Presentation Information
[4DSP-13]Towards efficient chromatography-based downstream process of adeno-associated virus vector particles using mechanistic models
○Shuichi Yamamoto1,2 (1. Yamaguchi University (Japan), 2. Manufacturing Technology Association of Biologics(MAB) (Japan))
Keywords:
chromatography,AAV,mechanistic model,anion-exchange chromatography
Bio-nanoparticles (BNPs) such as viruses, virus-like particles, exosomes (extracellular vesicles), RNAs and DNAs are important bio-materials as drugs, vaccines or vectors for gene and cell therapy. Although chromatography is essential for the downstream process (DSP) of BNPs, it is not easy to develop the efficient chromatography process due to the large size of BNPs, which lowers the mass transfer rate and the binding capacity.
Our national consortium (MAB) developed a DSP for purifying adeno-associated virus (AAV) vector particles from the suspended cell culture liquid. After cell lysis and enzymatic fragmentation of DNA, the liquid was clarified with membranes. Then, a capture step was carried out with an affinity chromatography column. As AAV particles recovered from the capture chromatography column contains both empty (uncomplete) and full (complete) particles, the full (F) particles have to be separated from the empty particles (E) during the polishing chromatography step. The full particle separation (EF separation) by anion-exchange chromatography (AIEC) was carried out with very large pore AIEC resins.
In this paper the EF separation process by AIEC was analyzed by using mechanistic models as it is an inefficient process. Linear gradient elution (LGE) experiments of AAV particles at various gradient slopes and flow-rates were carried out with a small AIEC column (0.3 mL). The two parameters were obtained by the Yamamoto method (the normalized gradient slope vs. the peak retention salt concentration). These data were employed for the numerical simulation of EF particle separations. The peak salt concentrations of AAV particles were much lower than the values compared with the standard proteins. The number of binding site values of AAV particles to AIEC were also not so high (<5) compared with typical values for proteins.
The numerical simulations by the mechanistic model were carried out in order to understand the EF particle separation mechanism, and to optimize the separation process. A 333 fold-scale up (from 0.3 mL to 100 mL) run for a 50 L cell culture liquid was successfully carried out with different gradient conditions, which was well described by the model simulations. The purity was over 90% and the recovery was over 80%.
As the AIEC resin used in this study has a very large pore (>1 μm), the perfusion effect may occur at high flow velocities. LGE curves of standard proteins as well as AAV particles did not change with the flow velocity. These results confirmed the perfusion effect.
Optimization of EF separation by LGE with AIEC was carried out by using numerical model simulations in order to reduce buffer consumption and separation time while the purity is maintained.
Our national consortium (MAB) developed a DSP for purifying adeno-associated virus (AAV) vector particles from the suspended cell culture liquid. After cell lysis and enzymatic fragmentation of DNA, the liquid was clarified with membranes. Then, a capture step was carried out with an affinity chromatography column. As AAV particles recovered from the capture chromatography column contains both empty (uncomplete) and full (complete) particles, the full (F) particles have to be separated from the empty particles (E) during the polishing chromatography step. The full particle separation (EF separation) by anion-exchange chromatography (AIEC) was carried out with very large pore AIEC resins.
In this paper the EF separation process by AIEC was analyzed by using mechanistic models as it is an inefficient process. Linear gradient elution (LGE) experiments of AAV particles at various gradient slopes and flow-rates were carried out with a small AIEC column (0.3 mL). The two parameters were obtained by the Yamamoto method (the normalized gradient slope vs. the peak retention salt concentration). These data were employed for the numerical simulation of EF particle separations. The peak salt concentrations of AAV particles were much lower than the values compared with the standard proteins. The number of binding site values of AAV particles to AIEC were also not so high (<5) compared with typical values for proteins.
The numerical simulations by the mechanistic model were carried out in order to understand the EF particle separation mechanism, and to optimize the separation process. A 333 fold-scale up (from 0.3 mL to 100 mL) run for a 50 L cell culture liquid was successfully carried out with different gradient conditions, which was well described by the model simulations. The purity was over 90% and the recovery was over 80%.
As the AIEC resin used in this study has a very large pore (>1 μm), the perfusion effect may occur at high flow velocities. LGE curves of standard proteins as well as AAV particles did not change with the flow velocity. These results confirmed the perfusion effect.
Optimization of EF separation by LGE with AIEC was carried out by using numerical model simulations in order to reduce buffer consumption and separation time while the purity is maintained.
Comment
To browse or post comments, you must log in.Log in