Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

Korecká, Lucie; Bı́lková, Zuzana; Holèapek, Michal; Královský, Josef; Beneš, Milan J.; Lenfeld, J; Minc, Nicolas; Cecal, Roxana; Viovy, Jean‐Louis; Przybylski, Michael

doi:10.1016/j.jchromb.2004.04.035

Cited by 37 publications

(25 citation statements)

References 19 publications

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“…The characterization of the post-translational modifications that occur on therapeutic proteins produced in mammalian cells has previously focused on the glycosylation profile because of their impact on efficacy (Hossler et al, 2009;Jefferis, 2009;Kawasaki et al, 2009), and to this end have usually involved digestion of the protein (Bailey et al, 2005;Bongers et al, 2000;Boyiadzis and Foon, 2008;Damen et al, 2009;Fernandez et al, 2001;Hooker et al, 1995;Korecka et al, 2004;Rousseaux et al, 1989;Schahs et al, 2007;Stigter et al, 2007) or cleavage of the glycan moiety (Fernandez et al, 2001;Jefferis, 2005;Kamoda et al, 2004;Majid et al, 2007;Mechref et al, 2005;Qian et al, 2007;Robinson et al, 1994;Yu et al, 2005) from the protein backbone or a combination of the two. To enable the simultaneous and rapid analysis of different PTMs, and to allow the investigation as to how processing conditions can affect this, LC-MS analysis of intact monoclonal antibodies was used in conjunction with USD bioprocessing mimics.…”

Section: Resultsmentioning

confidence: 99%

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale‐down study of the effect of harvesting by centrifugation on the post‐translational modification profile

Reid

Tait

Baldascini

et al. 2010

Biotech & Bioengineering

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With the trend towards the generation and production of increasing numbers of complex biopharmaceutical (protein based) products, there is an increased need and requirement to characterize both the product and production process in terms of robustness and reproducibility. This is of particular importance for products from mammalian cell culture which have large molecular structures and more often than not complex post-translational modifications (PTMs) that can impact the efficacy, stability and ultimately the safety of the final product. It is therefore vital to understand how the operating conditions of a bioprocess affect the distribution and make up of these PTMs to ensure a consistent quality and activity in the final product. Here we have characterized a typical bioprocess and determined (a) how the time of harvest from a mammalian cell culture and, (b) through the use of an ultra scale-down mimic how the nature of the primary recovery stages, affect the distribution and make up of the PTMs observed on a recombinant IgG(4) monoclonal antibody. In particular we describe the use of rapid whole antibody analysis by mass spectrometry to analyze simultaneously the changes that occur to the cleavage of heavy chain C-terminal lysine residues and the glycosylation pattern, as well as the presence of HL dimers. The time of harvest was found to have a large impact upon the range of glycosylation patterns observed, but not upon C-terminal lysine cleavage. The culture age had a profound impact on the ratio of different glycan moieties found on antibody molecules. The proportion of short glycans increased (e.g., (G0F)(2) 20-35%), with an associated decrease in the proportion of long glycans with culture age (e.g., (G2F)(2) 7-4%, and G1F/G2F from 15.2% to 7.8%). Ultra scale-down mimics showed that subsequent processing of these cultures did not change the post-translational modifications investigated, but did increase the proportion of half antibodies present in the process stream. The combination of ultra scale-down methodology and whole antibody analysis by mass spectrometry has demonstrated that the effects of processing on the detailed molecular structure of a monoclonal antibody can be rapidly determined early in the development process. In this study we have demonstrated this analysis to be applicable to critical process design decisions (e.g., time of harvest) in terms of achieving a desired molecular structure, but this approach could also be applied as a selection criterion as to the suitability of a platform process for the preparation of a new drug candidate. Also the methodology provides means for bioprocess engineers to predict at the discovery phase how a bioprocess will impact upon the quality of the final product.

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Section: Resultsmentioning

confidence: 99%

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale‐down study of the effect of harvesting by centrifugation on the post‐translational modification profile

Reid

Tait

Baldascini

et al. 2010

Biotech & Bioengineering

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“…The reaction conditions for protein fragmentation by trypsin reactors were optimized (Table 3), following an approach similar to this described by Korecká et al [51,52]. Briefly, the type of buffer, ions, pH, temperature, E:S ratio, digestion time, surfactant, and K M , as well as the composition of the storage solution were varied.…”

Section: Characterization Of Proteolytic Microreactorsmentioning

confidence: 99%

Functionalized magnetic micro‐ and nanoparticles: Optimization and application to μ‐chip tryptic digestion

et al. 2006

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The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.

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“…Toward this end, a large number of papers have illustrated integration of mass spectrometry detection directly from the end of the microdevices [3][4][5][6][7]. To exploit the potential capabilities of sample preparation microchips for protein analysis using MS, efforts have been directed toward demonstrating on-chip digestion, and separations that could be integrated with MS detection on a single device [8][9][10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Protein digestion and phosphopeptide enrichment on a glass microchip

Yue

Roper

Balchunas

et al. 2006

Analytica Chimica Acta

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This work describes an integrated glass microdevice for proteomics, which directly couples proteolysis with affinity selection. Initial results with standard phosphopeptide fragments from β-casein in peptide mixtures showed selective capture of the phosphorylated fragments using immobilized metal affinity chromatography (IMAC) beads packed into a microchannel. Complete selectivity was seen with angiotensin, with capture of only the phosphorylated form. On-chip proteolysis, using immobilized trypsin beads packed into a separate channel, was directly coupled to the phosphopeptide capture and the integrated devices evaluated using β-casein. Captured and eluted fragments were analyzed using both capillary electrophoresis (CE) and capillary liquid chromatography/mass spectrometry (cLC/MS). The results show selective capture of only phosphopeptide fragments, but incomplete digestion of the protein was apparent from multiple peaks in the CE separations. The MS analysis indicated a capture bias on the IMAC column for the tetraphosphorylated peptide fragment over the monophosphorylated fragment. Application to digestion and capture of a serum fraction showed capture of material; however, non-specific binding was evident. Additional work will be required to fully optimize this system, but this work represents a novel sample preparation method, incorporating protein digestion on-line with affinity capture for proteomic applications.

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Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

Cited by 37 publications

References 19 publications

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale‐down study of the effect of harvesting by centrifugation on the post‐translational modification profile

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale‐down study of the effect of harvesting by centrifugation on the post‐translational modification profile

Functionalized magnetic micro‐ and nanoparticles: Optimization and application to μ‐chip tryptic digestion

Protein digestion and phosphopeptide enrichment on a glass microchip

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