Peptide Separation by Reverse-Phase High-Performance Liquid Chromatography
Roger L. Lundblad in Chemical Reagents for Protein Modification, 2020
The original expectation in development of HPLC was that existing thin-layer and column chromatographic separation methods would be transferred to columns packed with small, uniform-sized, rigid particles. This implied a stationary phase of unmodified silica. However, the retention properties of a silica surface are very sensitive to moisture levels. Elution times may be difficult to reproduce, and long column equilibration times may be required following solvent changes. Attempts to improve reproducibility of retention properties by masking the surface silanol groups led to the development of packing materials having covalently bonded surface phases. The most successful of these have been the so-called C-18 or ODS (octadecylsilyl) packings, which have 18-carbon hydrocarbon chains bonded to the surface silanols. The use of these and other hydrophobic bonded-phase packings is known as “reverse-phase” chromatography because the stationary phase is nonpolar and the mobile phase polar, which happens to be the reverse of most older methods of partition liquid chromatography.
Drug Substance and Excipient Characterization
Dilip M. Parikh in Handbook of Pharmaceutical Granulation Technology, 2021
In addition to its application in the separation and identification of materials, chromatography is also employed to detect potential interactions between materials. Both thin-layer chromatography and liquid chromatography are commonly employed for this purpose. In thin-layer chromatography, the stationary phase consists of a powder adhered onto a glass, plastic, or metal plate. The powders commonly used are silica, alumina, polyamides, celluloses, and ion-exchange resins. Solutions of the drug, excipient, and drug–excipient mixture are prepared and spotted on the same baseline at one end of the plate. The plate is then placed upright in a closed chamber containing the solvent, which constitutes the mobile phase. As the solvent moves up the plate, it carries with it the materials. Those materials that have a stronger affinity for the stationary phase will move at a slower rate. The material is identified by its Rf value, which is defined as the ratio of the distance traveled by the material to the distance traveled by the solvent front. The position of the material on the plate is indicated by spraying the plate with certain reagents or exposing the plate to ultraviolet radiation. If there is no interaction between the drug and excipient, the mixture will produce two spots whose Rf values are identical to those of the individual drug and excipient. If there is interaction, the complex formed will produce a spot whose Rf value is different from those of the individual components.
Approaches for Identification and Validation of Antimicrobial Compounds of Plant Origin: A Long Way from the Field to the Market
Mahendra Rai, Chistiane M. Feitosa in Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
Ion exchange chromatography, affinity and molecular exclusion are efficient in promoting the purification of active proteins because they explore structural characteristics such as charge, biospecificity and size without promoting denaturation (Feng et al. 2021; Benedini et al. 2019). Reverse-phase chromatography is advisable only for small molecules such as AMPs since the hydrophobic matrix and organic eluents used can result in the denaturation of larger proteins by the interaction with nonpolar amino acid residues (Feng et al. 2021). The reverse-phase procedure that separates based on hydrophobicity and uses denaturation conditions is used to determine the impurity profile and prepare samples for mass spectrometry (Soares et al. 2011).
Influence of physiochemical properties on the subcutaneous absorption and bioavailability of monoclonal antibodies
Published in mAbs, 2020
Amita Datta-Mannan, Selina Estwick, Chen Zhou, Hiuwan Choi, Nicole E. Douglass, Derrick R. Witcher, Jirong Lu, Catherine Beidler, Rohn Millican
The global molecule hydrophobicity was determined using a hydrophobic interaction chromatography (HIC)-based method. The data were expressed as a relative hydrophobicity interaction percentage for each of the mAbs to allow for comparisons both within and across the three mAb platforms; larger hydrophobicity interaction percent (HIP) values indicate an increased affinity for the HIC matrix. The Platform 1 molecules show similar and relatively low HIP values; the HIP for mAb 1P and mAb 1RE were 1.3% and 0.7%, respectively. In contrast, both the Platform 2 and 3 molecules showed ~10- to ~100-times higher HIP values than the Platform 1 mAbs (Table 2). The Platform 2 constructs showed similar HIP values for mAb 2P and mAb 2RE of ~16% and ~20%, respectively. Platform 3 mAbs had the widest diversity of HIP, with mAb 3P and 3RE displaying values of ~100% and ~12%, respectively.
Update on Proteomic approaches to uncovering virus-induced protein alterations and virus –host protein interactions during the progression of viral infection
Published in Expert Review of Proteomics, 2020
Complex mixtures may be fractionated in any of a variety of ways. The two most general ways, each of which can resolve up to 100 fractions or more [9,10] in each dimension, are gel electrophoresis and HPLC (high-pressure liquid chromatography). Gel electrophoresis is one of the components of 2D-DIGE and is usually used to resolve proteins prior to their digestion to peptides. HPLC may be performed with intact proteins but usually gives better performance when resolving peptides [11]; thus, samples are usually digested prior to HPLC. Similar to 2D gel electrophoresis offering better resolution than 1D electrophoresis, the resolving capacity of LC may be enhanced by incorporating orthogonal separation strategies. This has led to development of multi-dimensional peptide identification techniques, including, for example, MudPIT [9]. For example, reverse-phase LC, which separates based on peptide hydrophobicity, may be coupled with an alternate strategy, such as affinity chromatography or may be performed under different pH conditions [12]. Thus, some of the quantitative strategies to be discussed below use one or more additional fractionation steps in order to delve deeper into complex cellular Proteomes.
An accelerated surface-mediated stress assay of antibody instability for developability studies
Published in mAbs, 2020
Marie R.G. Kopp, Adriana-Michelle Wolf Pérez, Marta Virginia Zucca, Umberto Capasso Palmiero, Brigitte Friedrichsen, Nikolai Lorenzen, Paolo Arosio
We next applied hydrophobic interaction chromatography (HIC) to determine protein hydrophobicity, which has been associated with aggregation,41 viscosity and in vivo clearance.78 The principle of the assay relies on modulating the interactions between mAbs and a hydrophobic stationary phase through the application of a salt gradient. Hydrophobic mAbs show longer retention times compared to less hydrophobic mAbs at increasing salt concentrations.79 We observed that the measured variant hydrophobicities correlate with their aggregation propensities under thermally stressed conditions, measured from the total amount of aggregates determined by SEC (rp = 0.70, rs = 0.70; p < 0.01, and rp = 0.75; p < 0.01, rs = 0.54; p = 0.06 in the first and second replicate runs, respectively). However, when comparing the HNSSA and HIC, the determined correlation was non-significant (rp = 0.42, rs = 0.48; p ≥ 0.1, Figure 5). This poor correlation might arise from the different hydrophobic materials used in both assays, and the fact that the hydrophobic nanoparticles used in the HNSSA carry a residual negative charge, which might play a role in the aggregation of the different variants. Moreover, we have observed that the nanoparticles not only trigger the adsorption of the protein on their surface but are also responsible for further aggregation events, which are not probed for with HIC.60
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