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Journal of Chromatography. B,... Nov 2020The field of affinity chromatography, which employs a biologically-related agent as the stationary phase, has seen significant growth since the modern era of this method... (Review)
Review
The field of affinity chromatography, which employs a biologically-related agent as the stationary phase, has seen significant growth since the modern era of this method began in 1968. This review examines the major developments and trends that have occurred in this technique over the past five decades. The basic principles and history of this area are first discussed. This is followed by an overview of the various supports, immobilization strategies, and types of binding agents that have been used in this field. The general types of applications and fields of use that have appeared for affinity chromatography are also considered. A survey of the literature is used to identify major trends in these topics and important areas of use for affinity chromatography in the separation, analysis, or characterization of chemicals and biochemicals.
Topics: Biochemistry; Biomedical Research; Biotechnology; Chromatography, Affinity; History, 20th Century; History, 21st Century; Humans
PubMed: 32871378
DOI: 10.1016/j.jchromb.2020.122332 -
Nature Communications Feb 2022Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein...
Transcription factors (TFs) interact with several other proteins in the process of transcriptional regulation. Here, we identify 6703 and 1536 protein-protein interactions for 109 different human TFs through proximity-dependent biotinylation (BioID) and affinity purification mass spectrometry (AP-MS), respectively. The BioID analysis identifies more high-confidence interactions, highlighting the transient and dynamic nature of many of the TF interactions. By performing clustering and correlation analyses, we identify subgroups of TFs associated with specific biological functions, such as RNA splicing or chromatin remodeling. We also observe 202 TF-TF interactions, of which 118 are interactions with nuclear factor 1 (NFI) family members, indicating uncharacterized cross-talk between NFI signaling and other TF signaling pathways. Moreover, TF interactions with basal transcription machinery are mainly observed through TFIID and SAGA complexes. This study provides a rich resource of human TF interactions and also act as a starting point for future studies aimed at understanding TF-mediated transcription.
Topics: Biotinylation; Chromatin; Chromatography, Affinity; Gene Expression Regulation; Gene Regulatory Networks; HEK293 Cells; Humans; Mass Spectrometry; NFI Transcription Factors; Protein Interaction Maps; Proteomics; Transcription Factors
PubMed: 35140242
DOI: 10.1038/s41467-022-28341-5 -
Antibody Therapeutics Apr 2021Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality. The downstream processing of this class of antibodies is therefore of... (Review)
Review
Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality. The downstream processing of this class of antibodies is therefore of crucial importance in ensuring that these products can be obtained with high purity and yield. Due to the various fundamental structural similarities between bsAbs and monoclonal antibodies (mAbs), many of the current bsAb downstream purification methodologies are based on the established purification processes of mAbs, where affinity, charge, size, hydrophobicity and mixed-mode-based purification are frequently employed. Nevertheless, the downstream processing of bsAbs presents a unique set of challenges due to the presence of bsAb-specific byproducts, such as mispaired products, undesired fragments and higher levels of aggregates, that are otherwise absent or present in lower levels in mAb cell culture supernatants, thus often requiring the design of additional purification strategies in order to obtain products of high purity. Here, we outline the current major purification methods of bsAbs, highlighting the corresponding solutions that have been proposed to circumvent the unique challenges presented by this class of antibodies, including differential affinity chromatography, sequential affinity chromatography and the use of salt additives and pH gradients or multistep elutions in various modes of purification. Finally, a perspective towards future process development is offered.
PubMed: 34056544
DOI: 10.1093/abt/tbab007 -
International Journal of Molecular... May 2020While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular...
While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular are a new class of small proteins marketed as bio-analytic reagents. They feature tailorable binding affinity, low immunogenicity, high tissue permeation, and high expression titer in bacterial hosts. This work presents the development of affibody-binding peptides to be utilized as ligands for their purification from bacterial lysates. Affibody-binding candidates were identified by screening a peptide library simultaneously against two model affibodies (anti-immunoglobulin G (IgG) and anti-albumin) with the aim of selecting peptides targeting the conserved domain of affibodies. An ensemble of homologous sequences identified from screening was synthesized on Toyopearl resin and evaluated via binding studies to select sequences that afford high product binding and recovery. The affibody-peptide interaction was also evaluated by in silico docking, which corroborated the targeting of the conserved domain. Ligand IGKQRI was validated through purification of an anti-ErbB2 affibody from an lysate. The values of binding capacity (~5 mg affibody per mL of resin), affinity (K ~1 μM), recovery and purity (64-71% and 86-91%), and resin lifetime (100 cycles) demonstrate that IGKQRI can be employed as ligand in affibody purification processes.
Topics: Amino Acid Sequence; Humans; Immunoglobulin G; Ligands; Molecular Docking Simulation; Peptide Library; Peptides; Recombinant Fusion Proteins; Serum Albumin, Human; Temperature
PubMed: 32471034
DOI: 10.3390/ijms21113769 -
Molecular Therapy. Methods & Clinical... Mar 2023The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for...
