Technical note More purification insight from less lentiviral vector sample: automated MiMode PuraBead® screening for LV polishing Demonstrated using the Beckman Coulter Biomek® i7 Workstation with scale verification in 200 µL RoboColumns®
Low-volume, automated plate screening was used to evaluate mixed-mode chromatography conditions for lentiviral vector polishing. In this workflow, run on the Beckman Coulter Biomek® i7 Workstation, the strongest recovery-clearance balance was observed with HX1 under the selected screening conditions. Verification in 200 µL RoboColumns® under the selected condition produced 77% lentiviral vector recovery and 90% DNA clearance, supporting progression from plate screening to further scale-verification studies.
Key takeaways: • Low-volume plate screening enabled parallel comparison of mixed-mode resins and buffer conditions using limited LV sample volume, helping PD teams generate more insight before committing material to larger-format studies. • The screen identified a preferred polishing condition that balanced LV recovery with residual DNA clearance, providing a data-supported candidate for follow-on development. • Verification in a 200 µL column format produced 77% LV recovery and 90% DNA clearance, supporting translation from plate screening to development-scale verification. • Automated execution reduced total workflow time from 4.5 hours manually to 3.5 hours while reducing hands-on intervention. • Repeatability testing showed well-to-well variability below 15% RSD for the plate formats evaluated, supporting confidence in screening consistency.
Introduction Interest in lentiviral vectors is expanding beyond ex vivo cell therapy into broader therapeutic applications, including emerging in vivo approaches. As these applications progress, downstream purification strategies must deliver stronger impurity reduction while preserving infectious vector activity. This is particularly challenging for lentiviral vectors, where product fragility, limited starting material and complex
impurity profiles place pressure on process development teams to identify suitable polishing conditions quickly and with minimal sample consumption. Residual host cell DNA remains a key impurity to control during lentiviral vector purification. Chromatography-based polishing can contribute to DNA reduction, but resin selection and buffer conditions must be carefully balanced to avoid unacceptable losses in functional vector recovery. For this reason, high-throughput, low-volume screening tools are valuable early in development: they allow teams to compare resin chemistries and buffer design space before committing scarce vector material to larger-format verification runs. MiMode PuraBead® 96-well plates provide a compact, automation-ready format for evaluating mixed-mode chromatography conditions using only 20 µL resin per well. When paired with an automated liquid-handling workstation, the workflow can reduce manual handling and analytical preparation and increase the number of conditions evaluated from limited LV sample volumes. This creates a practical bridge from early screening to verification: more resin and buffer conditions can be assessed using less high-value LV material, supporting better-informed polishing decisions before moving into larger formats. This application note demonstrates an automated platescreening workflow for LV polishing using the Beckman Coulter Biomek® i7 Workstation. The study identifies HX1 with 0.4–0.8 M arginine as the best balance of LV recovery and residual DNA clearance in the plate screen, then verifies the selected condition in 200 µL RoboColumns®, where the workflow achieved 77% LV recovery and 90% DNA clearance. The results show the workflow’s ability to identify promising polishing conditions, quantify the recovery-clearance trade-off and inform the next stage of scale-up or process confirmation.
Workflow overview
Key results and benefits
MiMode PuraBead 96-well plates were used to optimize a polishing workflow for removing residual DNA from an LV-rich post-capture fraction. The workflow was automated on a Beckman Coulter Life Sciences Biomek® i7 Liquid Handling Workstation to demonstrate compatibility with liquid-handling platforms, and the selected HX1 condition was then verified using 200 µL RoboColumns® with 20 mM Tris, 20 mM MgCl2, 0.4 M arginine, pH 7.2.
MiMode® resin and process-condition screen
®
A separate repeatability assessment was included to evaluate well-to-well variability in the MiMode® plates independently of the TU and PicoGreen assay variability. Model standard proteins were used to assess whether the plate format delivered consistent recovery across replicate wells.
The resin and process-condition screen evaluated four mixedmode resins across multiple arginine concentrations using limited LV sample volume. This allowed resin selection and buffer-condition assessment within the same automated plate workflow before committing material to larger-format testing. HX1 in the 0.4–0.8 M arginine range provided the strongest balance of LV recovery and residual DNA clearance, supporting its selection for follow-on polishing development and scale verification. Figure 3 shows how the MiMode® screening workflow was connected to downstream sample analysis. Automated steps, shown in green, were preceded by manual reagent and labware preparation, including LV production and buffer preparation.
