Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity

Zhang, Zichuan; Park, Jeehae; Barrett, Hannah; Dooley, Scott; Davies, Charlotte Aull; Verhagen, Marc F. J. M.

doi:10.1089/hum.2020.233

Cited by 29 publications

(20 citation statements)

References 27 publications

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“…Recent studies indicated the existence of an additional VP component. 20,24,26 In this study, the VP3 variant of which the translation started at M211, not at M203, was identified by CGE and LC-UV-MS, and the VP3 fragments of which the C-terminal region was cleaved off between the DP or DG sequence were detected by LC-UV-MS.…”

Section: Discussionmentioning

confidence: 71%

“…As the characterization methods of VPs have been developed, several researches suggested the presence of VP related components which were different from conventional VPs. 20,[24][25][26][27] However, these components were neither fully identified nor quantified. Appropriate quantification of VP related components as well as VP1, VP2, and VP3 is necessary for better quality control of AAV-based gene therapy products.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

et al. 2021

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Recombinant adeno-associated virus (rAAV) is a leading platform in human gene therapy.The AAV capsid is composed of three viral proteins (VPs): VP1, VP2, and VP3. To ensure the safety of AAV-based gene therapy products, the stoichiometry of VPs of AAV vector should be carefully monitored. Here, SDS-PAGE, capillary gel electrophoresis (CGE), and liquid chromatography-UV-mass spectrometry (LC-UV-MS) were performed to evaluate the VP components of AAV1, AAV2, and AAV6. Two types of VP3 related components, VP3 variant and VP3 fragment, were identified. The VP3 variant was the N-terminal shorter VP3 of which the translation started at M211, not at the conventional initiation codon, M203. The VP3 variant could be generated by leaky scanning of the first initiation codon of VP3. We also showed that the VP3 variant was identified in a minor peak prior to VP3 in CGE measurement. Meanwhile, the VP3 fragment was the C-terminal cleaved VP3 of which the sequence of VP3 ended at D590 or D626, indicating that cleavage occurred between D590 and P591, or D626 and G627. The cause of the cleavage of the DP or DG sequence was hydrolysis due to low pH of the mobile phase and high temperature of the column oven in the LC system which was necessary to clearly separate the peak of VPs. VP3 fragments detected only in LC-UV-MS in small amount account with less than 3% of total peak area, should be included in the quantification of VP3. Finally, the relationship of VP stoichiometry determined by the above three methods was discussed. From this study, we

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Section: Discussionmentioning

confidence: 71%

Section: Introductionmentioning

confidence: 99%

Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

et al. 2021

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“…If IACE were to be incorporated in sample preparation prior to sample introduction into the capillary or microchip, the entire system could be automated, reduce analysis time, and overcome limitations in poor detection sensitivity. We will emphasize the advantages for the determination of proteoforms when compared with other methods described to enhance the analytical sensitivity of analytes, such as field-amplified sample stacking, large-volume sample stacking, pH-mediated stacking, isotachophoreris (ITP), stacking techniques in micellar electrokinetic chromatography (MEKC), soli-phase extraction, and tagging target analytes with various chromophores and detecting the derivatized substances with sensitive detectors [123,151,152,[173][174][175][176][177][178][179][180][181][182][183][184][185][186][187][188][189][190]. In some instances, IACE can be used in combination with other methods to enhance analytical sensitivity even further.…”

Section: Capillary Electrophoresis As a Useful Technology For The Separation Of Structurally Related Small Molecular-weight Substances Anmentioning

confidence: 99%

Immunoaffinity Capillary Electrophoresis in the Era of Proteoforms, Liquid Biopsy and Preventive Medicine: A Potential Impact in the Diagnosis and Monitoring of Disease Progression

