Chromatography Process Development Using 96-Well Microplate Formats - One plate can test one adsorbent in two days for its optimum condition. Results are confirmed in a laboratory column. - BioPharm


Chromatography Process Development Using 96-Well Microplate Formats
One plate can test one adsorbent in two days for its optimum condition. Results are confirmed in a laboratory column.

BioPharm International

Chromatography Conditions

Table 3. Summary of buffers used to analyze protein binding and elution from microplates containing ion-exchange media. Sanitization was carried out using 0.5 M NaOH, and media were stored in 20% ethanol. The plate is depicted in Figure 1.
Microplate data do not necessarily represent a developed process; more assurance is required. We tested the optimum results from each plate in a traditional packed column. The elution conditions achieved to desorb a single protein, and then both proteins, as observed on the E-PAGE when relatively intense bands on the gel were seen, were applied to a traditional packed column, and run on an automated workstation.

The ion exchangers' parameters were scaled up to ensure good correlation of results between the 96-well chromatography microplate study and conventional column chromatography. Two elution conditions were chosen for each ion exchanger, based on the images from the E-PAGE experiment, which demonstrated the elution of a single protein and both proteins. The buffer conditions are summarized in Table 4. In all, eight experiments were performed (one for each elution condition) corresponding to the four 96-well microplate studies. The elution conditions applied for column chromatography are identified on the E-PAGE images by white circles.

Table 4. Optimum conditions for separating proteins as developed using microplates in conjunction with E-PAGE analysis. White circles in Figures 2-5 reflect the chosen points, which were then applied to these conventional packed chromatography columns.
From observing SDS PAGE analysis for all four ion exchangers (Figure 6) it can be seen that the elution profiles from column chromatography indicate that only IgG is eluted from the column when presented with the first elution buffer (lanes 4, 8, 12, and 16), exactly as was observed for the 96-well chromatography microplate study. We see that a small quantity of BSA eluted from the Q-ion exchange media from the first elution buffer, and this might indicate a requirement to elute the IgG under slightly less stringent conditions. In lanes 5, 9, 13, and 17, both proteins are eluted. Again, this result correlates with the 96-well chromatography microplate study indicated by the white circles in Figures 2 to 5. This correlation indicates that the separation of proteins in column chromatography compares favorably with the separation of proteins developed under the same conditions in the 96-well chromatography microplate study. Thus, we have shown the validity of using a microplate-based approach for initial process development activities.

Media Selection

Figure 7. Example composition and layout of 96-well microplate containing a range of adsorbents (Mimetic Ligand adsorbents, Prometic BioSciences) and two anion exchange media.
The microplate format also can be used for media selection. Using the microplate format would offer re-searchers the ability to investigate the purification capabilities of numerous affinity adsorbents in short time frames with a minimum quantity of representative process material. In our laboratories, we have been able to acquire extensive and informative purification data from a single plate containing up to 64 adsorbents within two days.

An example of a format (well layout) for this type of study is shown in Figure 7, which contains our complete range of Mimetic Adsorbents, and in this example, two anion exchange media. Depending on the nature of the target protein, plates with cation exchangers might be used. Each adsorbent is represented in four wells to provide greater confidence in any acquired data. Applying a representative feed stream across the plate followed by the analyses discussed earlier would readily point to any potential candidates that could be developed for use in a manufacturing situation.

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