Katja Walpurgis’s research while affiliated with Deutsche Sporthochschule Köln and other places

What is this page?


This page lists works of an author who doesn't have a ResearchGate profile or hasn't added the works to their profile yet. It is automatically generated from public (personal) data to further our legitimate goal of comprehensive and accurate scientific recordkeeping. If you are this author and want this page removed, please let us know.

Publications (54)


Testing for the erythropoiesis‐stimulating agent Sotatercept/ACE‐011 (ActRIIA‐Fc) in serum by means of Western blotting and LC‐HRMS
  • Article

November 2016

·

60 Reads

·

15 Citations

Drug Testing and Analysis

Katja Walpurgis

·

Andreas Thomas

·

·

[...]

·

Mario Thevis

Testing for the erythropoiesis-stimulating agent Sotatercept/ACE-011 (ActRIIA-Fc) in serum by means of Western blotting and LC-HRMS: Testing for Sotatercept/ACE-011 in serum by Western blotting and LC-HRMS

September 2016

·

80 Reads

·

27 Citations

Drug Testing and Analysis

Sotatercept (formerly ACE-011) is a glycosylated, dimeric fusion protein composed of the extracellular domain of the human activin receptor type IIA (ActRIIA) and the Fc region of human IgG1. The protein-based drug candidate acts as ligand trap which competitively binds to activin A and other members of the transforming growth factor beta superfamily, thus blocking signalling through ActRIIA. Since the inhibition of activin A was found to significantly increase bone formation and quality, Sotatercept was originally developed for the treatment of diseases involving bone loss. But as the protein therapeutic also stimulates erythropoiesis by a mechanism independent from the EPO receptor, it has been evaluated for the treatment of anaemia in rare blood diseases such as beta thalassemia. Due to its positive effects on erythropoiesis and bone formation, Sotatercept may also be misused as performance-enhancing agent in sports. Within this study, two complementary detection assays for Sotatercept and related ActRIIA-Fc fusion proteins in serum samples were developed. While the first assay combines affinity purification and Western blotting to generically detect ActRIIA-Fc fusion proteins irrespective of their amino acid sequence, the LC-HRMS method is highly specific for proteolytic peptides originating from the receptor and Fc domain of Sotatercept. Both approaches can readily be modified to include other pharmaceutical proteins such as therapeutic antibodies, and serve as proof-of-concept for the capability of the approach to detect TGF-β inhibitors and Fc fusion proteins in doping control serum samples.


Expanded test method for peptides >2 kDa employing immunoaffinity purification and LC-HRMS/MS

September 2015

·

135 Reads

·

36 Citations

Drug Testing and Analysis

Bioactive peptides with an approximate molecular mass of 2-12 kDa are of considerable relevance in sports drug testing. Such peptides have been used to manipulate several potential performance-enhancing processes in the athlete's body and include for example growth hormone releasing hormones (sermorelin, CJC-1293, CJC-1295, tesamorelin), synthetic/animal insulins (lispro, aspart, glulisine, glargine, detemir, degludec, bovine and porcine insulin), synthetic ACTH (synacthen), synthetic IGF-I (longR(3) -IGF-I) and mechano growth factors (human MGF, modified human MGF, 'full-length' MGF). A combined initial test method using one analytical procedure is a desirable tool in doping controls and related disciplines as requests for higher sample throughput with utmost comprehensiveness preferably at reduced costs are constantly issued. An approach modified from an earlier assay proved fit-for-purpose employing pre-concentration of all target analytes by means of ultrafiltration, immunoaffinity purification with coated paramagnetic beads, nano-ultra high performance liquid chromatography (UHPLC) separation, and subsequent detection by means of high resolution tandem mass spectrometry. The method was shown to be applicable to blood and urine samples, which represent the most common doping control specimens. The method was validated considering the parameters specificity, recovery (11-69%), linearity, imprecision (<25%), limit of detection (5-100 pg in urine, 0.1-2 ng in plasma), and ion suppression. The analysis of administration study samples for insulin degludec, detemir, aspart, and synacthen provided the essential data for the proof-of-principle of the method. Copyright © 2015 John Wiley & Sons, Ltd.