Guzman¹,

Guzman

2021

Biomolecules

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Over the years, multiple biomarkers have been used to aid in disease screening, diagnosis, prognosis, and response to therapy. As of late, protein biomarkers are gaining strength in their role for early disease diagnosis and prognosis in part due to the advancements in identification and characterization of a distinct functional pool of proteins known as proteoforms. Proteoforms are defined as all of the different molecular forms of a protein derived from a single gene caused by genetic variations, alternative spliced RNA transcripts and post-translational modifications. Monitoring the structural changes of each proteoform of a particular protein is essential to elucidate the complex molecular mechanisms that guide the course of disease. Clinical proteomics therefore holds the potential to offer further insight into disease pathology, progression, and prevention. Nevertheless, more technologically advanced diagnostic methods are needed to improve the reliability and clinical applicability of proteomics in preventive medicine. In this manuscript, we review the use of immunoaffinity capillary electrophoresis (IACE) as an emerging powerful diagnostic tool to isolate, separate, detect and characterize proteoform biomarkers obtained from liquid biopsy. IACE is an affinity capture-separation technology capable of isolating, concentrating and analyzing a wide range of biomarkers present in biological fluids. Isolation and concentration of target analytes is accomplished through binding to one or more biorecognition affinity ligands immobilized to a solid support, while separation and analysis are achieved by high-resolution capillary electrophoresis (CE) coupled to one or more detectors. IACE has the potential to generate rapid results with significant accuracy, leading to reliability and reproducibility in diagnosing and monitoring disease. Additionally, IACE has the capability of monitoring the efficacy of therapeutic agents by quantifying companion and complementary protein biomarkers. With advancements in telemedicine and artificial intelligence, the implementation of proteoform biomarker detection and analysis may significantly improve our capacity to identify medical conditions early and intervene in ways that improve health outcomes for individuals and populations.

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“…The method has been validated to be accurate and precise with a linear dynamic range of 8.0 × 10 7 -3.0 × 10 11 vector genomes per mL (vg/mL). The detection limit and quantitation limit were established to be 8.0 × 10 7 vg/mL and 4.2 × 10 8 vg/mL, respectively [30]. LIF is also a very sensitive detection method in micro total analytical systems (µTAS) owing to the good monochromaticity, strong collimation and high optical density of diodepumped solid-state lasers.…”

Section: Designing a Lif Detection Systemmentioning

confidence: 99%

Current State of Laser-Induced Fluorescence Spectroscopy for Designing Biochemical Sensors

Taylor

Lai

2021

Chemosensors

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Laser-induced fluorescence (LIF) has been a valuable analytical technique since the 1970s that has only been made more useful through advances in other scientific fields such as biochemistry. Moreover, advances in laser and detector technology have seen a decrease in LIF detector costs and an increase in their ease of use. These changes have allowed for LIF technology to be widely adopted for various sensor designs in combination with advanced instruments. With advances in biochemistry necessitating the detection of complex metabolites, labelling with fluorescent chemical reagents may be necessary to improve detection sensitivity. Furthermore, advances made in fluorescent labeling technologies have allowed for the use of LIF in the detection of nanoparticles as well as for imaging techniques using nanoparticles as signal amplifiers. This technology has become invaluable in the detection of environmental pollutants, monitoring of biological metabolites, biological imaging, and cancer diagnosis, making it one of the most valuable analytical science techniques currently available.

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Capillary Electrophoresis-Sodium Dodecyl Sulfate with Laser-Induced Fluorescence Detection as a Highly Sensitive and Quality Control-Friendly Method for Monitoring Adeno-Associated Virus Capsid Protein Purity

Cited by 29 publications

References 27 publications

Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

Characterization of Adeno-Associated Virus Capsid Proteins with Two Types of VP3-Related Components by Capillary Gel Electrophoresis and Mass Spectrometry

Immunoaffinity Capillary Electrophoresis in the Era of Proteoforms, Liquid Biopsy and Preventive Medicine: A Potential Impact in the Diagnosis and Monitoring of Disease Progression

Current State of Laser-Induced Fluorescence Spectroscopy for Designing Biochemical Sensors

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