference and the weakness of the Raman effect mean that it can never be a panacea. In particular, Raman-based methods are intrinsically limited by the chemical complexity and wide analyte-concentration-profiles of cell culture media/bioprocessing broths which limit their use for quantitative analysis. Nevertheless, with appropriate foreknowledge of these limitations and good experimental design, robust analytical methods can be produced. In addition, new technological developments such as time-resolved detectors, advanced lasers, and plasmonics offer potential of new Raman-based methods to resolve existing limitations and/or provide new analytical insights.

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“…For (poly)peptides, liquid chromatography (LC) is by far the most convenient technique for hyphenation with MS via an ESI source. Capillary electrophoresis (CE) has unique advantages over LC for the separation of peptides and proteins (Haselberg, de Jong, & Somsen, 2013;Robledo & Smyth, 2014) but is less common. We will therefore focus on LC as separation technique.…”

Section: Separation Of Complex Samples: Lc-ms/msmentioning

confidence: 99%

Bacterial Electron Transfer Chains Primed by Proteomics

Wessels¹,

Almeida²,

Kartal³

et al. 2016

Advances in Bacterial Electron Transport Systems and Their Regulation

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Electron transport phosphorylation is the central mechanism for most prokaryotic species to harvest energy released in the respiration of their substrates as ATP. Microorganisms have evolved incredible variations on this principle, most of these we perhaps do not know, considering that only a fraction of the microbial richness is known. Besides these variations, microbial species may show substantial versatility in using respiratory systems. In connection herewith, regulatory mechanisms control the expression of these respiratory enzyme systems and their assembly at the translational and posttranslational levels, to optimally accommodate changes in the supply of their energy substrates. Here, we present an overview of methods and techniques from the field of proteomics to explore bacterial electron transfer chains and their regulation at levels ranging from the whole organism down to the Ångstrom scales of protein structures. From the survey of the literature on this subject, it is concluded that proteomics, indeed, has substantially contributed to our comprehending of bacterial respiratory mechanisms, often in elegant combinations with genetic and biochemical approaches. However, we also note that advanced proteomics offers a wealth of opportunities, which have not been exploited at all, or at best underexploited in hypothesis-driving and hypothesis-driven research on bacterial bioenergetics. Examples obtained from the related area of mitochondrial oxidative phosphorylation research, where the application of advanced proteomics is more common, may illustrate these opportunities.

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CE-MS for the analysis of intact proteins

Cited by 14 publications

References 141 publications

Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography

Fast, selective and quantitative protein profiling of adenovirus-vector based vaccines by ultra-performance liquid chromatography

Applications of Raman Spectroscopy in Biopharmaceutical Manufacturing: A Short Review

Bacterial Electron Transfer Chains Primed by Proteomics

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