Myostatin inhibitors in sports drug testing: Detection of myostatin-neutralizing antibodies in plasma/serum by affinity purification and Western blotting

July 2015

·

135 Reads

·

10 Citations

PROTEOMICS - CLINICAL APPLICATIONS

Myostatin is a key regulator of skeletal muscle growth and inhibition of its signaling pathway results in an increased muscle mass and function. The aim of this study was to develop a qualitative detection assay for myostatin-neutralizing antibodies for doping control purposes by using immunological approaches. To detect different types of myostatin-neutralizing antibodies irrespective of their amino acid sequence, an immunological assay specific for antibodies directed against myostatin and having a human Fc domain was established. Affinity purification and western blotting strategies were combined to allow extracting and identifying relevant analytes from 200 μL of plasma/serum in a non-targeted approach. The assay was characterized regarding specificity, linearity, precision, robustness, and recovery. The assay was found to be highly specific, robust, and linear from 0.1 to 1 μg/mL. The precision was successfully specified at three different concentrations and the recovery of the affinity purification was 58%. Within this study, an immunological detection assay for myostatin-neutralizing antibodies present in plasma/serum specimens was developed and successfully characterized. The presented approach can easily be modified to include other therapeutic antibodies and serves as proof-of-concept for the detection of antibody-based myostatin inhibitors in doping control samples. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.


Isolation, Enrichment, and Analysis of Erythropoietins in Anti-Doping Analysis by Receptor-Coated Magnetic Beads and Liquid Chromatography–Mass Spectrometry

November 2014

·

71 Reads

·

16 Citations

Analytical Chemistry

Human erythropoietin (hEPO) is an erythropoiesis stimulating hormone frequently employed in anti-anemia therapy. Its capability to increase the amount of red blood cells however makes hEPO and its derivatives also attractive to dishonest athletes aiming at an artificial and illicit enhancement of their endurance performance. A major objective of the international anti-doping fight is the elimination of drug misuse and prevention of severe adverse effects caused by non-therapeutic administrations of highly potent drugs. The emergence of novel and innovative erythropoietin-mimetic agents (EMAs) has been continuously growing in the last years, and the option of using dedicated monoclonal antibodies (MAb) for analytical and sample preparation approaches is gradually reaching limits. In the present study the common ability and property of all EMAs - to bind on the human erythropoietin receptor (hEPOR) - is therefore exploited. An alternative methodology to isolate and analyze EMAs, in particular endogenous EPO and the recombinant forms EPOzeta, Darbepoetin alfa and C.E.R.A., from human urine is described, employing conventional ultrafiltration for preconcentration of the target analytes followed by EMA-specific isolation via hEPOR-bound magnetic beads. Analytical data were generated by means of gel-based electrophoretic analysis and nanoliquid chromatography / high resolution/high accuracy tandem mass spectrometry. Limits of detection enabled by the established sample preparation protocols were approximately 20 pg/mL for EPOzeta, 30 pg/mL for Darbepoetin alfa and 80 pg/mL for C.E.R.A.


Detection of an unknown fusion protein in confiscated black market products

November 2014

·

34 Reads

·

14 Citations

Drug Testing and Analysis

Even without clinical approval, many performance‐enhancing drugs are available on the black market and can therefore be easily obtained by cheating athletes. The misuse of these preparations can be associated with unforeseeable health risks – either due to a poor quality of the drugs or as a result of an insufficient clinical assessment. Moreover, confiscated black market products have frequently been shown to contain ingredients other than those declared on the label as well as additional by‐products or compounds with a modified molecular structure. This communication describes the identification of an unknown fusion protein observed in several unlabelled black market products obtained from independent sources. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis of the confiscated preparations indicated the presence of an 18‐kDa fusion protein consisting of the bacterial redox protein thioredoxin‐1 (Trx, 12 kDa) and a 6‐kDa peptide of unassigned composition. Trx has no relevance as performance enhancing agent but is routinely used as solubility tag for recombinant protein production. Further evaluation of the acquired MS/MS data revealed both an additional His tag and a thrombin cleavage site between the tags and the presumed bioactive peptide. However, thrombin cleavage of the fusion protein and LC‐MS/MS analysis of the resulting peptide fragment finally suggested that the unknown protein is only the product of an empty expression vector without the DNA insert of interest. These findings are a further alarming example for the high level of risk that athletes take when misusing drugs obtained from the black market. Copyright © 2014 John Wiley & Sons, Ltd.


