Explore chapters and articles related to this topic
Conjugation and Other Methods in Polymeric Vaccines
Published in Mesut Karahan, Synthetic Peptide Vaccine Models, 2021
Chromatography is known as an important biophysical technique that can separate, identify, and purify the blend segments for subjective and quantitative investigation. Proteins can be purified based on properties such as size and shape, total charge, hydrophobic groups present on the surface, and the ability to bind to the stationary phase. It is based on molecular properties and interaction type use mechanism, four separation technologies, ion exchange, dispersion, surface adsorption, and size exclusion. Column chromatography is one of the most used and common techniques for protein purification methods. This technique is basically used to purify biological molecules. The application of the method can be summarized as follows. The sample is separated on the column (stationary phase) and then the wash buffer is added to the column (mobile phase). It flows through the column material placed on the fiberglass support. With the help of the wash buffer, the samples are accumulated at the bottom of the column chromatography instrument, based on time and volume (Coskun 2016). Column chromatography is a powerful purification and separation process that is closely controlled to the hydrodynamic diameters of the macromolecules depending on the diameter of the pores in the filling material (see in HPLC Method) (Acar 2006; Fornaguera and Solans 2018).
Biologic Drug Substance and Drug Product Manufacture
Published in Anthony J. Hickey, Sandro R.P. da Rocha, Pharmaceutical Inhalation Aerosol Technology, 2019
Ajit S. Narang, Mary E. Krause, Shelly Pizarro, Joon Chong Yee
Chromatographic purification is the mainstream technology for most of the protein purification steps. While improving purity, chromatographic steps applied in sequence tend to reduce the yield. A minimalistic approach that assures adequate product purity is, therefore, adopted on a case-by-case basis.
Overview of Drug Development
Published in Mark Chang, John Balser, Jim Roach, Robin Bliss, Innovative Strategies, Statistical Solutions and Simulations for Modern Clinical Trials, 2019
Mark Chang, John Balser, Jim Roach, Robin Bliss
Following ’hits’, the lead compounds are purified using chromatographic techniques and their chemical compositions identified via spectroscopic and chemical means. Structures may be elucidated using X-ray or nuclear magnetic resonance (NMR) methods. Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Protein purification is an important step in the characterization of the function, structure and interactions of the protein of interest.
Mass Spectrometry-based Biomarkers for Knee Osteoarthritis: A Systematic Review
Published in Expert Review of Proteomics, 2021
Mirella J.J. Haartmans, Kaj S. Emanuel, Gabrielle J.M. Tuijthof, Ron M. A. Heeren, Pieter J. Emans, Berta Cillero-Pastor
Post-translational modifications of proteins could also be of interest while searching for biomarkers. Top-down proteomics allows the detection of specific post-translational modifications such as phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation or acetylation, and typically involves purification steps. Glycoprotein modifications in the cartilage for instance have been described [61]. In many cases these modifications cannot be detected. Chromatographic separation or previous protein purification are often needed. In addition, protocols that disrupt collagen fibers are recommended since the access to proteins with specific roles and PTMs can be better digested then. For example, Hsueh et al. used a strong guanidine extraction buffer for protein extraction in cartilage [62].
Peptidomics and proteogenomics: background, challenges and future needs
Published in Expert Review of Proteomics, 2021
Rui Vitorino, Manisha Choudhury, Sofia Guedes, Rita Ferreira, Visith Thongboonkerd, Lakshya Sharma, Francisco Amado, Sanjeeva Srivastava
The proteogenomics and peptidomics involve identification of peptides, usually based on advances in basic techniques of gel electrophoresis, mass spectrometry, ionization, or chromatography. These technologies, in conjunction with advanced sequencing techniques, form the basis of modern peptidomics and proteogenomics technology. 2-DE polyacrylamide gel electrophoresis (PAGE) was the first and most suitable technique for protein purification [14]. In this technique, proteins are first separated according to their isoelectric point (pI) and then according to their molecular weight using a sodium dodecyl sulfate gel PAGE (SDS-PAGE). The ability to analyze complete proteins in terms of their post-translational modifications (PTMs) is of great theoretical and practical interest in proteomics. To maximize the biological relevance of the method, proteomics should ideally be able to analyze complete, native, folded proteins with their PTMs and prosthetic groups [15,16,17]. However, gel-based proteomics has low sensitivity, low throughput, and poor predictive power. Further, protein spots/bands identified by gel-based methods are subjected to mass spectrometric techniques for protein identification [18]. To overcome several challenges associated with gel-based proteomics now there is more emphasis on mass spectrometry-based shotgun proteomics.
Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis
Published in mAbs, 2020
Rachel Liuqing Shi, Gang Xiao, Thomas M. Dillon, Margaret S. Ricci, Pavel V. Bondarenko
As a conventional and nondenaturing technique, ion-exchange chromatography (IEX) has been widely used to separate and isolate protein charge variants during protein purification and for subsequent characterization.22-24 Upon the separation of charge variants by IEX, current strategies to determine the effects of modifications on specific charge variant peak involve isolating the peak of interest followed by various mass spectrometric analyses, such as intact mass analysis, peptide mapping, and glycan analysis.10,25 In addition to being limited by time and resources, this two-step approach may overlook the minor species that do not exhibit distinctive UV peaks and introduce artifacts because of the lengthy sample preparation processes.26 Therefore, the ability to directly couple IEX to high-resolution mass spectrometry (MS) is highly desirable to enable sensitive MS detection, which can significantly improve efficiency for charge heterogeneity characterization.