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

ADVERTISEMENT

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


Process Development


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.


blog comments powered by Disqus

ADVERTISEMENT

ADVERTISEMENT

Mallinckrodt to Acquire Questcor Pharmaceuticals
April 16, 2014
EMA Warns of Falsified Herceptin Vials
April 16, 2014
PhRMA Report Reveals Growth Trajectories and Policy Factors Affecting Biopharmaceutical Growth
April 11, 2014
American CryoStem and Rutgers University File Joint Patent on Stem Cell Platform
April 11, 2014
Center for Biologics Evaluation and Research Relocates
April 11, 2014
Author Guidelines
Source: BioPharm International,
Click here