Direct Liquid Chromatography-mass Spectrometry Research Articles

A Systematic Workflow for Fragmentation Identification of Therapeutic Antibodies by Liquid Chromatography-Mass Spectrometry.

Fragmentation is a phenomenon ubiquitously observed during research and development of therapeutic antibodies. The clear identification of the cleavage site is vital for comprehending fragmentation mechanisms and optimizing processes. Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) is now widely used to detect and quantify fragments, while its peak identification is hindered by immature capillary electrophoresis-sodium dodecyl sulfate coupled with mass spectrometry techniques. In this study, we developed a systematic workflow for fragment characterization, which integrated direct identification, fragment enrichment, and fragmentation confirmation using multiple techniques, such as microscale peptide mapping (PM), PM of N-termini labeled sample, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in-gel extraction for molecular weight (MW) and PM measurements. By employing this innovative workflow, we successfully identified the cleavage sites of two therapeutic antibodies. In the first case, through direct liquid chromatography-mass spectrometry (LC-MS) characterization, the cleavages leading to the loss of biological function were identified on the linker of a bispecific antibody. In the second case involving a tetravalent antibody, direct LC-MS analysis failed. However, more sophisticated analysis nailed down the critical cleavage at the LC/HC: G105-R106 site in the CDR3 region of the antibody. Our systematic workflow provides a clear and accessible method for identifying cleavage sites with broad applicability across pharmaceutical laboratories.

Direct measurement of pregnanediol 3-glucuronide (PDG) in dried urine spots by liquid chromatography-mass spectrometry to detect ovulation

BackgroundNon-invasive self-testing using an objective chemical method to detect ovulation is valuable for women planning conception, practising contraception or undergoing infertility investigations or treatment. MethodsBased on luteal phase secretion of progesterone (P4) and excretion of its major metabolite, pregnanediol glucuronide (PDG), we developed a novel direct liquid chromatography-mass spectrometry (LCMS) method to measure PDG and other steroid glucuronides in urine and in dried urine spots (DUS) without deconjugation or derivatization. Urine PDG by LCMS and immunoassay (P3G) and P4 by immunoassay with and without adjustment for creatinine were evaluated in daily first void urine samples from 10 women through a single menstrual cycle in which ovulation was confirmed by serial transvaginal ultrasound. ResultsUrine PDG with and without creatinine adjustment was stable during the follicular phase with the expected striking rise in the luteal phase peaking at 5 days after ovulation. Using a single spot urine sample (100 μL) or a DUS (<20 μL urine) and an optimal threshold to distinguish pre- from post-ovulatory samples, in ROC analysis urine PDG adjusted for creatinine accurately identified ovulation in 92 % of samples was comparable with P3G immunoassay and superior to urine P4 with or without adjustment for creatinine. Extending the analysis to two or three consecutive daily samples reduced the false negative rate from 8% to 2.6 % for two and 1.9 % for three urine samples. ConclusionsThis method holds promise as a non-invasive self-test method for women to determine by an objective chemical method their ovulatory status.

LC-MS Compatible Antiadsorption Diluent for Peptide Analysis.

Analytical method development for peptides often proves challenging since these molecules can adsorb to the plastic or glass consumables used in the analysis. This adsorption causes considerable loss and unreliable results, especially in the lower concentration range. Therefore, a variety of antiadsorption strategies have previously been developed to cope with this adsorption, often however incompatible with direct liquid chromatography-mass spectrometry (LC-MS) analysis. Here, a novel antiadsorption diluent is introduced, based on controlled hydrolysis and precipitation of bovine serum albumin. This diluent considerably decreases the adsorption of certain peptides to glass. Moreover, it is LC-MS compatible and can also be used in combination with formic acid and/or acetonitrile addition.

Detection of dichlorvos adducts in a hepatocyte cell line.

The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying noncholinesterase targets. Here we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography-mass spectrometry (LC-MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues. The MS data have been deposited to the ProteomeXchange with identifier PXD001107.

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.

Blood cells RNA biomarkers as a first long‐term detection strategy for EPO abuse in horseracing

Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping control. In this context, gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been conducted on seven thoroughbreds treated with Eprex. This functional genomic method has been performed from total blood cells collected from each animal to assess the mRNA expression consecutive to rHuEPO injections. Sample pooling was chosen as a powerful, cost-effective, and rapid means of identifying the most common and specific changes in terms of gene expression profile and to eliminate individual variation. Consequently, three SAGE libraries were constructed, before, during, and after Eprex treatment. More than 71 440 mRNA signatures were observed and subjected to statistical analysis; 49 differentially expressed genes were identified and analyzed by qPCR. From the selected gene list, were defined as potential biomarkers in terms of their low inter-individual variation and capacity as strong markers of rHuEPO administration up to 60 days after the beginning of the doping period. In this paper, a new strategy is proposed to the horseracing industry to prevent rHuEPO abuse.