Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins

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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“…It can be utilized in optimizing information regarding the constituents of a polar mixture, especially of small molecules [1][2][3][4][5].…”

Section: Introductionmentioning

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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“…Methods based on alternative separation strategies (CE, HILIC, etc.) were developed also recently [29][30][31].…”

Section: Introductionmentioning

confidence: 99%

Simplifying and expanding the screening for peptides <2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry

Thomas

Görgens

Guddat

et al. 2015

J of Separation Science

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The analysis of low-molecular-mass peptides in doping controls has become a mandatory aspect in sports drug testing and, thus, the number of samples that has to be tested for these analytes has been steadily increasing. Several peptides <2 kDa with performance-enhancing properties are covered by the list of prohibited substances of the World Anti-Doping Agency including Desmopressin, LH-RH, Buserelin, Triptorelin, Leuprolide, GHRP-1, GHRP-2, GHRP-3, GHRP-4, GHRP-5,GHRP-6, Alexamorelin, Ipamorelin, Hexarelin, ARA-290, AOD-9604, TB-500 and Anamorelin. With the presented method employing direct urine injection into a liquid chromatograph followed by ion-mobility time-of-flight mass spectrometry, a facile, specific and sensitive assay for the aforementioned peptidic compounds is provided. The accomplished sensitivity allows for limits of detection between 50 and 500 pg/mL and thus covers the minimum required performance level of 2 ng/mL accordingly. The method is precise (imprecision <20%) and linear in the estimated working range between 0 and 10 ng/mL. The stability of the peptides in urine was tested, and -20°C was found to be the appropriate storage temperature for sports drug testing. Finally, proof-of-concept was shown by analysing elimination study urine samples collected from individuals having administered GHRP-6, GHRP-2, or LHRH.

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“…Since the term hydrophilic interaction chromatography (HILIC) was coined in 1990 by Alpert [1], it has been an established chromatographic technique for separation of highly polar analytes that have poor retention on common reverse phase liquid chromatography (RPLC) [2][3][4][5][6][7]. Along with increasing popularity of HILIC, the development of novel HILIC stationary phases (SPs) has received much attention [2][3][4][5][6].…”

Section: Introductionmentioning

confidence: 99%

A hyperbranched polyethylenimine functionalized stationary phase for hydrophilic interaction liquid chromatography

et al. 2016

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A hyperbranched stationary phase for hydrophilic interaction liquid chromatography (HILIC) has been prepared by grafting polyethylenimine (PEI) onto silica gel (termed as PEI-Sil). Rich primary, secondary, and tertiary amino groups associated with PEI render its good hydrophility. More importantly, the hyperbranched structure of PEI molecule is greatly helpful in improving interaction with polar analytes. For several kinds of model polar compounds, including organic acids, nucleosides, nucleic acid bases, amino acids, cephalosporins, and non-reducing sugars, PEI-Sil demonstrated excellent separation performance in terms of running stability, reproducibility, and separation efficiency (e.g., plate count ~74,000/m). In addition, PEI-Sil also exhibited much better separation selectivity toward inorganic anions when operated in the mode of ion chromatography relative to a commercial amino propyl-bonded column.

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Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins

Cited by 66 publications

References 66 publications

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

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

Simplifying and expanding the screening for peptides <2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry

A hyperbranched polyethylenimine functionalized stationary phase for hydrophilic interaction liquid chromatography

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