The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype. Here, we demonstrate that the POROS CaptureSelect AAVX affinity resin efficiently captures a panel of 15 divergent AAV serotypes, including the commonly used AAV2, AAV8, AAV9, PHP.B, and Anc80. We also find that AAVX resin can be regenerated repeatedly without loss of efficiency or carry-over contamination. While AAV preps purified with AAVX showed a higher fraction of empty capsids than preps purified using iodixanol ultracentrifugation, the potency of the AAVX purified vectors was comparable with that of iodixanol purified vectors both and . Finally, optimization of the purification protocol resulted in a process with an overall efficiency of 65%-80% across all scales and AAV serotypes tested. These data establish AAVX affinity chromatography as a versatile and efficient method for purification of a broad range of AAV serotypes.
PubMed: 36654797
DOI: 10.1016/j.omtm.2022.12.009 -
Frontiers in Chemistry 2019Important information on chemical processes in living systems can be obtained by the rates at which these biological interactions occur. This review will discuss several... (Review)
Review
Important information on chemical processes in living systems can be obtained by the rates at which these biological interactions occur. This review will discuss several techniques based on traditional and high-performance affinity chromatography that may be used to examine the kinetics of biological reactions. These methods include band-broadening measurements, techniques for peak fitting, split-peak analysis, peak decay studies, and ultrafast affinity extraction. The general principles and theory of each method, as applied to the determination of rate constants, will be discussed. The applications of each approach, along with its advantages and limitations, will also be considered.
PubMed: 31681727
DOI: 10.3389/fchem.2019.00673 -
Molecules (Basel, Switzerland) Nov 2020CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of...
CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein ( = 38.71nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.
Topics: Aptamers, Nucleotide; Breast Neoplasms; Chromatography, Affinity; Exosomes; Female; Humans; Immunoenzyme Techniques; SELEX Aptamer Technique; Tetraspanin 30; Tumor Cells, Cultured
PubMed: 33261145
DOI: 10.3390/molecules25235585 -
ACS Omega Sep 2022Affinity chromatography is a well-known method dependent on molecular recognition and is used to purify biomolecules by mimicking the specific interactions between the... (Review)
Review
Affinity chromatography is a well-known method dependent on molecular recognition and is used to purify biomolecules by mimicking the specific interactions between the biomolecules and their substrates. Enzyme substrates, cofactors, antigens, and inhibitors are generally utilized as bioligands in affinity chromatography. However, their cost, instability, and leakage problems are the main drawbacks of these bioligands. Biomimetic affinity ligands can recognize their target molecules with high selectivity. Their cost-effectiveness and chemical and biological stabilities make these antibody analogs favorable candidates for affinity chromatography applications. Biomimetics applies to nature and aims to develop nanodevices, processes, and nanomaterials. Today, biomimetics provides a design approach to the biomimetic affinity ligands with the aid of computational methods, rational design, and other approaches to meet the requirements of the bioligands and improve the downstream process. This review highlighted the recent trends in designing biomimetic affinity ligands and summarized their binding interactions with the target molecules with computational approaches.
PubMed: 36157742
DOI: 10.1021/acsomega.2c03530 -
Biomolecules Jun 2022Antibodies have become an important class of biological products in cancer treatments such as radiotherapy. The growing therapeutic applications have driven a demand for... (Review)
Review
Antibodies have become an important class of biological products in cancer treatments such as radiotherapy. The growing therapeutic applications have driven a demand for high-purity antibodies. Affinity chromatography with a high affinity and specificity has always been utilized to separate antibodies from complex mixtures. Quality chromatographic components (matrices and affinity ligands) have either been found or generated to increase the purity and yield of antibodies. More importantly, some matrices (mainly particles) and affinity ligands (including design protocols) for antibody purification can act as radiosensitizers or carriers for therapeutic radionuclides (or for radiosensitizers) either directly or indirectly to improve the therapeutic efficiency of radiotherapy. This paper provides a brief overview on the matrices and ligands used in affinity chromatography that are involved in antibody purification and emphasizes their applications in radiotherapy to enrich potential approaches for improving the efficacy of radiotherapy.
Topics: Antibodies; Chromatography, Affinity; Ligands
PubMed: 35740946
DOI: 10.3390/biom12060821 -
Recent Advances in Supramolecular Affinity Separations: Affinity Chromatography and Related Methods.Advances in Chromatography 2021Affinity chromatography is a technique that uses a stationary phase based on the supramolecular interactions that occur in biological systems or mimics of these systems....
Affinity chromatography is a technique that uses a stationary phase based on the supramolecular interactions that occur in biological systems or mimics of these systems. This method has long been a popular tool for the isolation, measurement, and characterization of specific targets in complex samples. This review discusses the basic concepts of this method and examines recent developments in affinity chromatography and related supramolecular separation methods. Topics that are examined include advances that have occurred in the types of supports, approaches to immobilization, and binding agents that are employed in this method. New developments in the applications of affinity chromatography are also summarized, including an overview on the use of this method for biochemical purification, sample preparation or analysis, chiral separations, and biointeraction studies.
PubMed: 36186535
DOI: 10.1201/9781003223405-1