Journal: Analytical Chemistry

Article Title: Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

doi: 10.1021/acs.analchem.5c03104

Figure Lengend Snippet: Charge heterogeneity analysis and fractionation of intact AAV9 capsids. (A) Triplicate injections of AAV9 CMV-GFP (blue) and empty AAV9 capsids (orange) are shown in overlay, detected using native fluorescence (excitation = 280 nm, emission = 325 nm, using an 80s exposure). A clear pI difference is seen between full and empty AAV9 capsids. (B) Analysis of area under the curve for triplicate injections of AAV9 CMV-GFP and empty capsids shows reproducible icIEF analysis of AAV capsids. Unfractionated AAV9-CMV-GFP (C) and empty AAV9 (D) were compared to the respective fractionation series. Each AAV9 reference sample was fractionated into five fractions for (E) AAV9-CMV-GFP for (F) empty AAV9 capsids. Fractions are labeled based on their relative pI position to the center of the unfractionated capsids, where A2 is the most acidic peak, M is the middle peak, and B2 is the most basic peak.

Article Snippet: For fractionation, 15 μL of AAV9 CMV-GFP or empty AAV9 capsids (Virovek, 2 × 10 13 VP/mL) was mixed into a final sample solution containing 0.35% methylcellulose, 2.5% Pharmalyte 5–8, 1.5% Pharmalyte 8–10.5, 20% SimpleSol, 25 mM arginine, and pI markers of 7.0 and 8.4.

Techniques: Fractionation, Fluorescence, Labeling

Journal: Analytical Chemistry

Article Title: Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

doi: 10.1021/acs.analchem.5c03104

Figure Lengend Snippet: Fractionated AAV9 capsid protein analysis by Jess CE instrument, as described in  . (A) Comparison of reference samples (diluted 1:800) using an anti-VP1/2/3 antibody for detection. While similar levels of VP1, VP2, and VP3a are seen in the two samples, more VP3 is observed in the empty AAV9 capsids. (B) Comparison of the fractions (diluted 1:20) to reference material shows fractions have similar capsid proteins levels compared to the unfractionated samples. The molecular weight ( M W ) of the samples is calibrated by the M W ladder run in ref channel. (C) Quantitation of the data, represented as a stacked bar graph of the % peak areas of the three capsid proteins and the VP3a variant. (D) Capsid protein analysis of the reference samples and fractions, represented as total proteins present in a 60-mer.

Article Snippet: For fractionation, 15 μL of AAV9 CMV-GFP or empty AAV9 capsids (Virovek, 2 × 10 13 VP/mL) was mixed into a final sample solution containing 0.35% methylcellulose, 2.5% Pharmalyte 5–8, 1.5% Pharmalyte 8–10.5, 20% SimpleSol, 25 mM arginine, and pI markers of 7.0 and 8.4.

Techniques: Comparison, Molecular Weight, Quantitation Assay, Variant Assay

Journal: Analytical Chemistry

Article Title: Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

doi: 10.1021/acs.analchem.5c03104

Figure Lengend Snippet: VP3 deamidation profiling. Fractions of AAV9 CMV-GFP and empty AAV9 capsids were analyzed for VP3 by denatured icIEF-immunoassay. The y -axis is the ratio of deamidated VP3 peak area to amidated VP3 peak area, normalized by the capsid protein levels present in each fraction.

Article Snippet: For fractionation, 15 μL of AAV9 CMV-GFP or empty AAV9 capsids (Virovek, 2 × 10 13 VP/mL) was mixed into a final sample solution containing 0.35% methylcellulose, 2.5% Pharmalyte 5–8, 1.5% Pharmalyte 8–10.5, 20% SimpleSol, 25 mM arginine, and pI markers of 7.0 and 8.4.

Techniques:

Journal: Analytical Chemistry

Article Title: Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

doi: 10.1021/acs.analchem.5c03104

Figure Lengend Snippet: Capsid Content CE-Immunoassay. (A) AAV9 capsid protein analysis and (B) DNA analysis using CE-immunoassay detection of the AAV9 CMV-GFP reference sample. (C) Capsid protein analysis of the fractions and (D) DNA analysis of the fractions. As described in and the same as in , the M W of the samples are calibrated by the M W standard ladder run in the first channel in the method. ( E ) Normalized DNA levels of the capsid fractions. The DNA signal for each sample was divided by the sum peak area of the capsid proteins for the same sample.

Article Snippet: For fractionation, 15 μL of AAV9 CMV-GFP or empty AAV9 capsids (Virovek, 2 × 10 13 VP/mL) was mixed into a final sample solution containing 0.35% methylcellulose, 2.5% Pharmalyte 5–8, 1.5% Pharmalyte 8–10.5, 20% SimpleSol, 25 mM arginine, and pI markers of 7.0 and 8.4.

Techniques:

Journal: Analytical Chemistry

Article Title: Insights into Adeno-Associated Virus Capsid Charge Heterogeneity

doi: 10.1021/acs.analchem.5c03104

Figure Lengend Snippet: CD-MS mass histograms of reference materials (top) for (A) AAV9-CMV-GFP and (B) AAV9-Empty all normalized to 1 for ions detected within the capsid mass range. Beneath each CD-MS mass histogram of the reference are the spectra for each fractionation series, shown from acidic to basic fractions. (C) Statistics from CD-MS mass histograms of reference material and fractions. Full refers to the CMV-GFP genome, while Full+ refers to packaging of the CMV-GFP genome plus other genome content (scDNA of CMV-GFP copy). Although all fractions were buffer exchanged, they might contain trace amount of SimpleSol solvent since it was added in the fractionation sample as described in . The impact of the SimpleSol to the CD-MS results is minimum as shown in Figure S2 .

Article Snippet: For fractionation, 15 μL of AAV9 CMV-GFP or empty AAV9 capsids (Virovek, 2 × 10 13 VP/mL) was mixed into a final sample solution containing 0.35% methylcellulose, 2.5% Pharmalyte 5–8, 1.5% Pharmalyte 8–10.5, 20% SimpleSol, 25 mM arginine, and pI markers of 7.0 and 8.4.

Techniques: Fractionation, Solvent