Quantitative Bottom Up Analysis of Infliximab in Serum Using Protein A Purification and Integrated μLC-Electrospray Chip Ionkey MS/MS Technology

Kleinnijenhuis, Anne J.; Ingola, Martha; Toersche, Jan; Holthoon, Frédérique L. van; Dongen, W. D. van

doi:10.4155/bio-2015-0015

Cited by 34 publications

(12 citation statements)

References 30 publications

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“…Finally, the experimental ratios were assessed. With matching settings between non‐labeled analyte peptides and their SIL equivalents, the theoretical signal ratio between analyte and SIL IS for the 80 μg/mL calibration sample was calculated to be 9.2 according to the formula for absolute recovery 29 . This theoretical ratio only applies when the monitored product ions contain all the labels or none of the labels, see also the earlier discussion about the experimental analyte/SIL IS ratios for transitions with products ions that are partially labeled.…”

Section: Resultsmentioning

confidence: 99%

Reliable and generic liquid chromatography/mass spectrometry quantification of short peptides using a stable‐isotope‐labeled labeling agent

Kleinnijenhuis¹,

Vergauwen²,

Holthoon³

et al. 2020

Rapid Comm Mass Spectrometry

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Rationale It is important to investigate the behavior of protein hydrolysate components in both in vitro and in vivo studies, to support the elucidation of their biological functions. As protein hydrolysates and biological matrices are highly complex mixtures, it is essential to apply fully reliable and flexible analytical approaches. Methods A novel and generic Liquid Chromatography/Mass Spectrometry methodology was developed to analyze short peptides. A stable‐isotope‐labeled labeling agent 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (13C3) was synthesized and used to prepare internal standards from non‐labeled analyte peptides. The amino acid and peptides p, pG, Pp, GPp and PpG (where p stands for hydroxyproline) were used for proof of principle. Results The method showed acceptable performance in solvent, in simulated gastrointestinal fluid and in serum. The (linear) dynamic range expanded to over four orders of magnitude, which is very useful when multiple analytes are analyzed in a biological matrix, due to the large differences in concentrations observed for endogenous and protein hydrolysate components. The method provides absolute‐quantitative results and is fully accountable on the single‐sample and single‐component level. Conclusions The methodology can be applied to reliably quantify protein hydrolysate nutraceutical components at various stages during their in vivo processing. Internal standards can also be synthesized for other short peptides whenever they are expected to have biological relevance and require quantification. Overall this provides an excellent analytical tool to support the elucidation of the biological functions of protein hydrolysate components.

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Section: Resultsmentioning

confidence: 99%

Reliable and generic liquid chromatography/mass spectrometry quantification of short peptides using a stable‐isotope‐labeled labeling agent

Kleinnijenhuis¹,

Vergauwen²,

Holthoon³

et al. 2020

Rapid Comm Mass Spectrometry

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“…The technique is a greener alternative too, as solvent waste is also much reduced, with one application reported as better than a 20-fold improvement against conventional flow [21]. There have been several recent reported applications of microflow [18,[20][21][22][23][24] and although not there in abundance as yet, usage is fully expected to increase as the technology becomes better accepted and its benefits fully realized across the industry.…”