Figure 1: Automated MiMode® screening workflow and main consumables. A Automation workstation with integrated positive pressure capability. B MiMode PuraBead® Mixed-H 96well plate. C MiMode PuraBead® HX1 200 µL RoboColumns® for scale verification. Figure 3: Screening and downstream analysis flowchart. TU stands for transduction unit assay.
Figure 2: Resin layout; MiMode PuraBead® Mixed-H 96-well plate.
Figure 4: Distribution of buffers in the product selection screen. (A) Buffers in a quarter modular reservoir. (B) Layout of buffers in half a MiMode PuraBead® Mixed-H 96-well plate. (C) Concentration of arginine used in each well. Samples were measured in quadruplicate.
Figure 5 shows the recovery of LV and clearance of DNA. Comparison of the LV recovery values across the arginine concentration range (Figures 5A and 5B) identify an improvement of > 20% at 0.4 M (in blue) and 0.8 M arginine (in yellow) over the 0.2 M condition (in purple). This applied to every resin except for HX2 where recoveries were similar throughout the arginine concentration range. The highest recoveries, at 0.4 M and 0.8 M arginine, were seen for HL4 (84% and 82%, respectively) and HX1 (77% and 75%, respectively). DNA clearance did not show a clear dependence on arginine concentration across the conditions tested. Average DNA clearance was highest for HX1 (86%), followed by HL3 (71%) and HX2 (57%), while HL4 showed no measurable DNA removal under these conditions. The best balance between LV recovery and DNA clearance was achieved with HX1 in the 0.4–0.8 M arginine range, identifying HX1 as the preferred resin for further process investigation and scale verification.
MiMode® HX1 process-design investigation: salt screen Salt additives were evaluated on MiMode PuraBead® HX1 to explore whether LV recovery could be improved further while maintaining DNA clearance. The results showed that selected low-concentration salt additives influenced LV recovery under the tested conditions, but the effect was condition-specific and did not establish a broadly optimized salt-additive condition.
Figure 5: Purification performance of the resins in the MiMode PuraBead® Mixed-H 96-well plate. (A) LV recovery. (B) Average LV recovery over the sample replicates. (C) DNA clearance. (D) Average DNA clearance over the sample replicates. A red square identifies HX1 as the selected product. Compared with the no-salt condition, 0.2 M acetate and chloride salts produced a modest increase in LV recovery, while calcium chloride and sodium iodide substantially reduced LV recovery in the non-bound fraction. These results indicate that salt additives can affect LV recovery on HX1, but the benefit was specific to the tested salt identity and concentration. Although salt additives produced marked differences in LV recovery, DNA removal was similar across the conditions tested.
Figure 6: Salt screen on the MiMode PuraBead® HX1 96-well plate. (A) Layout on a half plate colored in gradient from darker (highest recovery) to white (lowest recovery). (B) Relative recovery of LV against the no added salt condition.
Automation benefits of the MiMode® workflow The workflow demonstrates how compact plate-based screening can be integrated with automated downstream analytical preparation while maintaining short overall process times. In this study, automation supported consistent execution, parallel condition screening and structured sample preparation for downstream analysis. Method
Figure 8: Automated vs. manual times.
Automated
Manual
5 min
5 min
1h 40 min
1 h 20 min
Reagent/labware prep
5 min
20 min
TU assay
40 min
1h
Reagent/labware prep
5 min
17 min
PicoGreen assay
55 min
1 h 25 min
Total
3.5 h
4.5 h
Reagent/labware prep *Screening
* Note: screening time includes 1 hour plate shaking time of the sampleresin mixture at 1100 rpm.
Table 1: Automated vs. manual times.
Reducing manual intervention during sample loading, incubation, pressure-driven processing and analytical preparation can help teams manage higher screening demand with less repetitive handling.
Scale verification of the selected MiMode® condition in RoboColumns® The selected HX1 condition was verified in 200 µL RoboColumns® to assess whether the MiMode® plate result translated to a larger development-scale format. RoboColumns® verification using 20 mM Tris, 20 mM MgCl2, 0.4 M arginine, pH 7.2 produced 77% LV recovery and 90% DNA clearance, closely matching the MiMode® plate screen and supporting the use of MiMode® plates for identifying conditions suitable for further scale verification.
Figure 7: Process chart for the product selection screen. (A) Automated process. Note, activities in parenthesis were done by hand. (B) Manual process. LV RoboColumns®
DNA
HCP
Concentration (TU/mL)
Recovery (%)
Concentration (ng/mL)
Clearance (%)
Concentration (ng/mL)
Clearance (%)
Load
NB
Load
NB
NB
Load
NB
NB
451
15
87
6794
490
71
776
14
93
8346
428
80
436
11
90
7840
305
84
NB
1
1.2E+08 2.3E+07
81
2
1.1E+08 2.3E+07
80
3
1.1E+08 1.9E+07
70
Av.