Fig. 1. Dilution linearity and correlation analysis with LC-MS/MS measurements. (A) The dilution linearity of the assay with three different native rabbit serum samples in relation to recombinant rabbit IGF-I (r-rbIGF-I) and recombinant human (r-hIGF-I) standard curves. (B) The correlation of rabbit IGF-I measurements obtained using either the immunoassay or LC-MS/MS by using Passing–Bablock regression. Rabbit IGF-I concentrations were on average approximately twofold higher when measured by using LC-MS/MS (Spearman's ρ, 0.813; P<0.0001; Passing–Bablock: slope 0.684 and intercept −78.3). The solid line shows the regression fit; the dotted lines show the 95% confidence limits of the regression fit.  
Fig. 2. Serum IGF-I and IGFBPs in rabbits. (A) Serum IGF-I concentrations in male (blue) and female (red) rabbits from 13-week-old NZW and CB rabbits (males, n=7; females, n=8 for each strain). Data were analyzed by using non-parametric Mann–Whitney-U tests, *significant differences between male and female or between New Zealand White (NZW) and Chinchilla Bastard (CB) rabbits. Data are presented as box plots, center lines represent the median, boxes represent the middle 50% of data (interquartile range) and whiskers the 2.5-97.5 percentiles (*P<0.05, **P<0.01). (B) Analysis of serum IGFBPs by using quantitative western ligand blotting in male (blue) and female (red) rabbits from 13- week-old NZW and CB rabbits (males, n=7; females, n=8 for each strain). The analyses revealed that, in rabbits, the IGFBP-2 concentrations are quantitatively most abundant among the serum IGFBPs measured. With respect to sex and genetic background, IGFBP-2 concentrations were significantly (P<0.05) higher in 13- week-old NZW rabbits when compared with age-matched male CB rabbits. IGFBP-3 and IGFBP-4 were not significantly affected by sex or genetic background. Data are presented as means±s.e.m. (*P<0.05).  
Fig. 3. Serum IGF-I and IGFBP-2 in 1-to 6-month-old rabbits. (A) Serum IGFBP-2 concentrations (being the most abundant circulating IGFBP in rabbits) and (B) serum IGF-I concentrations in 1-, 2-, 3-, 4-and 6-month-old male (blue dots) and female (red dots) NZW rabbits (n=10 per sex and age group). Each dot represents one serum sample; the black lines indicate means±s.e.m. for each sex and age group, respectively (means with the s.e.m. bars 'up', females; means with the s.e.m. bars 'down', males). For illustrative purposes, the statistical comparison of age groups by using ANOVA is shown in the respective section of the results only.  
Fig. 4. Effect of treatment with recombinant GH or a GHA on serum IGF-I and IGFBP-2. (A) Concentrations of serum IGF-I and (B) circulating concentrations of the growth hormone antagonist Pegvisomant (GHA) in GHA-treated rabbits. The green arrows indicate the injection time points (1 mg/kg of body weight). Controls received equal volumes of PBS. Serum samples were collected before injection at baseline (0 h) and 24, 48, 72, 96, 120, 144 and 168 h after the first GHA injection. Concentrations of (C) serum IGF-I and (D) circulating concentrations of recombinant human growth hormone (rhGH) in rabbits that had been treated with recombinant human GH. The green arrows indicate the injection time points (1 mg/kg of body weight). Controls received equal volumes of PBS. Serum samples were collected before injection at baseline (0 h) and 4, 8, 24, 48, 56 and 72 h after the first injection of recombinant human GH. (E) Concentrations of circulating IGFBP-2 in rabbits that had been treated with recombinant human GH (red line), GHA (blue line) or PBS (black line). Data were analysed by using two-way ANOVA with subsequent Bonferroni correction for multiple-comparison post-hoc tests. Data are presented as means±s.e.m. ( # P=0.054, *P<0.05, **P<0.01).  
Fig. 5. Changes in the body weight of rabbits before the start of treatment with recombinant human GH or a growth hormone antagonist (GHA) and after termination of the treatment protocols. Data were analysed by using ANOVA with subsequent Bonferroni correction for multiple-comparison post-hoc tests. Data are presented as means±s.e.m. (*P<0.05, **P<0.01).  
Validation of serum IGF-I as a biomarker to monitor the bioactivity of exogenous growth hormone agonists and antagonists in rabbits
  • Article
  • Full-text available

November 2014

·

620 Reads

·

15 Citations

Disease Models and Mechanisms

The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, the most sensitive pharmacodynamic marker for the action of GH in humans, shows no response to treatment with recombinant human GH and there is little evidence for the effects of GHAs, except when administered at very high doses or when overexpressed. As an alternative, more suitable model, we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for the analysis of rabbit serum and tested precision, sensitivity, linearity and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with recombinant human GH or the GHA Pegvisomant. For a subset of samples, we used LC-MS/MS to measure IGF-I, and quantitative western ligand blot to analyze IGF-binding proteins (IGFBPs). Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, our results show that the sensitivity, precision (1.7–3.3% coefficient of variation) and linearity (90.4–105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I concentration in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple injections of recombinant human GH (IGF-I: 286±22 versus 434±26 ng/ml; P<0.01) and were highly correlated (P<0.0001). Treatment with the GHA lowered IGF-I levels from the fourth injection onwards (P<0.01). In summary, we demonstrated that the IDS-iSYS IGF-I immunoassay can be used in rabbits. Similar to rodents, rabbits display variations in IGF-I depending on sex, age and genetic background. Unlike in rodents, the IGF-I response to treatment with recombinant human GH or a GHA closely mimics the pharmacodynamics seen in humans, suggesting that rabbits are a suitable new model to test human GH agonists and antagonists.

