12 Buyers’ Guide 2021


two traces, however, this assumption needs to be verified with further experiments. In addition, the higher temperature denaturation step also caused an apparent resolution decrease between the VP3 and VP3’ peaks, possibly due to decomposition related processes.


The limit of detection (LOD) and limit of quantitation (LOQ) values were assessed for the AAV8 sample with UV detection using the industry standard dilution series method [14]. The LOD was 1 x1012


GC/mL (peak to noise ratio 5) and the LOQ was 5 x1012 GC/mL (peak


to noise ratio 15), with the linear detection response of r2 range of 5 x1011


GC/mL.


=0.9991 in the concentration – 1 x1014


SDS-CGE-LIF detection workflow


While UV detection during the SDS-CGE separation of intact adeno-associated virus capsid proteins provided an adequate quantification limit (as shown above), in-process control during manufacturing would require higher sensitivity, preferably ≤1 x1010


GC/mL (~50 ng/mL), which is the


typical AAV concentration in gene therapy. To increase detection sensitivity, laser induced fluorescent detection was used after fluorophore labelling of the capsid proteins with the 3-2-(furoyl quinoline-2- carboxaldehyde) (FQ) dye. The sample preparation procedure including the FQ labelling reaction and the quenching step took less than one hour. The resulting electropherograms for the AAV8 and AAV2 samples are shown in Figure 3. Again, baseline separation of all three viral capsid proteins was obtained with the VP3 : VP2 : VP1 ratio of 8 : 1 : 1 and 7 : 1 : 1, respectively, i.e., the fluorophore labelling did not change the overall peak distribution profiles.


The detection and quantitation linearities were evaluated as in the UV detection method, but in the 1 x1010


– 1.6 x1014 GC/


mL range. The linear detection response result was r2


values of 1 x1010 for the VP3 peak.


= 0.9989 with the LOD and LOQ GC/mL and 3 x1010


GC/mL


quantification. In this paper, sodium dodecyl sulphate capillary gel electrophoresis was employed for ultrahigh sensitivity AAV capsid protein analysis using UV detection for intact and LIF for fluorophore labelled forms. Both approaches resulted in excellent size separation of the VP1, VP2 and VP3 proteins along with the resolution of a small VP3 impurity peak (VP3’).


Figure 3: SDS-CGE-LIF separation of the AAV2 (trace A, 1 x1010 (trace B, 1 x1010


GC/mL) and AAV8 GC/mL) capsid protein samples labelled with the FQ dye. Conditions


were the same as in Figure 1, but with LIF detection (488 nm excitation, 600 nm emission filter).


The migration time reproducibility for both methods was <0.34% RSD, while the corrected peak area reproducibility was <0.75% RSD for SDS-CGE-UV and < 5% RSD for the SDS-CGE-LIF analysis of the fluorophore labelled capsid proteins. The linearity of detection ranged over two orders of magnitude for UV detection (LOD = 1 x1012


GC/mL) and four orders of magnitude with LIF detection (LOD =1 x1010


GC/mL). Acknowledgment


The authors greatly acknowledge the fruitful discussion with Elliott Jones, Sahana Mollah and Fang Wang.


References


[1.] J.C. Grieger, R.J. Samulski, Methods Enzymol, 507 (2012) 229-254.


[2.] R. dos Santos Coura, N. Beyer Nardi, Genetics and Molecular Biology, 31 (2008) 1-11.


Conclusions


As adeno-associated viruses are more and more extensively considered by the biopharmaceutical industry as delivery vehicles for gene therapy, reliable and quantitative assays are critical for their proper characterisation and impurity


[3.] H.J. Nam, B.L. Gurda, R. McKenna, M. Potter, B. Byrne, M. Salganik, N. Muzyczka, M. Agbandje-McKenna, J Virol, 85 (2011) 11791-11799.


[4.] L. Hajba, A. Guttman, Current Molecular Medicine, 20 (2020).


doi: 10.2174/1566524020999200730181042


[5.] T. Li, T. Gao, H. Chen, P. Pekker, A. Menyhart, A. Guttman, Current Molecular Medicine, 20 (2020).


DOI: 10.2174/1566524020666200915105456


[6.] M. Penaud-Budloo, A. Francois, N. Clement, E. Ayuso, Mol Ther Methods Clin Dev, 8 (2018) 166-180.


[7.] K.M. Van Vliet, V. Blouin, N. Brument, M. Agbandje-McKenna, R.O. Snyder, Methods Mol Biol, 437 (2008) 51-91.


[8.] S. Daya, K.I. Berns, Clin Microbiol Rev, 21 (2008) 583-593.


[9.] A. Srivastava, Curr Opin Virol, 21 (2016) 75-80.


[100.] C.E. Sanger-van de Griend, Electrophoresis, 40 (2019) 2361-2374.


[11.] B. Bosma, F. du Plessis, E. Ehlert, B. Nijmeijer, M. de Haan, H. Petry, J. Lubelski, Gene Therapy, 25 (2018) 415-424.


[12.] L. Lisowski, S.S. Tay, I.E. Alexander, Curr Opin Pharmacol, 24 (2015) 59-67.


[13.] B. Mary, S. Maurya, S. Arumugam, V. Kumar, G.R. Jayandharan, The FEBS Journal, 286 (2019) 4964-4981.


[14.] O. Salas-Solano, B. Tomlinson, S. Du, M. Parker, A. Strahan, S. Ma, Analytical Chemistry, 78 (2006) 6583-6594.


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