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Vaccine Aug 2019Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams.... (Review)
Review
Affinity chromatography is among the most powerful separation techniques, achieving the finest separation with high yields even in the most challenging feed streams. Incorporating affinity chromatography in vaccine purification has long been attempted by researchers to improve unit yield and purity with the secondary goal of reducing the number of downstream process operations. Despite the success in laboratory-scale proof of concept, implementation of this technique in pilot or cGMP manufacturing has rarely been realised due to technical and economic challenges in design and manufacturing of ideal ligands as well as availability of high-productivity chromatography media. This paper reviews evolving technologies in engineered ligands and chromatography media that are encouraging companies to re-visit the possible use of affinity chromatography in larger scale vaccine purification. It is postulated that commercial-scale implementation of high throughput single-use affinity chromatography can significantly simplify process architecture, improve productivity and flexibility, and reduce cost of goods.
Topics: Automation; Bioreactors; Chromatography, Affinity; Ligands; Vaccines
PubMed: 29627235
DOI: 10.1016/j.vaccine.2018.02.090 -
Journal of Chromatography. A May 2022The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products....
The purification of extracellular vesicles (EVs) remains a major hurdle in the progression of fundamental research and the commercial application of EV-based products. In this study, we evaluated the potential of heparin affinity chromatography (HAC) to purify neural stem cell-derived EVs as part of a multistep process. Bind-elute chromatography, such as HAC, is an attractive method of purification because it is highly scalable, robust and can be automated. Our findings support an interaction between EVs and heparin. The recovery of EVs using HAC based on particle counts was a minimum of 68.7%. We found HAC could remove on average 98.8% and 99.0% of residual protein and DNA respectively. In addition to EV purification, HAC was used to separate EVs into three populations based on their affinity to the heparin column. Within these populations, we detected differences in the expression of the exosome-associated protein TSG101 and the tetraspanin immunophenotype. However, the significance of these observations is not clear. Overall HAC shows promise as a potential purification method to capture EVs and this study proposes a novel application of HAC for EV fractionation. Moving forward, a better understanding of the heparin-EV interaction would be required before HAC can be more widely adopted for these applications.
Topics: Chemical Fractionation; Chromatography, Affinity; Exosomes; Extracellular Vesicles; Heparin
PubMed: 35367893
DOI: 10.1016/j.chroma.2022.462987 -
Cell Reports Methods Aug 2022In living systems, a complex network of protein-protein interactions (PPIs) underlies most biochemical events. The human protein-protein interactome has been surveyed... (Review)
Review
In living systems, a complex network of protein-protein interactions (PPIs) underlies most biochemical events. The human protein-protein interactome has been surveyed using yeast two-hybrid (Y2H)- and mass spectrometry (MS)-based approaches such as affinity purification coupled to MS (AP-MS). Despite decades of systematic investigations and collaborative multi-disciplinary efforts, there is no "gold standard" for documenting PPIs. A surprisingly large fraction of the human interactome remains uncharted, which we refer to as the "dark interactome." In this review, we highlight the complexity of the human interactome and discuss the current status of the human reference interactome maps. We discuss why a large proportion of the human interactome has remained refractory to traditional approaches. We propose an experimental model that can enable the identification of the dark interactome in a cell-type-specific manner. We also propose a framework to implement when embarking on studies designed to rigorously identify and characterize protein interactions.
Topics: Humans; Protein Interaction Mapping; Proteins; Chromatography, Affinity; Saccharomyces cerevisiae
PubMed: 36046620
DOI: 10.1016/j.crmeth.2022.100275 -
Journal of Chromatography. B,... Oct 2014Cellular membrane affinity chromatography stationary phases have been extensively used to characterize immobilized proteins and provide a direct measurement of multiple... (Review)
Review
Cellular membrane affinity chromatography stationary phases have been extensively used to characterize immobilized proteins and provide a direct measurement of multiple binding sites, including orthosteric and allosteric sites. This review will address the utilization of immobilized cellular and tissue fragments to characterize multiple transmembrane proteins co-immobilized onto a stationary phase. This approach will be illustrated by demonstrating that multiple transmembrane proteins were immobilized from cell lines and tissue fragments. In addition, the immobilization of individual compartments/organelles within a cell will be discussed and the changes in the proteins binding/kinetics based on their location.
