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ABSTRACT: The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose, and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper, a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic compact-disc (CD) platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified, and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme α1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and, simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel, and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.
Journal of the American Society for Mass Spectrometry 04/2013; · 4.00 Impact Factor
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ABSTRACT: Optimal glycosylation with respect to the efficacy, serum half-life time, and immunogenic properties is essential in the generation of therapeutic antibodies. The glycosylation pattern can be affected by several different parameters during the manufacture of antibodies and may change significantly over cultivation time. Fast and robust methods for determination of the glycosylation patterns of therapeutic antibodies are therefore needed. We have recently presented an efficient method for the determination of glycans on therapeutic antibodies using a microfluidic CD platform for sample preparation prior to matrix-assisted laser-desorption mass spectrometry analysis. In the present work, this method is applied to analyse the glycosylation patterns of three commercially available therapeutic antibodies and one intended for therapeutic use. Two of the antibodies produced in mouse myeloma cell line (SP2/0) and one produced in Chinese hamster ovary (CHO) cells exhibited similar glycosylation patterns but could still be readily differentiated from each other using multivariate statistical methods. The two antibodies with most similar glycosylation patterns were also studied in an assessment of the method's applicability for quality control of therapeutic antibodies. The method presented in this paper is highly automated and rapid. It can therefore efficiently generate data that helps to keep a production process within the desired design space or assess that an identical product is being produced after changes to the process.
Journal of pharmaceutical and biomedical analysis 05/2012; 70:47-52. · 2.45 Impact Factor
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ABSTRACT: Recombinant therapeutic antibodies have shown a great potential in the treatment of several severe medical conditions such as cancer and autoimmune diseases. Glycosylation plays a critical role in biological activity and immunogenic properties of these compounds. The analysis of glycan profiles is therefore necessary in many steps of the development and manufacturing process from early development to quality control of the final product. In this paper, a fast, parallel, and robust sample preparation platform for glycosylation profiling using a microfluidic compact disc (CD) is presented. A sequential process including selective capture of antibody from a crude cell supernatant using protein A beads, enzymatic release of glycans, purification with a graphitized carbon black column, and crystallisation for MALDI-TOF analysis were performed on the CD. Glycosylation profiles of an antibody intended for therapeutic use produced in two different cell lines were compared.
Analytical and Bioanalytical Chemistry 02/2011; 399(4):1601-11. · 3.78 Impact Factor
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ABSTRACT: A microfluidic structure is presented where selective capture of proteins in complex samples, followed by clean-up, enzymatic processing, and MALDI-MS sample preparation of peptides generated, can be performed. The structure uses an affinity column to capture the protein while all other components in the sample are disposed of. The protein of interest is then eluted from the affinity column and captured on a second column on which the enzymatic processing is performed. Salts and hydrophilic contaminants are then removed before the products from the enzymatic reaction are eluted together with a suitable MALDI matrix and the solvent evaporated in a designated MALDI target structure. All steps can be performed automatically in 54 parallel microstructures on a microfluidic compact disc. The process is demonstrated by the selective capture and tryptic digest of recombinant IgG molecules from samples containing other proteins: an excess of bovine serum albumin or spent cell culture media.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 10/2010; 878(28):2803-10. · 2.78 Impact Factor
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ABSTRACT: Pharmaceuticals are constantly dispersed into the environment and little is known of the effects on non-target organisms. This is an issue of growing concern. In this study, Baltic Sea blue mussels, Mytilus edulis trossulus, were exposed to diclofenac, ibuprofen and propranolol, three pharmaceuticals that are produced and sold in large quantities and have a widespread occurrence in aquatic environments. The mussels were exposed to pharmaceuticals in concentrations ranging from 1 to 10,000 microg l(-1). The pharmaceuticals were added both separately and in combination. Mussels exposed to high concentrations of pharmaceuticals showed a clear response compared to controls. Firstly, they had a significantly lower scope for growth, which indicates that the organisms had a smaller part of their energy available for normal metabolism, and secondly, they had lower byssus strength and lower abundance of byssus threads, resulting in reduced ability to attach to the underlying substrate. Mussels exposed to lower concentrations showed tendencies of the same results. The concentration of diclofenac and propranolol was quantified in the mussels using both liquid chromatography coupled to mass spectrometry (LC-MS). The measurements showed a significantly higher concentration in the organisms as compared to the water the mussels were exposed to; the uptake reached concentrations two orders of magnitudes higher than found in sewage treatment plant effluents. This study showed that common pharmaceuticals are taken up and negatively affect the physiology of a non-target species at levels of two to three orders of magnitudes higher than found in sewage treatment plant effluents.
