Journal: ACS omega

Article Title: Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy.

doi: 10.1021/acsomega.1c01443

Figure Lengend Snippet: Figure 1. Scheme of the two analytical focuses of this study. (a) Empty and filled AAV8 VLP particles are analyzed via nES GEMMA and AFM. (b) AF4 liquid-phase separation enables AAV8 VLP oligomer analysis by means of nES GEMMA and AFM after fractionation.

Article Snippet: The capillary is manually cut and tapered with a homebuilt grinding machine based on the work of Tycova et al.15 AF4 experiments were performed on an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA, auto sampler, pump, and detector), which consisted of an auto sampler, HPLC pumps, an AF4 separation device (Wyatt Technology, Santa Barbara, CA, USA), and a fluorescence detector (λex/em = 280/340 nm).

Techniques: Gas Phase Electrophoretic Molecular Mobility Analysis, Fractionation

Journal: ACS omega

Article Title: Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy.

doi: 10.1021/acsomega.1c01443

Figure Lengend Snippet: Figure 2. Implications of the buffer exchange treatment. (a) nES GEMMA spectra of the sample before (black trace) and after (red trace) desalting. Nonvolatile salts and other components (3−15 nm) are drastically reduced. The inset shows the shift toward small EM diameter of the AAV8 VLP’s peak. (b) AF4 fractogram of the sample with (red trace) or without (blue trace) a buffer exchange procedure. Both nES GEMMA spectra (a) and (b) show reduced signal intensity for the AAV8 VLP’s peak due to sample loss during the desalting treatment.

Article Snippet: The capillary is manually cut and tapered with a homebuilt grinding machine based on the work of Tycova et al.15 AF4 experiments were performed on an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA, auto sampler, pump, and detector), which consisted of an auto sampler, HPLC pumps, an AF4 separation device (Wyatt Technology, Santa Barbara, CA, USA), and a fluorescence detector (λex/em = 280/340 nm).

Techniques: Gas Phase Electrophoretic Molecular Mobility Analysis

Journal: ACS omega

Article Title: Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy.

doi: 10.1021/acsomega.1c01443

Figure Lengend Snippet: Figure 4. Stressing procedure and fractionation of the sample. (a) AF4 fractogram of control (blue trace) and heat/mechanical-stressed (magenta trace) sample. (b) The red vertical lines mark the collected fractions: 16.5−19.2 min monomer fraction; 19.2−20.6 min dimer and trimer fraction; 20.6−25.0 min higher oligomeric fraction.

Article Snippet: The capillary is manually cut and tapered with a homebuilt grinding machine based on the work of Tycova et al.15 AF4 experiments were performed on an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA, auto sampler, pump, and detector), which consisted of an auto sampler, HPLC pumps, an AF4 separation device (Wyatt Technology, Santa Barbara, CA, USA), and a fluorescence detector (λex/em = 280/340 nm).

Techniques: Fractionation, Control

Journal: ACS omega

Article Title: Adeno-associated Virus Virus-like Particle Characterization via Orthogonal Methods: Nanoelectrospray Differential Mobility Analysis, Asymmetric Flow Field-Flow Fractionation, and Atomic Force Microscopy.

doi: 10.1021/acsomega.1c01443

Figure Lengend Snippet: Figure 5. nES GEMMA spectra and AFM images of the three fractions collected with the AF4 technique (see Figure 4b). (a, b) Monomer (M) fraction; (c, d) dimer (D) and trimer (T) fraction; and (e, f) higher oligomeric (O) fraction. nES GEMMA spectra compare signals obtained for VLP-containing samples (red traces) and blanks recorded for a NH4OAc blank (black traces), respectively. nES GEMMA signals below 20 nm EM diameter putatively correspond to incompletely removed, aggregating AF4 buffer components. In (f), the arrow indicates an AAV8 VLP higher oligomer with a height of 35 nm.

Article Snippet: The capillary is manually cut and tapered with a homebuilt grinding machine based on the work of Tycova et al.15 AF4 experiments were performed on an Agilent 1200 system (Agilent Technologies, Santa Clara, CA, USA, auto sampler, pump, and detector), which consisted of an auto sampler, HPLC pumps, an AF4 separation device (Wyatt Technology, Santa Barbara, CA, USA), and a fluorescence detector (λex/em = 280/340 nm).

Techniques: Gas Phase Electrophoretic Molecular Mobility Analysis