Topics: Binding Sites; Chromatography, Affinity; Humans; Immobilized Proteins; Kinetics; Ligands; Membrane Proteins; Protein Binding
PubMed: 24780640
DOI: 10.1016/j.jchromb.2014.04.005 -
Analytical and Bioanalytical Chemistry Jan 2016Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum...
Ultrafast affinity extraction and a two-dimensional high performance affinity chromatographic system were used to measure the free fractions for various drugs in serum and at typical therapeutic concentrations. Pooled samples of normal serum or serum from diabetic patients were utilized in this work. Several drug models (i.e., quinidine, diazepam, gliclazide, tolbutamide, and acetohexamide) were examined that represented a relatively wide range of therapeutic concentrations and affinities for human serum albumin (HSA). The two-dimensional system consisted of an HSA microcolumn for the extraction of a free drug fraction, followed by a larger HSA analytical column for the further separation and measurement of this fraction. Factors that were optimized in this method included the flow rates, column sizes, and column switching times that were employed. The final extraction times used for isolating the free drug fractions were 333-665 ms or less. The dissociation rate constants for several of the drugs with soluble HSA were measured during system optimization, giving results that agreed with reference values. In the final system, free drug fractions in the range of 0.7-9.5% were measured and gave good agreement with values that were determined by ultrafiltration. Association equilibrium constants or global affinities were also estimated by this approach for the drugs with soluble HSA. The results for the two-dimensional system were obtained in 5-10 min or less and required only 1-5 μL of serum per injection. The same approach could be adapted for work with other drugs and proteins in clinical samples or for biomedical research.
Topics: Chromatography, Affinity; Diabetes Mellitus; Humans; Pharmaceutical Preparations
PubMed: 26462924
DOI: 10.1007/s00216-015-9082-7 -
Current Protein & Peptide Science 2021Phosphorylation is arguably the most important post-translational modification that occurs within proteins. Phosphorylation is used as a signal to control numerous... (Review)
Review
Phosphorylation is arguably the most important post-translational modification that occurs within proteins. Phosphorylation is used as a signal to control numerous physiological activities ranging from gene expression to metabolism. Identifying phosphorylation sites within proteins was historically a challenge as it required either radioisotope labeling or the use of phospho-specific antibodies. The advent of mass spectrometry (MS) has had a major impact on the ability to qualitatively and quantitatively characterize phosphorylated proteins. In this article, we describe MS methods for characterizing phosphorylation sites within individual proteins as well as entire proteome samples. The utility of these methods is illustrated in examples that show the information that can be gained using these MS techniques.
Topics: Amino Acid Sequence; Chromatography, Affinity; Chromatography, Liquid; Humans; Peptide Mapping; Phosphopeptides; Phosphoproteins; Phosphorylation; Protein Processing, Post-Translational; Proteome; Proteomics; Tandem Mass Spectrometry
PubMed: 33231146
DOI: 10.2174/1389203721999201123200439 -
Methods (San Diego, Calif.) Aug 2018Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these... (Review)
Review
Many biological processes involve solute-protein interactions and solute-solute competition for protein binding. One method that has been developed to examine these interactions is zonal elution affinity chromatography. This review discusses the theory and principles of zonal elution affinity chromatography, along with its general applications. Examples of applications that are examined include the use of this method to estimate the relative extent of solute-protein binding, to examine solute-solute competition and displacement from proteins, and to measure the strength of these interactions. It is also shown how zonal elution affinity chromatography can be used in solvent and temperature studies and to characterize the binding sites for solutes on proteins. In addition, several alternative applications of zonal elution affinity chromatography are discussed, which include the analysis of binding by a solute with a soluble binding agent and studies of allosteric effects. Other recent applications that are considered are the combined use of immunoextraction and zonal elution for drug-protein binding studies, and binding studies that are based on immobilized receptors or small targets.