Aquatic toxicology (Amsterdam, Netherlands) 08/2010; 99(2):223-31. · 3.12 Impact Factor
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ABSTRACT: A simple platform for combining solid phase extraction (SPE) and surface-assisted laser desorption ionization mass spectrometry (SALDI-MS) of extracted analytes, using disks prepared by embedding graphitized carbon black (GCB-4) particles in a network of polytetrafluoroethylene (PTFE), is presented. The system provides a convenient approach for rapid SALDI-MS screening of substances in aqueous samples, which can be followed by robust quantitative and/or structural analyses by liquid chromatography (LC)/MS/MS of positive samples. The extraction discs are easily transferred between gaskets where the sample extraction and desorption of selected samples is performed and the mass spectrometer. The SPE and SALDI properties of the new GCB-4 disc have been characterized for 15 pesticides with varying chemical properties, and the screening strategy has been applied to the analysis of pesticides in agricultural drainage water. Atrazine and atrazine-desethyl-2-hydroxy were detected in the sampled water by SALDI-MS screening and subsequently confirmed and quantified using LC/MS/MS.
Analytical Chemistry 12/2009; 82(1):290-6. · 5.86 Impact Factor
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ABSTRACT: The signal intensity of low-molecular-weight compounds analyzed using surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF-MS) was significantly enhanced when oxidized graphitized carbon black (GCB) particles were used as the desorption/ionization surface. The surface of oxidized GCB contains more carboxylic acid groups than non-oxidized GCB. Carboxylic acid groups enhance the efficiency of the ionization process and the desorption of more hydrophobic compounds. A common pharmaceutical compound, propranolol, was successfully extracted from Baltic Sea blue mussels and quantified using oxidized GCB as the SALDI surface, whereas deuterated propranolol was used as the internal standard. The calibration curve showed a wide linear dynamic range of response (0.1-20 microg/mL) and good reproducibility (RSD < 10%). It was not possible to detect propranolol in Baltic Sea blue mussels when non-oxidized GCB was used as the SALDI surface.
Journal of the American Society for Mass Spectrometry 02/2009; 20(6):1207-13. · 4.00 Impact Factor
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ABSTRACT: A parallel nanoliter microfluidic analysis system based on capillary action, centrifugal force, and hydrophobic barriers is described. The precision of 112 parallel volume definition operations is determined to 0.75% CV at 200 nL using the individual sample introduction structure. For 20 nL, the actual measurement error is the dominating factor, with a combined error of 1.9% CV. Individual dispensing as well as dispensing through a common distribution channel is described. The volume definition precision for the common distribution channel is 1.6% CV for 200 nL. Unlike the dominating forces in microliter-sized channel systems, we describe hysteresis effects as exerting a major influence, which needs to be considered in order to control the operation and design of discrete nanoliter fluidics. Hydrophobic patches at the corners of the rectangular channel control corner-enhanced wicking. Excellent flow control of 1 and 2 nL/s is achieved using a predefined spin program.
Analytical Chemistry 07/2007; 79(11):4022-30. · 5.86 Impact Factor
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Clinical Chemistry 11/2005; 51(10):1985-7. · 7.91 Impact Factor
