A single monoclonal antibody as probe to detect the entire set of native and partially unfolded rhEPO glycoforms

Amadeo, Ignacio; Oggero, Marcos; Zenclussen, María Laura; Robles, Lucía; Pereira, Dardo; Kratje, Ricardo; Etcheverrigaray, Marina

doi:10.1016/j.jim.2004.08.003

Cited by 12 publications

(9 citation statements)

References 33 publications

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“…Although the pAb had a high affinity for rhEPO and DAR, pAb-bound IA columns yielded poor (25-40%) recovery of rhEPO and DAR from spiked horse plasma. The recovery values observed in this study were similar to that obtained for plasma extracted using other EPO antibodies [24]. Albumin-depletion is commonly used to prevent the albumin-induced suppression of the target protein extraction by IA columns.…”

Section: Discussionsupporting

confidence: 81%

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Singh

Gupta

2007

Proteomics Clinical Apps

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Darbepoietin (DAR) and recombinant human erythropoietin (rhEPO) stimulate erythropoiesis, leading to an increase in red blood cells. Along with their legitimate clinical use, rhEPO and DAR are also misused in racing horses for performance enhancement. To control the illegal use of DAR and rhEPO, it is important to develop analytical methods for the detection and confirmation of these proteins in plasma. Analysis of rhEPO and DAR in plasma is challenging due to the presence of a number of high abundance proteins including albumin that interferes with their extraction. The present study showed that the extraction of rhEPO or DAR from plasma using anti-EPO-antibody coupled immunoaffinity (IA) extraction yielded low (25-40%) recovery. Albumin-depletion using antialbumin-antibody coupled IA columns also depleted the target proteins and further reduced their recovery. Pre-extraction of spiked plasma using hydroxyapatite (HTP)-ProGel or ConA columns followed by the IA column yielded 65 to 70% recovery. The extracted samples were (i) analyzed directly with or without SDS-PAGE for intact proteins and (ii) analyzed after trypsin hydrolysis, with or without SDS-PAGE, for peptide fingerprinting using MALDI-TOF. Trypsin and enolase were used as internal calibrators for intact protein analysis and a peptide EYEATLEECCAK was used as internal calibrator for fragment analysis. Analysis of extracted sample without SDS-PAGE yielded, along with the target proteins (rhEPO and DAR), albumin and other related proteins. SDS-PAGE separated the target proteins with albumin and yielded clean samples. Inclusion of internal calibrators resulted in a linear dose-response relationship for both intact protein and digested fragments and allowed quantification of the target peptides. Thus, extraction of plasma using a combination of ConA and IA extractions yielded approximately 70% recovery of target proteins with a small amount of albumin and other proteins. SDS-PAGE improved the quality of the MALDI-TOF results. Minimum detection limits for digested fragments were lower than those for intact proteins.

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Section: Discussionsupporting

confidence: 81%

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Singh

Gupta

2007

Proteomics Clinical Apps

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“…Synthetic peptides corresponding to the N-and C-terminal sequences of EPO and NESP have been selected in several instances as immunogens. As the sequences of both glycoproteins in these regions are identical, antibodies that are currently available do not discriminate between EPO and NESP [6,[13][14][15][16][17][18][19].…”

Section: Discussionmentioning

confidence: 99%

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

et al. 2007

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Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87-90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81-95), NESP (81-95), and NESP (86-104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide-OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81-95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84-106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86-91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.

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“…Recombinant human EPO concentration was evaluated by sandwich ELISA. 18 Briefly, ELISA microplates were coated with 400 ng of anti‐rhEPO mAb per well diluted in coating buffer (1 h at 37°C). The plates were washed with washing buffer and then blocked with PBS‐BSA 1% (w/v) to prevent nonspecific binding (1 h at 37°C).…”

Section: Methodsmentioning

confidence: 99%

Determination of robustness and optimal work conditions for a purification process of a therapeutic recombinant protein using response surface methodology

Amadeo

Mauro²,

Ortí³

et al. 2011

Biotechnology Progress

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A typical chromatographic purification step has numerous operating parameters that can impact its performance. As it is not feasible to evaluate the influence of each one, the current practice in biopharmaceutical industry is to apply risk analysis approach to identify process parameters that should be examined during process characterization. Once these parameters are identified, a response surface study can be run to help understand the relationship between critical inputs and outputs. We performed a study comprising optimization and robustness determination for a Blue-Sepharose purification step of rhEPO, a well-known therapeutic glycoprotein. Initially, risk analysis was fulfilled to identify key parameters. A small-scale model was created and qualified before its use in experimental studies, given by a Box-Behnken design with three factors. This method proved to be a very useful tool in bioprocess validation studies in which many input variables can affect product quality and safety.

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A single monoclonal antibody as probe to detect the entire set of native and partially unfolded rhEPO glycoforms

Cited by 12 publications

References 33 publications

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Analysis of recombinant human erythropoietin and darbepoietin in spiked plasma

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Determination of robustness and optimal work conditions for a purification process of a therapeutic recombinant protein using response surface methodology

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