Topics: Binding Sites; Chromatography, Affinity; Protein Binding; Thermodynamics
PubMed: 29409783
DOI: 10.1016/j.ymeth.2018.01.020 -
Journal of Chromatography. A Sep 2019Protein purification by affinity chromatography relies primarily on the interaction of a fused-tag to the protein of interest. Here, we describe a tag-free affinity...
Protein purification by affinity chromatography relies primarily on the interaction of a fused-tag to the protein of interest. Here, we describe a tag-free affinity method that employs functional selection interactions to a broad range of proteins. To achieve this, we coupled human DNA-clamp proliferating cell nuclear antigen (PCNA) that interacts with over one hundred proteins to an agarose resin. We demonstrate the versatility of our PCNA-Agarose column at various chromatographic steps by purifying PCNA-binding proteins that are involved in DNA Replication (DNA polymerase δ, flap endonuclease 1 and DNA ligase 1), translesion DNA synthesis (DNA polymerases eta, kappa and iota) and genome stability (p15). We also show the competence of the PCNA-Agarose column to purify non-PCNA binding proteins by fusing the PCNA-binding motif of human p21 as an affinity tag. Finally, we establish that our PCNA-Agarose column is a suitable analytical method for characterizing the binding strength of PCNA-binding proteins. The conservation and homology of PCNA-like clamps will allow for the immediate extension of our method to other species.
Topics: Buffers; Chromatography, Affinity; DNA Polymerase III; DNA Repair; DNA Replication; Humans; Proliferating Cell Nuclear Antigen; Protein Binding; Recombinant Proteins; Resins, Synthetic; Sepharose
PubMed: 31204039
DOI: 10.1016/j.chroma.2019.06.008 -
Analytical and Bioanalytical Chemistry Aug 2021The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense...
The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses. The oligonucleotide-based stationary phase was applied for the retention studies. Unmodified oligonucleotides of different complementarity to the molecule attached as a stationary phase, as well as antisense oligonucleotides, were tested. The comparative study upon complex optimization of oligonucleotide analysis in different liquid chromatography modes was performed. Results have shown that this stationary phase may be applied for oligonucleotide analysis in hydrophilic interaction liquid chromatography and ion exchange chromatography, but no unique sequence-based selectivity was obtained. Contrary results were observed for affinity chromatography, which allowed for specific separation of the complementary strands based on hydrogen bonding and stacking interactions, where the temperature was the main factor influencing the selectivity of the separation. Furthermore, the oligonucleotide-based stationary phase may be applied for comparative antisense oligonucleotide hybridization studies to a specific RNA sequence. All of the results have shown that affinity chromatography with oligonucleotide-based stationary phases is a powerful technique for the specific base recognition of polynucleotides.
Topics: Chromatography, Affinity; Chromatography, Ion Exchange; DNA; DNA, Antisense
PubMed: 34165593
DOI: 10.1007/s00216-021-03473-7 -
Scientific Reports May 2021Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the...
Extracellular vesicles (EVs) have recently gained growing interest for their diagnostic and therapeutic potential. Despite this, few protocols have been reported for the isolation of EVs with preserved biological function. Most EV purification methods include a precipitation step that results in aggregation of vesicles and most available techniques do not efficiently separate the various types of EVs such as exosomes and ectosomes, which are involved in distinct biological processes. For this reason, we developed a new two-step fast performance liquid chromatography (FPLC) protocol for purification of large numbers of EVs. The method comprises size exclusion chromatography followed by immobilized metal affinity chromatography, which is enabled by expression of poly-histidine tagged folate receptor α in the parental cells. Characterisation and comparison of the EVs obtained by this method to EVs purified by differential centrifugation, currently the most common method to isolate EVs, demonstrated higher purity and more selective enrichment of exosomes in EV preparations using our FPLC method, as assessed by comparison of marker proteins and density distribution. Our studies reveal new possibilities for the isolation of defined subpopulations of EVs with preserved biological function that can easily be upscaled for production of larger amounts of EVs.
Topics: Chromatography, Affinity; Chromatography, Gel; Extracellular Vesicles; Humans; Proteins
PubMed: 34006937
DOI: 10.1038/s41598-021-90022-y