Download


Validation of serum IGF-I as a biomarker to monitor exogenous growth hormone agonist and antagonist bioactivity in rabbits

September 2014

·

356 Reads

·

15 Citations

Disease Models and Mechanisms

The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, the most sensitive pharmacodynamic marker for the action of GH in humans, shows no response to treatment with recombinant human GH and there is little evidence for the effects of GHAs, except when administered at very high doses or when overexpressed. As an alternative, more suitable model, we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for the analysis of rabbit serum and tested precision, sensitivity, linearity and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with recombinant human GH or the GHA Pegvisomant. For a subset of samples, we used LC-MS/MS to measure IGF-I, and quantitative western ligand blot to analyze IGF-binding proteins (IGFBPs). Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, our results show that the sensitivity, precision (1.7–3.3% coefficient of variation) and linearity (90.4–105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I concentration in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple injections of recombinant human GH (IGF-I: 286±22 versus 434±26 ng/ml; P


Identification of black market products and potential doping agents in Germany 2010-2013

August 2014

·

298 Reads

·

97 Citations

European Journal of Clinical Pharmacology

Purpose: The desire to increase the athletic performance, to 'optimize' an individual's appearance, and to complement but also to arguably substitute exercise by means of drugs and drug candidates has generated a considerable (illicit) market for compounds such as anabolic-androgenic steroids, stimulants, growth promoting peptide hormones, and so on. Genuinely developed for therapeutic use, their abuse/misuse generates enormous health risks, which has necessitated comprehensive controls of compound trafficking by customs and anti-doping authorities. Methods: From 2012 to 2013, the Bureau of Customs Investigation confiscated products containing anabolic-androgenic steroids (AAS; 259 kg), stimulants (13 kg), selective estrogen receptor modulators (SERMs; 24 kg), and human growth hormone (hGH; 3500 ampules). In cooperation with the Bureau and under the umbrella of the European Monitoring Center for Emerging Doping Agents (EuMoCEDA), the Cologne Anti-Doping Laboratory analyzed an additional 337 (black market) products between 2010 and 2013, allowing to monitor developments in drug use and, hence, the anticipation of new challenges in sports drug testing. Main tools utilized in characterizing confiscated materials were liquid chromatography-high resolution mass spectrometry (LC-HRMS), gas chromatography-high resolution mass spectrometry (GC-HRMS), and polyacrylamide gel electrophoresis (PAGE) with subsequent bottom-up identification of peptidic compounds using nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS). Results: Among the 337 substances analyzed in the doping control laboratory in Cologne, 67 active ingredients were found, 49 of which being categorized as doping agents by the World Anti-Doping Agency (WADA). A total of 83.7 % accounted for steroidal substances (predominantly testosterone, trenbolone, and nandrolone and corresponding esters), 12.8 % accounted for peptide hormones and growth factors (predominantly hGH and growth hormone releasing peptides (GHRPs)), 3.2 % of the products contained hormones and metabolic modulators, and 0.3 % accounted for diuretic agents. Outstanding findings were the detection of the selective androgen receptor modulator (SARM) LGD-4033, the thymic hormone thymosin β4, and a fusion protein of unknown biological activity. Conclusions: Trafficking of considerable amounts of arguably performance and/or body-enhancing compounds has been observed during the past 4 years, the majority of which is categorized as relevant to sports drug testing. Several substances are of fake/non-approved nature and represent enormous health risks to the 'customer'.


Citations (44)


... Therefore, automating most processes in doping testing is desirable as well. There are previous studies on the application of DBSs in doping testing, such as the targeting of steroids and other compounds [17][18][19][20], genetic polymorphisms [9,21,22], plasmids via the spike-in test [23], hEPO proteins with mutations [24], and mRNAs [25]. Although these previous studies described the affinity and future promise of DBSs in doping testing, there was only one report of an automated testing process [18]. ...

