2.3 Anion Exchange Chromatography
To prepare the AEX load, 1.5 mL of affinity eluate was diluted 8-fold (to 15 mL) with 20 mM Bis-tris-propane (BTP) to adjust pH to 9.0 and to reduce the load conductivity to approximately 4 mS/cm. The AEX experiments were performed on a 1-mL strong-base, CIMmultus-QA monolith (Lot: 20-GE03-005-001A-P1-2A-8-MI-2-MQ17, BIA Separations) with a nominal channel size of 2 μm, using three basic buffering formats:
Linear-gradient Elution (NaCl, with and without MgCl2) – 10 mL of the diluted affinity eluate was loaded on AEX media pre-equilibrated with pH-matched equilibration buffer (NaCl buffer: 20 mM BTP, 25 mM NaCl, 0.001% (w/v) Pluronic F-68, pH 9.0). After washing with 10 column volumes (CV) of equilibration buffer, the column-bound material was eluted with a linear gradient from 25 to 182.5 mM NaCl generated over 90 CVs. AEX chromatography experiments utilizing Mg-containing buffers were performed identically except for the constituents of the equilibration (Buffer A) and end-point buffer (Buffer B) contained 2 mM MgCl2. AEX chromatography experiments utilizing 10% (v/v) isopropyl alcohol buffers were performed identically except for the constituents of the Buffer A and Buffer B contained 10% (v/v) isopropyl alcohol. The eluate was collected in fractions and fractions corresponding to “Empty” and “Full” peaks were pooled accordingly.
Linear-gradient Elution (QA, with and without MgCl2) – 10 mL of the diluted affinity eluate was loaded on AEX media pre-equilibrated with pH-matched equilibration buffer (QA buffer: 20 mM BTP, 25 mM QA, 0.001% (w/v) Pluronic F-68, pH 9.0). After washing with 10 CVs of equilibration buffer, the column-bound material was eluted with a linear gradient from 25 to 340 mM QA generated over 90 CVs. AEX chromatography experiments utilizing Mg-containing buffers were performed identically except for the constituents of the Buffer A and Buffer B contained 0.2, 1, 5, or 10 mM MgCl2. The eluate was collected in fractions and fractions corresponding to “Empty” and “Full” peaks were pooled accordingly.
Isocratic Wash and Elution – 10 mL of the diluted affinity eluate was loaded on AEX media pre-equilibrated with pH-matched equilibration buffer (TEA-Ac buffer: 20 mM BTP, 25 mM TEA-Ac, 0.001% (w/v) Pluronic F-68, pH 9.0). After washing with 10 CVs of equilibration buffer, the column was washed with 190–200 mM TEA-Ac and the corresponding concentration of MgCl2 (either 0, 1, or 5 mM). The QA buffer was then removed in a second wash step (NaCl buffer: 20 mM BTP, 25 mM NaCl, 2 mM MgCl2, 0.001% (w/v) Pluronic F-68, pH 9.0). Elution was carried out in two manners: 1) a linear gradient was generated over 30 CVs from 25 to 200 mM NaCl; or 2) a step elution was employed using a buffer containing 20 mM BTP, 117 mM NaCl, 2 mM MgCl2, 0.001% (w/v) Pluronic F-68, pH 9.0. In gradient operation, the eluate was collected in fractions and fractions corresponding to “Empty” and “Full” peaks were pooled accordingly. In isocratic mode, the full elution peak was collected, which typically corresponded to a 3 CVs pool.
Following elution, the column was then washed with 10 CVs of a high-salt strip solution (20 mM BTP, 2 M NaCl, pH 9.0), followed by 10 CVs of a sanitation solution (3 M NaCl, 1 M NaOH), and subsequently 10 CVs of a column-storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH 7.5). The column is refrigerated in 2–8 °C after use.
