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.
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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.
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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.
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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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