Reference:

Detection of Gene Doping Using Dried Blood Spots from a Mouse Model with rAAV9 Vector-Mediated Human Erythropoietin Expression as a Pilot Study
Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing
  • Citing Article
  • November 2023

Drug Testing and Analysis

... Psychological interventions such as mental skills training and biofeedback enhance focus and stress management (Lindsay et al., 2023). Recovery technologies like cryotherapy (Poignard et al., 2023) and Normatec systems (Gras, 2023) accelerate recovery, and genetic research explores the potential of gene editing and epigenetics in sports performance (Naumann et al., 2023). Data analytics and machine learning aid in performance analysis and injury prevention, while VR (Virtual Reality) (Richlan et al., 2023b) and AR (Augmented Reality) (Cossich et al., 2023) technologies create immersive training environments. ...

Analysis of Potential Gene Doping Preparations for Transgenic DNA in the Context of Sports Drug Testing Programs

International Journal of Molecular Sciences

... Anti-doping authorities use a variety of analytical techniques, including urine and blood tests, to detect insulin abuse in athletes. These methods aim to measure insulin levels and identify the presence of insulin analogs [7]. ...

Insulin-mimetic peptides in sports drug testing
  • Citing Article
  • September 2023

Drug Testing and Analysis

... Endogenous metabolites such as organic acids, hormones, and metabolites from dietary compounds, drugs or pollutants can be detected in DBS samples [10]. Several applications of metabolomics and lipidomics have been developed and validated for the analysis of biofluids collected in this way [11][12][13]. ...

DropWise: current role and future perspectives of dried blood spots (DBS), blood microsampling, and their analysis in sports drug testing
  • Citing Article
  • August 2022

Critical Reviews in Clinical Laboratory Sciences

... To assess the plausibility of AAFs caused by intimate contact in general, information on the approximate concentration ranges of substances commonly abused in sports such as anabolic agents in sf would be helpful [13,14]. In this study, two different substances from the class of anabolic agents are investigated as to their appearance and concentrations in sf, with LGD-4033 representing a selective androgen receptor modulator (SARM) recently discussed in the context of an AAF related to intimate contact, and stanozolol (Stan) as a typical representative of anabolic steroids [5,15]. ...

Androgens, sports, and detection strategies for anabolic drug use
  • Citing Article
  • December 2021

Best Practice & Research: Clinical Endocrinology & Metabolism

... International sports drug testing procedures should take into account the ability of some drugs to improve muscle size and function as well as mitochondrial biogenesis since the abuse of these compounds calls for caution and regulation (51) . This includes the following: to the target analyte, other potential metabolites and degradation products are identified using this technique on the concentrated supernatant. ...

Peptidic drugs and drug candidates in sports drug testing: agents affecting mitochondrial biogenesis or preventing activin receptor II activation
  • Citing Article
  • December 2019

Current Opinion in Endocrine and Metabolic Research

... SCUBE2 [24], MEGF8 [25]) and TGF-β inhibitory factors (e.g. FST [26] and THSD4 [27]) were significantly upregulated by exposure to rapamycin alone (Fig. 5D). Moreover, the pro-survival factor GAS6 [28] was significantly overexpressed at the gene (Fig. 5E) and protein (Fig. 5F) level in AD-MSCs following rapamycin addition independently of IFN-γ, thus revealing its therapeutic interest. ...

Detection of follistatin-based inhibitors of the TGF-β signaling pathways in serum/plasma by means of LC-HRMS/MS & Western blotting

Drug Testing and Analysis

... Three studies analyzed the elimination profiles of SARMs. [89][90][91] Similar methods, aims, and outcomes were investigated. Each studied the SARM elimination profile after a single dose and multiple repeated doses, which were substantially below the therapeutic dosages assessed in clinical trials. ...

Elimination profiles of microdosed ostarine mimicking contaminated products ingestion

Drug Testing and Analysis

... 77 Recent human investigations into the metabolism and excretion patterns of microdosed SARMs can even help determine situations of supplements contaminated with trace amounts of SARMs versus intentional abuse. 81,[89][90][91][92] The growing popularity of SARMs among athletes may imply that athletes believe SARMs to be superior or ''safer'' than other performance-enhancing drugs. 8,27,85 Potential reasons for use include oral formulations, ease of online purchase, abundant biased misinformation on social media promoting safe SARM use, and its gray-area legal status. ...

Dietary Supplement and Food Contaminations and Their Implications for Doping Controls

Foods

... In the case of well-equipped high-end instruments (especially HRMS instruments), there are no issues in finding concentrations below 1 ng/mL. The main issue with such compounds is their stability and the excretion time in urine [14][15][16]. The main sample preparation technique for such compounds, like GHRP-2 and GHRP-6, is solid-phase extraction on SCX or WCX sorbents of different volumes. ...

Fully automated dried blood spot sample preparation enables the detection of lower molecular mass peptide and non-peptide doping agents by means of LC-HRMS

Analytical and Bioanalytical Chemistry