2.4 Analysis of Chromatography Fractions
Quantitative Polymerase Chain Reaction (qPCR) – The quantity of encapsulated vector genome was determined using a qPCR method applicable to rAAV samples that contain the SV40 Poly-A sequence. To quantify only encapsulated vector genome, rAAV samples were treated with salt-activated nuclease (SAN) to remove non-encapsulated polynucleotides. The SAN were then inactivated and the rAAV samples were treated with Proteinase K to digest the capsid protein. This is followed by qPCR using the primer/probe set that is specific to the SV40 Poly-A sequence of the rAAV genomes in the rAAV therapeutic products prepared at Ultragenyx. The qPCR is performed on an Applied Biosystems Flex 7 instrument and quantification is made using the QuantStudio 7 software. For a given experiment (e.g., alkyl series study or anion series study) the qPCR measurements were performed on the same multi-well plate.
Gyrolab xPand – The quantity of rAAV capsid particles was determined using the Gyrolab xPand (Gyros Protein Technologies) nanoliter-scale, high-throughput immunoassay system, in combination with the Gyrolab AAVX Titer kit (Gyros Protein Technologies, P0020695). The AAVX ligand included in Gyrolab AAVX Titer kit is based on the selective affinity ligands developed with CaptureSelect technology (Thermo Fisher Scientific). Typically, a dilution plate containing rAAV samples, calibrants required to generate a standard curve, controls, and wash buffers are prepared ahead of time. The xPand’s liquid handling system transfers these solutions into microfluidic channels that contain biotin-labelled CaptureSelect AAVX ligand functionalized on streptavidin-coated particles. As the solutions pass through the channels, rAAV capsid particles are bound to the capture media. Then, fluorescent-conjugated material is passed across the channel to generate a fluorescence signal in the form of response units. The signal obtained from the calibration solutions are then fitted with a four-parameter logistic regression to generate a standard curve to convert response units to particle titer units [vp mL−1]. For a given study (e.g., alkyl series study or anion series study) particle titer measurements were performed on the same Gyrolab disc.
Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) – The capsid content in rAAV preparations and AEX elution fractions were quantified by SV-AUC using absorption optics at 230 nm. SV-AUC was performed using the ProteomeLab LX-A analytical ultracentrifuge from Beckman Coulter (Indianapolis, IN). Samples were loaded into AUC cells equipped with sapphire windows and 12 mm double-sector charcoal-filled EPON centerpieces. 420 μL of sample was loaded in the sample sector, whereas 430 μL of the buffer matrix was loaded in the reference sector. AUC cells containing the samples were equilibrated at 20 °C for 1.5 h before the rotor was brought up to 12,000 rpm. UV scans were collected at 230 nm every 20 s at a radial step-size of 3 μm. Sedimentation coefficients and relative amounts of each species present in solution were determined by processing the data with SEDFIT (v15.01b) using a c(s) model. The empty, intermediate, and full species were identified using established reference sedimentation coefficients.[34]
3. RESULTS
3.1 Effect of Quaternary Ammonium Salt in Preparative AEX Chromatography
The AEX salt LGE runs were performed on CIMmultus-QA column with either baseline condition (using NaCl) and QA condition (using tetramethylammonium chloride, TMAC). As shown in Figure 1A and 1B , in both conditions, the empty peak elutes earlier than the full peak, which is then followed by a third peak (attributed as empty capsids or capsid debris,[23]). As TMAC requires a higher ionic strength (191 mM) to elute rAAV than that of NaCl (124 mM), TMAC salt is considered a weaker eluent than NaCl. In terms of chromatographic separation, the AEX run with TMAC has higher empty and full peak resolution (1.06) than that of the NaCl run (0.75), which is consistent with a previous report.[21] However, the work from Yang et al . was conducted using 0.1-mL CIMac column with analytical loading and high operational pressure to ensure high resolution. In contrast, we employed a 1-mL CIMmultus-QA column with preparative loading and low operational pressure (<50 psi). As 1-mL CIMmultus-QA column is a qualified scale-down model for up to 8000-mL CIMmultus-QA (internal data, not shown), the AEX method established in this work is appropriately scalable.