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
Figure 5. E-PAGE showing elution profiles from DEAE PuraBead HF ion exchange media.
In our procedure for process development, we filled 64 of the 96 wells with the same adsorbent and then explored optimum elution
conditions for separating commonly encountered proteins, as detailed in Table 2. Four different plates were used to illustrate
their capability in small-scale process development, containing ProMetic BioSciences Q PuraBead HF, DEAE PuraBead HF, SP PuraBead
HF, and CM PuraBead HF ion exchange media, which we abbreviate as Q, DEAE, SP, CM respectively. Mock feedstocks were prepared
in equilibration buffers as described in Table 3. These feedstocks are composed of two proteins that can be readily distinguished
by E-PAGE, and challenged to plates under binding conditions. Figure 1 displays the trends in pH and salinity, moving down
and across the microplate respectively, and also states the anticipated, differing responses from anion and cation exchange
media.
Table 2. Proteins used to develop elution strategies from ion-exchange media. Anion exchangers (Q, DEAE) were challenged with
a mix of polyclonal IgG and BSA. Cation exchangers were challenged with a mix of lysozyme and polyclonal IgG.
Total protein was measured using Bradford's Protein Assay at the following stages: flow-through, wash, elution, and sanitization.
Significant quantities of protein were only observed in elution and sanitization fractions. After pH and salinity gradients
were applied across the plate, elution profiles were also monitored using the E-PAGE 96 Protein Electrophoresis System as
shown in summary in Figures 2 to 5. The white circles represent elution conditions that were later compared for verification
using a conventional column format. These conditions are listed in Table 4.
Figure 6. SDS PAGE of challenge and elution materials from ion exchangers. Lane 1 (SeeBlue standard); lanes 3–5 (SP Load,
1st elution, 2nd elution); lanes 7–9 (CM Load, 1st elution, 2nd elution); lanes 11–13 (Q Load, 1st elution, 2nd elution);
and lanes 15–17 (DEAE Load, 1st elution, 2nd elution). Laboratory notebook reference BG/NB8/1.
Four 96-well plates were used to illustrate elution and separation of proteins. Each plate contained uniquely one ion-exchanger
of these four: SP, CM, Q and DEAE, all on PuraBead HF 6% agarose. The results of eluting the bound protein from each well
of each plate were illustrated by the E-PAGE system. The eluate from each well was transferred to the corresponding location
in the gel, and scrutiny of these E-PAGE profiles readily identified the originating well. Therefore, conditions that were
applied to facilitate separation of IgG and BSA (in the case of the anion exchangers) or IgG and lysozyme (in the case of
the cation exchangers) could be determined by referring back to the pH and NaCl concentrations applied to that particular
well.
