Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry

Liu, Anita P.; Patel, Shailin K.; Xing, Tao; Yan, Yuetian; Wang, Shunhai; Li, Ning

doi:10.1016/j.jpba.2020.113481

Cited by 39 publications

(87 citation statements)

References 29 publications

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“…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%

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: 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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“…Another study observed Asp isomerization and cyclization, as well as Asn deamidation, on AAV8 capsids (Liu et al, 2020).…”

Section: Deamidation Isomerization Racemization and Formation Of Stable Succinimide For Asparagine And Aspartate Residuesmentioning

confidence: 96%

“…The oxidation of Met residues is a commonly observed PTM. A mass spectrometry study of AAV detected levels as high as 23.9% at M212 on the AAV8 capsids (Liu et al, 2020). It is currently unclear how capsid protein oxidation might impact the potency of AAV-based DPs, although it is possible efficacy may be altered by inhibition of the recognition of cellular receptors.…”

Section: Oxidationmentioning

confidence: 99%

Assessment of quality attributes for adeno‐associated viral vectors

Tustian

Bak

2021

Biotech & Bioengineering

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There is a strong and growing interest in the development and production of gene therapy products, including those utilizing adeno-associated virus (AAV) particles. This is evident with the increase in the number of clinical trials and agency approvals for AAV therapeutics. As bioproduction of AAV viral vectors matures, a quality by design (QbD) approach to process development can aid in process robustness and product quality. Furthermore, it may become a regulatory expectation. The first step in any QbD approach is to determine what physical, chemical, biological, or microbiological property or characteristic product attributes should be controlled within an appropriate limit, range, or distribution to ensure the desired product quality.Then predefined goals are set to allow proactive process development to design in quality. This review lists typical quality attributes used for release testing of AAV viral vectors and discusses these and selected attributes important to extended characterization studies in terms of safety, efficacy, and impact upon the patient immune response. K E Y W O R D S adeno-associated virus, analytics, critical quality attribute, gene therapy, quality by design 1 | INTRODUCTION Adeno-associated virus (AAV)-based viral vectors have shown enormous potential in recent years for use in gene therapy as a therapeutic modality to treat a wide range of genetic diseases, with more than 150 clinical trials specific to AAV (Penaud-Budloo et al., 2018).AAV treatments first gained regulatory approval in the European Union, with alipogene tiparvovec's (Glybera, uniQure) approval in 2012 for lipoprotein lipase deficiency. The first US regulatory ap-

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“…19,20 Zhang 21 , Pedrali 22 , Lauber 23 and Periat 24 et al discussed the unique selectivity of HILIC in the separation of glycoforms of sub-units and intact glycoproteins. These results have drawn attention to the use of HILIC-MS, with recent applications reporting high-resolution separations of neoglycoproteins 25 , biopharmaceuticals [26][27][28][29] , biotechnological products 30 and serum immunoglobins 31 . For these proteins, HILIC resolved proteoforms that would co-elute in RPLC.…”

Section: Introductionmentioning

confidence: 99%

Poly(acrylamide-co-N,N’-methylene bisacrylamide) monoliths for high peak capacity hydrophilic interaction chromatography high-resolution mass spectrometry of intact proteins at low trifluoroacetic acid content

Passamonti

Roos

Schoenmakers

et al. 2021

Preprint

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In this study, we optimized a polymerization mixture to synthesize polyacrylamide-co-N,N-methylene bisacrylamide monolithic stationary phases for hydrophilic-interaction chromatography (HILIC) of intact proteins. Thermal polymerization was performed, and the effects of varying the amount of crosslinker and the porogen composition on the separation performance of the resulting columns were studied. The homogeneity of the structure and the different porosities were examined through scanning electron microscopy. Further characterization of the monolithic structure revealed a permeable (K_f between 2.5 × 10-15 and 1.40 × 10-13 m2) and polar stationary phase suitable for HILIC. The HILIC separation performance of the different columns was assessed using gradient separation of a sample containing four intact proteins, with the best performing stationary phase exhibiting a peak capacity of 51 in a gradient of 25 min. Polyacrylamide-based materials were compared with a silica-based particulate amide phase (2.7 μm core-shell particles). The monolith has no residual silanol sites and, therefore, fewer sites for ion-exchange interactions with proteins. Thus, it required lower concentrations of ion-pair reagent in HILIC of intact proteins. When using 0.1% of trifluoroacetic acid (TFA) the peak capacities of the two columns were similar (31 and 36 for the monolithic and packed column, respectively). However, when decreasing the concentration of TFA to 0.005%, the monolithic column maintained its separation performance and selectivity (peak capacity 26), whereas the packed column showed greatly reduced performance (peak capacity 7), lower selectivity, and inability to elute all four reference proteins. Finally, using a mobile phase containing 0.1% formic acid and 0.005% TFA the HILIC separation on the monolithic column was successfully hyphenated with high-resolution mass spectrometry. Detection sensitivity for protein and glycoproteins was increased and the amount of adducts formed was decreased in comparison with separations performed at 0.1% TFA.

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Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry

Cited by 39 publications

References 29 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

Assessment of quality attributes for adeno‐associated viral vectors

Poly(acrylamide-co-N,N’-methylene bisacrylamide) monoliths for high peak capacity hydrophilic interaction chromatography high-resolution mass spectrometry of intact proteins at low trifluoroacetic acid content

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