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

Figure 2. E-PAGE analysis showing elution profiles from SP PuraBead HF ion exchange media. Proteins are labeled based on the profiles observed in the calibration part of the E-PAGE (lane 10). White circles represent elution conditions that were later verified using a conventional column.
Our laboratories have been using robotics in combination with 96-well microplate formats for many years to screen libraries of ligand candidates, which, on identification of promising results, can be developed into adsorbents used in the full-scale manufacture of biopharmaceuticals. Using 96-well microplates significantly increases the number of ligand candidates that can be screened as potential adsorbents in a given time frame and correspondingly, the chance of project success and its quality may also be improved. In addition, the small quantity of media used in each well means that relatively small quantities of test material are required to screen a high number of adsorbents. These reduced quantity requirements are helpful because often, representative feed material is in short supply, particularly at the onset of biopharmaceutical development campaigns.

Microplates for Chromatography

We made specially adapted micro-plates that have an outlet in the base beneath each well. These allow the passage of fluid through the well and the adsorbent, while a polypropylene frit in the base of the well retains the adsorbent within the well. The dimensions and characteristics of these adapted 96-well plates appear in Table 1.

Figure 3. E-PAGE showing elution profiles from CM PuraBead HF ion exchange media.
When these plates are used, known quantities of chromatography media are aliquoted into 64 of the wells, and the remaining 32 wells are reserved for calibrations, standards, and controls. In this way, flow-through, elution, and sanitization samples all can be generated for further analysis.

We routinely apply various analytical techniques to qualify and quantify both target proteins and impurities that have been challenged by the multitude of candidate ligand libraries stored in the plates. Our most common combination is Bradford's Protein Assay and Invitrogen's E-PAGE 96 Protein Electrophoresis System. This way, binding proteins are identified and can be compared with non-binding proteins to pinpoint the purification capability of any adsorbent. A positive aspect about microplate format is that additional analytical procedures (such as ELISA)can be readily applied to obtain more information from any well.

Using Plates for Media Selection and Process Development

Figure 4. E-PAGE showing elution profiles from Q PuraBead HF ion exchange media.
We have been investigating the potential use of microplate formats in two key areas: media selection and process development. In order to develop this tool as a product rather than an in-house technique, we have made some minor modifications to the composite of the microplate, frits, and adsorbent of the plates that are used in-house. Specifically, adding an upper frit to the wells means that the chromatography media is not unduly disrupted during shipping. Controlling the extent to which this frit compresses the media enables control over the flowrate of fluids passing through the resultant mini-column. In effect, the amount of compression controls residence time, and we have developed protocols that allow both selection and good reproducibility of residence times in the region of 100 to 300 seconds. The upper range of these residence times reflects what might typically be used in a large process-scale column.

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