New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications

Fekete, Szabolcs; Veuthey, Jean‐Luc; Guillarme, Davy

doi:10.1016/j.jpba.2012.03.024

Cited by 130 publications

(74 citation statements)

References 210 publications

(247 reference statements)

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“…The bigger the pore size, the more proteins can bind to the RP material. This underlines that the pore size of RP material is an important parameter to be considered for separation of proteins and peptides51, 52 and can be exploited for HLA‐I peptide isolation.…”

Section: Discussionmentioning

confidence: 99%

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

et al. 2018

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For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS3‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS3 detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E711–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.

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Section: Discussionmentioning

confidence: 99%

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

et al. 2018

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“…Higher temperature generally enhances mass transfer for proteins, leading to higher separation performance. In addition, temperature is a common parameter in chromatographic method development since selectivity and resolution can be tuned by changing this variable [25,27,29,31,39]. For the analysis of proteins under RPLC conditions, temperature affects solute adsorption on the stationary phase.…”

Section: Recovery Of Mab Species In Hilic Conditionsmentioning

confidence: 99%

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Bobály

D’Atri

Beck³

et al. 2017

Journal of Pharmaceutical and Biomedical Analysis

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a b s t r a c tHydrophilic interaction liquid chromatography (HILIC) is a well-established technique for the separation and analysis of small polar compounds. A recently introduced widepore stationary phase expanded HILIC applications to larger molecules, such as therapeutic proteins. In this paper, we present some generic HILIC conditions adapted for a wide range of FDA and EMA approved recombinant monoclonal antibody (mAb) species and for an antibody-drug conjugate (ADC). Seven approved mAbs possessing various isoelectric point (pI) and hydrophobicity as well as a cysteine conjugated ADC were used in this study. Samples were digested by IdeS enzyme and digests were further fragmented by chemical reduction. The resulting fragments were separated by HILIC. The main benefit of HILIC was the separation of polar variants (glycovariants) in a reasonable analysis time at the protein level, which is not feasible with other chromatographic modes. Three samples were selected and chromatographic conditions were further optimized to maximize resolution. A commercial software was used to build up retention models. Experimental and predicted chromatograms showed good agreement and the average error of retention time prediction was less than 2%. Recovery of various species and sample stability under the applied conditions were also discussed.

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“…In the biopharmaceutical field, LC is used for both the assessment of protein batch purity, protein modification (e.g. glycosylation) and aggregation [4,56]. In combination with MS, precise and complementary information is generated.…”

Section: Direct Analytical Characterization Of the Productsmentioning

confidence: 99%

“…8.0: 01/2008:0951) [58]. In CE, the separation is based on charge differences [59], in our case a loss of one positive charge of lysine with the simultaneous addition of negative charges of NOTA, whereas in RP HPLC mainly the decrease in hydrophobicity due to the attachment of NOTA leads to the separation [56]. Overall, the 1:1 and 3:1 samples show similar degrees of somatropin modification as measured with CE-MS compared to LC-MS (Fig.…”

Section: Direct Analytical Characterization Of the Productsmentioning

confidence: 99%

Analytical characterization of NOTA-modified somatropins

Bracke

Wynendaele

D׳Hondt

et al. 2014

Journal of Pharmaceutical and Biomedical Analysis

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Highlights• NOTA-labeled somatropins were synthesized using different NOTA:protein ratios.• Direct LC-MS and CE-MS approaches indicated multiple substitution degrees.• Bottom-up approaches gave structural insights and information on the labeling yield.• Lys-70 (in silico calculated pKa of 8.3) is the NOTA-modification hotspot.• We report a synthesis procedure for the production of target-specific radiopharmaceuticals.

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New trends in reversed-phase liquid chromatographic separations of therapeutic peptides and proteins: Theory and applications

Cited by 130 publications

References 210 publications

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

Analysis of recombinant monoclonal antibodies in hydrophilic interaction chromatography: A generic method development approach

Analytical characterization of NOTA-modified somatropins

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