A high-throughput study of sodium hydroxide cleaning efficiency for protein A resin

Screening of cleaning conditions using PreDictor™ plates

The methodology consists of three parts.

• First, we fouled the resin in the PreDictor™ plate by repeated load with concentrated mAb harvest followed by wash, elution, and optional acidic strip without any cleaning.
• Secondly, we added different NaOH concentrations to the fouled resin in the wells for different contact times. After incubation for a defined time, we removed the cleaning agents.
• The final step was to analyze the residual impurities remaining on the resin after cleaning. Identified CIP concentration and contact times could then be verified in a column lifetime study.

Fouling of MabSelect PrismA™ resin in PreDictor™ plates

PreDictor™ plates prefilled with 20 µL of MabSelect PrismA™ resin were used. To resuspend any particles of resin sticking to the top seal, the PreDictor™ plate was inverted several times in a controlled way according to the instruction (1).

In each step below, the PreDictor™ plate was mixed briefly on a microplate shaker at 1100 rpm. The PreDictor™ plate was kept on a collection plate throughout the workflow to avoid direct contact between the PreDictor™ plate outlets (the drips on the bottom) and any surface. During mixing, the PreDictor™ plate and the collection plate were fixed to each other and secured to the microplate shaker. Removal of liquid from the PreDictor™ plates between each step below was done using centrifugation (500 × g for 1 min). Before starting the cycling procedure, the storage solution (20% ethanol) was removed.

We performed fouling of resin and screening of cleaning conditions in a manual workflow using multipipettes and manual centrifugation.

1. Equilibration: 200 µL of PBS, pH 7.4 was added per well. This was performed three times. After centrifugation or vacuum filtration in the last equilibration step, the bottom of the PreDictor™ plate was blotted on a soft paper tissue to remove any drops of equilibration buffer that may have accumulated.
2. Feed application: 300 µL clarified CHO cell culture containing mAb at a concentration of 3.5 g/L was added to the wells. We performed incubation for 30 min on a microplate shaker at 1100 rpm*.
3. Wash: 200 µL PBS, pH 7.4 was added per well.
4. Elution: 200 µL of 0.05 M sodium acetate, pH 3.5 was added per well^† and the procedure repeated.
5. Acidic strip: 200 µL of 0.1 M sodium acetate, pH 2.9 was added per well^‡ in half of the plate and 200 µL of PBS, pH 7.4 was added per well in the second half of the plate.

Steps 1 to 5 were repeated 10 times corresponding to 10 chromatography cycles and we discarded all liquid fractions. No cleaning was included in the cycling procedure. After elution in the last cycle, the PreDictor™ plate was re-equilibrated three times with 200 µL of PBS per well and then washed once with 200 µL of ultrapure water before storage in 200 µL of ultrapure water until the screening procedure.

^* The top of the PreDictor™ plate was covered with microplate foil and secured on a collection plate on the microplate shaker using rubber bands.
^† The elution buffer/pH normally used in the process.
^‡ The strip buffer/pH normally used in the process.

Fig 2. Fouling of resin in PreDictor™ plates by repeated cycling of mAb harvest. Steps 1 to 5 were repeated 10 times, corresponding to 10 chromatography cycles. In each step, mixing was performed, and we used centrifugation for liquid removal.

Screening of NaOH cleaning efficiency for protein A resin

We evaluated five different NaOH concentrations up to 1.0 M and one control (wash with PBS) for cleaning efficiency.

In each step below, the PreDictor™ plate was shaken briefly on a microplate shaker at 1100 rpm. The PreDictor™ plate was kept on a collection plate throughout the workflow to avoid direct contact between the plate outlets (the drips on the bottom) and any surface. During mixing, the PreDictor™ plate and the collection plate were fixed to each other and secured to the microplate shaker. We removed liquid from the PreDictor™ plates between each step below using centrifugation (500 × g for 1 min). Before starting the screening procedure, we removed the ultrapure water from the fouled PreDictor™ plate filled with MabSelect PrismA™ resin. No tumbling of the plate was performed since the plate had been stored horizontally, with the top up, after the fouling procedure.

1. Wash: 200 µL of ultrapure water was added per well. This was repeated. After centrifugation or vacuum filtration in the last wash step, we blotted the bottom of the PreDictor™ plate on a soft paper tissue to remove drops that may have accumulated on the bottom of the PreDictor™ plate.

2. CIP step 1: We added 300 µL of cleaning solutions to wells according to Table 1. Incubation was carried out for 7.5, 15, and 30 min on a microplate shaker at 1100 rpm^†.

Table 1. Distribution of NaOH concentration in the PreDictor™ plates

3. Wash: 300 µL of PBS was added per well and this step was repeated three times.

4. Wash with ultrapure water: 300 µL of ultrapure water was added per well and we repeated this step three times.

All liquid fractions in the steps above were discarded.

Fig 3. Screening of CIP conditions. Different NaOH concentration (from 0.0 to 1.0 M) were applied to the fouled resins in the wells. Incubation contact times were 7.5, 15, and 30 min, and mixing was performed in each step. The top of the PreDictor™ plate was covered with microplate foil and secured on a collection plate on the microplate shaker using rubber bands.