77
Table 2: RoboColumns® performance parameters.
RSD
7.7
Av.
90
RSD
3.3
Av.
RSD
78
8.6
MiMode® plate repeatability investigation Repeatability testing showed consistent well-to-well performance across the MiMode® plate formats evaluated. In the Mixed-H plate, BSA recovery across 24 replicates per resin produced coefficients of variation in the 11–14% range. In the single-resin HL4 plate, IgG recovery across 96 replicates showed 6.4% well-to-well variability, supporting confidence in screening consistency.
Conclusion The automated plate workflow provides a low-volume format for LV polishing development, allowing resin and buffer-condition options to be evaluated while conserving valuable vector material. In this study, the selected polishing condition delivered the strongest balance of LV recovery and residual DNA clearance, with 200 µL column-format verification showing 77% LV recovery and 90% DNA clearance. Automation supported consistent plate processing, parallel condition screening and standardized sample preparation for downstream analysis. Together, these results demonstrate a practical route from high-throughput screening to further scale-verification studies, helping process-development teams select polishing conditions with a clearer data-driven rationale.
Experimental workflow and methods
Figure 9: Recovery of BSA. (A) Recovery expressed in µg. (B) Recovery expressed in percentage. Columns on the right show the identity of the resin, the average recovery and the coefficients of variation. Recovery values are colored in gradient from high (dark shades) to low (light shades). In a second experiment, the single-resin MiMode PuraBead® HL4 96-well plate (one resin, 96 replicates) was evaluated with IgG loaded at 30 mg/mL of adsorbent. The total amount of IgG recovered is shown in Figure 10, with well-to-well variability of 6.4%.
LV was obtained by primary capture from harvested feedstock from HEK 293 T packaging cell line. The LVV in this example had high DNA content in the elution pool (865 ng/mL) as the feed was only treated for 1 hour with 5 U/mL of endonuclease instead of 1 hour with 50 U/mL. The final concentration in the flowthrough pool was ~30 ng/mL. LV primary capture was performed with a 1 mL LentiHERO® 1 capsule and eluted with 20 mM Tris, 20 mM MgCl2, 0.8 M arginine, pH 7.2. The eluate from this step was the LV-rich fraction used as the load in the plate screen. An average of 4.4E+07 TU/mL of adsorbent was loaded. Binding buffers consisted of 20 mM Tris, 20 mM MgCl2, pH 7.2 supplemented with arginine to final concentrations of 0 M, 0.2 M, 0.4 M and 0.8 M. Binding buffers were used for load conditioning, resin equilibration and post-load wash. Stripping and cleaning-in-place were performed with 50 mM citriccitrate buffer pH 3 and 0.5 M NaOH, respectively. During automated MiMode® plate processing, buffers and samples were transferred to the appropriate labware, binding buffers were dispensed to pre-condition and equilibrate the resins, and conditioned LV samples were loaded onto the plates and incubated for 60 minutes. Liquid was driven through the resins under controlled positive pressure, with flow-through and post-load wash collected as the non-bound fraction for LV and residual DNA analysis.
Figure 10: Recovery of IgG. (A) Recovery expressed in µg. (B) Recovery expressed in percentage. Columns on the right show the identity of the resin, the average recovery and the coefficients of variation. Recovery values are colored in gradient from high (dark shades) to low (light shades).
For the salt screen, the 0.4 M arginine binding buffer was independently supplemented with ammonium acetate, ammonium sulfate, calcium chloride, tetramethylammonium chloride, sodium acetate, sodium sulfate, sodium chloride, sodium citrate, sodium iodide, sodium phosphate or lithium chloride at 0.1 M and 0.2 M final concentration.
Collected non-bound and load fractions were prepared for LV titer analysis by TU assay and residual DNA analysis by PicoGreen assay. TU assay plates were incubated for 3 days at 37 °C and 5% CO2 before infective titer measurement by flow cytometry. PicoGreen plates were incubated for 5 minutes at room temperature protected from light before fluorescence reading in a microplate reader. Repeatability testing used standard proteins to assess plate consistency independently of TU and PicoGreen assay variability. BSA was prepared in 20 mM Tris pH 8.0 at 0.9 mg/mL and IgG was prepared in phosphate-buffered saline at 1 mg/mL. Protein concentrations in load and non-bound fractions were measured by A280, and non-bound recovery was calculated from the resulting absorbance measurements.
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