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11


SDS-CGE is one of the popular AAV capsid protein analysis methods in the cell and gene therapy sector. SDS-CGE offers automated protein separation with excellent resolution and quantitation capabilities [10]. The method can utilise both UV and laser induced fluorescent (LIF) detectors. The former does not require pre-separation labelling but has lower sensitivity. LIF detection, on the other hand, necessitates fluorophore labelling of the capsid proteins but features several order of magnitude higher sensitivities. In this paper two SDS- CGE workflows are introduced using 1) UV detection for unlabelled capsid proteins and 2) LIF detection after covalent dye labelling.


Experimental


Materials: Sodium dodecyl sulphate, methanol, N-ethylmaleimide (NEM) and 2-mercaptoethanol were obtained from Sigma-Aldrich (St. Louis, MO, USA). The Amicon Ultra-0.5 Centrifugal Filters with 10,000 and 30,000 NMWL were purchased from EMD Millipore (Billerica, MA, USA). The ATTO-TA FQ (3-2-(furoyl quinoline- 2-carboxaldehyde) Amine-Derivatisation Kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The SDS-MW Analysis Kit was from SCIEX (Framingham, MA, USA) including the SDS-MW gel buffer and the SDS-MW sample buffer of 100 mM Tris-HCl (pH 9.0) with 1% SDS. Packaged AAV2 of pAV-CMV-GFP (titre 2.24 x1013


GC/


mL, (genome copies per millilitre), AAV8 of pAV-CMV-GFP (titre 3.99 x1013


GC/mL) and


packaged AAV8 of pAV-CMV-GFP (titre 1.57 X1014


GC/mL) were purchased from Vigene Biosciences (Rockville, MD, USA). The three samples were kept in Phosphate Buffered Saline (PBS, pH 7.5) with 0.001% pluronic F68 storage solution and were diluted in the same storage buffer to the desired concentrations. This latter was added to minimise sticking of AAV to hydrophobic plastic surfaces.


Sample Preparation: For UV absorbance detection, 5 µL of AAV solution (salt concentration of < 40 mM) was mixed with 5 µL of 1% SDS and 1.5 µL of 2-mercaptoethanol and incubated for 10 min at 50 °C to fully denature the sample proteins. After the denaturation step, 90 µL of DI water was added to the sample mixture. Buffer exchange was necessary if the salt concentration in the AAV sample was higher than that of 40 mM. For FQ labelling and LIF detection, 10 µL of AAV sample solution was mixed with 1.2 µL of 4% SDS in 150 mM NEM solution in a microfuge tube and incubated for 5 minutes at 70 °C followed by mixing with 1.5 µL of 2.5 mM FQ dye working solution and 1 µL of 30 mM


KCN solution. The labelling reaction mixture was incubated for 10 minutes at 70°C. The reaction was quenched by the addition of 28 µL of 1% SDS, kept for an additional 5 min at 70°C. After the quenching step, 20 µL of DI water was added to the diluted reaction mixture. The labelled samples were immediately used for SDS-


Figure 2. Effect of the denaturation temperature on the stability of the AAV8 capsid proteins. Separation conditions were the same as in Figure 1.


CGE-LIF separation. Capillary Electrophoresis


The PA 800 Plus Pharmaceutical Analysis system was equipped with UV and LIF detectors. For all separations a 500 V/cm electric field strength was applied. The EZ-CE pre-assembled capillary cartridge (bare fused-silica, 50 µm I.D., 30 cm total length, 20 cm effective length, SCIEX) was filled with the polymer based SDS-MW gel-buffer system. Detection: UV: 214 nm, LIF: 488 nm excitation wavelength with a 600 nm / 80 nm emission bandpass filter. The separation temperature was set to 25ºC. Stacking injection: water pre-injection for 0.4-0.6 min at 20 psi followed by sample injection for 1 min at 5-10 kV. Data acquisition, processing and analysis were performed employing the 32 Karat Software 10 package.


Results and Discussion


Various serotype adeno-associated viruses are recently becoming one of the most frequently utilised gene delivery vehicles. Therefore, their purity analysis is of high importance, both from manufacturing and regulatory points of views. The sodium dodecyl sulphate capillary gel electrophoresis workflows introduced in this paper for high sensitivity purity analysis of the capsid proteins utilise both UV and laser induced fluorescent detection, this latter requiring covalent fluorophore tagging.


SDS-CGE-UV detection workflow


First, the AAV8 and AAV2 serotypes were analysed by SDS-CGE using UV detection in the 1012


- 1014 GC/mL concentration range. The upper (A) and lower (B) traces in Figure 1


depict the separation of the AAV8 and AAV2 serotypes, respectively. As one can observe, baseline separation of all three capsid proteins was obtained for both sample types. The VP3 : VP2 : VP1 ratio for the AAV8 sample was approximately 8 : 1 : 1, while for the AAV2 sample it was 7 : 1 : 1. Albeit, the theoretical ratio is 10 : 1 : 1, in practice the ratios do not necessarily match to that exactly. Multiple factors may affect the VP ratios, such as the recombination design of the viral proteins for different tropism, the production process conditions, etc. [11, 12]. The slight differences observed in the ratio measurements could influence the shape of the two viruses examined.


In both traces a small peak is visible in front of the VP3 peak (depicted as VP3’), which is probably a small portion of the VP3 capsid protein with altered post translational modification as suggested in [13]. The migration time and relative peak area reproducibility values were less than 0.34% RSD and 0.75% RSD (n=8), respectively.


One of the important aspects of SDS- CGE sample preparation is the incubation temperature during the denaturation step. This process is usually accomplished at 90ºC for 3-5 minutes to ensure full denaturation of the proteins. However, the temperature should be reduced for sensitive proteins to avoid possible decomposition during the denaturation step. Figure 2 compares the SDS-CGE-UV traces of the AAV8 serotype sample after 10 minutes of 90ºC (upper trace) and 70ºC (lower trace) incubation. As can be seen, the higher temperature incubation resulted in the appearance of some decomposition products denoted as D1 and D2 in the upper electropherogram. These decomposition products may have originated from the VP3 protein since that peak appeared to decrease between the


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