Sample preparation for chip electrophoresis analysis

We prepared samples for analysis using chip electrophoresis by centrifuging the PreDictor™ plates (500 × g for 15 min) to remove all liquid from the resin. Afterwards, the drained resin samples (gel plugs) were transferred from the PreDictor™ plate to an empty collection plate by placing the collection plate very precisely upside down on the filter plate. The two plates were secured with rubber bands, inverted, and the resin in the wells transferred from the filter plate to the collection plate (Fig 4, Fig 5A) using centrifugation at 1000 × g for 10 min.

Fig 4. Centrifugation for transfer of resin from filter plate to collection plate for sample preparation.

Analysis of residual protein impurities on the resin after cleaning

Buffer preparation

We dissolved 3 g of Tris in 40 mL of ultrapure water. The pH was adjusted to 7.5 with glacial acetic acid. The final volume was adjusted to 50 mL by adding ultrapure water. SDS (0.5 g) was dissolved in 5 mL of the Tris buffer and the volume was adjusted to 50 mL by adding ultrapure water. We prepared the sample buffer by mixing 25 mL of SDS buffer and 1.39 mL of 1 M DTT and the volume was adjusted to 50 mL by adding ultrapure water.

Sample buffer (100 µL) was added to the resin in each well in the collection plate (Fig 5A and B). The plate was covered with microplate foil and mixed briefly at 1100 rpm on a microplate shaker before heating the plate in a heating chamber or heating block at 96°C for 10 min. The collection plate was mixed again briefly at 1100 rpm before it was centrifuged at 500 × g for 2 min to spin down the chromatography resin. 40 µL of the supernatant, which now contained the protein impurities from the resin, was automatically transferred to a PCR-plate using a pipetting robot for analysis by chip electrophoresis.

Fig 5. Sample preparation for analysis of residual impurities on the chromatography resin. (A) Fouled and cleaned resin is transferred to collection plate. (B) We prepared samples for chip electrophoresis by adding an SDS/DTT containing sample buffer and heating the collection plate in a heating chamber or heating block. The supernatant, which now contained the protein impurities from the resin, was removed and analyzed.

Analysis of residual protein impurities on the resin

We used LabChip GXII touch HT protein characterization system for the chip electrophoresis because of the large number of samples to analyze, its capability of automation, and high-throughput capability of the analytical method. With LabChipGX reviewer software (Revvity), it is possible to generate semi-quantitative data, which is convenient when evaluating hundreds of data points. If no chip electrophoresis system is available, it is also possible (but more time-consuming) to analyze the residual impurities using traditional SDS-PAGE.

The Protein Express assay chip (Revvity) was prepared according to the instructions. The chip was run on the electrophoresis system and the data were evaluated using standard system software.

Results

Screening of cleaning conditions using a high-throughput format

We screened NaOH cleaning efficiency for protein A resin in a high-throughput study in PreDictor™ 96-well filter plates, which enable evaluation of several conditions in one study and save time in process development. The PreDictor™ 96-well filter plate was prefilled with MabSelect PrismA™ resin, 20 µL resin per well, to screen the cleaning efficiency of an acidic strip, five different NaOH concentrations and one reference with PBS, and different contact times. The resin in the wells was fouled with concentrated mAb feed 10 times (corresponding 10 cycles) followed by incubation in different NaOH concentrations and contact times. Half of the wells were exposed to an acidic strip.

Chip electrophoresis was used for high-throughput analysis of protein residuals from the mAb feed left on the resin after cleaning with different NaOH concentration and contact times. The results show that the main protein foulant is the mAb. It is represented by light chains (approx. relative molecular mass of M_r 30 k [30 kDa]) and heavy chains (approx. M_r 60 000 [60 kDa]) in the gel picture (Fig 6) and electropherogram (Fig 7). From highest to lowest intensity lane in the virtual gel (and corresponding electropherogram) represents no CIP to 1.0 M of NaOH.

Fig 6. Example of a chip electrophoresis gel image (7.5 min contact time, no strip) representing the mAb residuals left on MabSelect PrismA™ resin after cleaning with different NaOH concentration. Light chains are shown at M_r 25 to 30 k (25 to 30 kDa) and heavy chains at M_r 60 to 70 k (60 to 70 kDa) in the graph.

Fig 7. Electropherogram overlays (7.5 min contact time, no strip) over chip electrophoresis analysis of mAb residuals left on MabSelect PrismA™ resin. Light chains are shown at M_r 25 to 30 k (25 to 30 kDa) and heavy chains at M_r 60 to 70 k (60 to 70 kDa) in the electropherogram.

Effect of different NaOH concentration and cycling with and without strip

Our recommendation in a mAb purification process is to use an acidic strip step between elution and CIP. Here, the fouling process was tested, with all three-contact times for CIP, with and without the acidic strip.

Figure 8 (CIP contact time 7.5 min) shows that an acidic strip as a part of the process results in lower concentration of mAb residuals on the resins. The same results are seen regardless of contact time and NaOH concentration.

Fig 8. Resin cleaning effect of acidic strip and different NaOH concentrations at 7.5 min contact time.

Figure 9 shows that higher NaOH concentrations in combination with longer contact time is the most efficient way for cleaning the protein A resin.

Fig 9. Resin cleaning effect of different NaOH concentration and three different contact times.

^* Values for 30 min treatment with 0.5 and 1.0 M of NaOH < limit of quantitation (LOQ).

The protein concentration determined by the chip electrophoresis analysis is directly correlated to the CIP efficiency of the protocol.