Section: Scaling Down Lc-ms With Benefits Scaling Upmentioning

confidence: 99%

Instrumental and Technical Evolution Over the Past Decade in Bioanalysis

MacNeill

2019

Bioanalysis

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In the domain of LC-MS, it has been an exciting and memorable 10 years of progress in quantitative bioanalytical techniques since the inception of Bioanalysis. Several innovative technologies have seen commercial manifestations, of wonderful value to the pharmaceutical and biotech industries. As such, we have seen drive and success in improving sensitivity of detection, a perennial pursuit. We have seen the creation of more avenues to obtain selectivity, the most pivotal property of a truly robust quantitative methodology. We have also significantly brought forward and established new ways to reliably use notably less matrix in fully quantitative applications. In this article, I have detailed my pick of the most compelling developments in the past decade. Matrix quantity basis reduction with innovationOn the note of reduction of analytical matrix volumes, the launch of this journal occurred just as a formidable wave of interest was gaining strength in dried blood spotting (DBS). There was a pronounced focus on a quantitative LC-MS end point as shown in the first of such reports [1,2] and for which there have been some informative and insightful reviews over the years [3,4]. The technique of DBS, an example of microsampling, involves minimally invasive drawings typically in the order of 10 μl, resulting in several practical and analytical advantages. The dramatic reduction in the blood volume required for a given toxicokinetic time point gives a concomitant reduction in animal number requirements, particularly important for the likes of rodents. Indeed, full toxicokinetic (TK) profiles can be acquired from a single animal, bestowing far greater confidence in concentration data and the derived statistical data and associated decisions from such. The card-based sample collection also has profound cost-saving implications, from the perspective of ease of storage and transport [5]. In addition, there are much reduced analyte stability concerns in the dried sample [6,7]. Supporting the reliability of the technique, there are reports of concentration data obtained from DBS correlating well with those data obtained from analysis of liquid samples [8]. As it stands, DBS has cemented itself as an option in the regulated laboratory, despite reliability-related question marks over aspects such as the impact of differing hematocrit [9] and uncertainty over best means of internal standard addition [10]. In any case, DBS has not just preclinical [11] but naturally also clinical application [2,12], the amount of blood required being no more than that obtained from a finger prick, and in this manner there are positive implications for the likes of pediatric studies.In the same microsampling vein, the acquisition and storage of liquid samples in this order of volume also gained great prominence on the heels of DBS, and with the same 3R (reduction, replacement and refinement) benefits for research involving animals. Liquid microsampling has overwhelmingly involved capillaries of 1-25 μl in the drawing and storage of blood samples [...

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“…In recent years, the use of LC‐–MS/MS to quantify therapeutic proteins using a surrogate peptide after proteolytic digestion is growing in popularity quickly as a mainstream bioanalytical technique. Its advantages over LBAs are the capability to detect and quantify peptides in specific regions of the proteins for specificity, ability to resist interference from specific matrix components such as antidrug antibodies, feasibility of multiplex analysis, wider linear range, and rapid method development (particularly saving time in preparing antigens) (Kleinnijenhuis, Ingola, Toersche, van Holthoon, & van Dongen, 2016).…”

Section: Introductionmentioning

confidence: 99%

Method development and validation of LC–MS/MS‐based assay for the simultaneous quantitation of trastuzumab and pertuzumab in cynomolgus monkey serum and its application in pharmacokinetic study

Gui

Dong

et al. 2020

Biomedical Chromatography

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We present a simple and robust LC–MS/MS assay for the simultaneous quantitation of an antibody cocktail of trastuzumab and pertuzumab in monkey serum. The LC–MS/MS method saved costs, decreased the analysis time, and reduced quantitative times relative to the traditional ligand‐binding assays. The serum samples were digested with trypsin at 50°C for 60 min after methanol precipitation, ammonium bicarbonate denaturation, dithiothreitol reduction, and iodoacetamide alkylation. The tryptic peptides were chromatographically separated using a C18 column (2.1 × 50 mm, 2.6 μm) with mobile phases of 0.1% formic acid in water and acetonitrile. The other monoclonal antibody, infliximab, was used as internal standards to minimize the variability during sample processing and detection. A unique peptide for each monoclonal antibody was simultaneously quantified using LC–MS/MS in the multiple reaction monitoring mode. Calibration curves were linear from 2.0 to 400 μg/mL. The intra‐ and inter‐assay precision (%CV) was within 8.9 and 7.4% (except 10.4 and 15.1% for lower limit of quantitation), respectively, and the accuracy (%Dev) was within ±13.1%. The other validation parameters were evaluated, and all results met the acceptance criteria of the international guiding principles. Finally, the method was successfully applied to a pharmacokinetics study after a single‐dose intravenous drip administration to cynomolgus monkeys.

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Quantitative Bottom Up Analysis of Infliximab in Serum Using Protein A Purification and Integrated μLC-Electrospray Chip Ionkey MS/MS Technology

Cited by 34 publications

References 30 publications

Reliable and generic liquid chromatography/mass spectrometry quantification of short peptides using a stable‐isotope‐labeled labeling agent

Reliable and generic liquid chromatography/mass spectrometry quantification of short peptides using a stable‐isotope‐labeled labeling agent

Instrumental and Technical Evolution Over the Past Decade in Bioanalysis

Method development and validation of LC–MS/MS‐based assay for the simultaneous quantitation of trastuzumab and pertuzumab in cynomolgus monkey serum and its application in pharmacokinetic study

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