Chromatography Process Development Using 96-Well Microplate Formats

Developing biopharmaceutical manufacturing unit operations inevitably involves screening chromatographic adsorbents that offer high selectivity and yield highly purified materials. Simultaneously, materials must meet the diverse and rigorous requirements for use in an economically viable cGMP process.

When considering the technical performance of chromatography media, the selection process might involve preparing numerous packed columns, followed by a target protein challenging various candidate adsorbents to allow scrutiny of protein recovery, yield, purity, and activity. Then the capacity, affinity, and selectivity of an adsorbent are considered in the context of process conditions such as binding and elution buffers, or response to repeated clean-in-place (CIP) procedures.

Packed columns have been commonly used in such process development activities. Using packed columns, however, limits the range of media that can be appraised because packing the media in small-scale columns is onerous and may be hindered by the limited availability of (sometimes costly) automated systems that will perform method development or scouting experiments. As a result, optimal adsorbents or even process conditions may not always be realized or implemented.

However, using 96-well microplates containing chromatography media to facilitate high throughput experimentation may rapidly enhance adsorbent selection, thereby allowing a greater opportunity to identify an optimum adsorbent and set of operating conditions. An investigation can facilitate the appraisal of a range of adsorbents in a single plate, or develop the process conditions for a single adsorbent by challenging it with various bind and elute conditions.

Table 1. Summary of dimensions and characteristics of 96-well microplates used for adsorbent selection and process development.

In this article we discuss adapting the high throughput ex-perimental advantage of 96-well microplate technologies for process selection and development. We highlight various aspects that need to be considered in developing a robust and trustworthy system of adsorbent appraisal.

96-Well Microplates

Microtiter plates in a 96-well format have been used for many years in analytical research and clinical diagnostic laboratories because of their high throughput of samples. Examples of their application include the enzyme-linked immunosorbent assay (ELISA) and high-throughput screening of pharmaceutical candidates. The speed, accuracy, and efficiency of these activities is often further enhanced by using robotic systems which can dispense and aspirate relevant fluids into specified areas of the plate, and manage multiple plates simultaneously.

Figure 1. Summary of anticipated and observed effects when ion exchangers are exposed to pH and salinity gradients.

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.

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.

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

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.

Figure 4. E-PAGE showing elution profiles from Q PuraBead HF ion exchange media.

Process Development

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.

Figure 5. E-PAGE showing elution profiles from DEAE PuraBead HF ion exchange media.

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.

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.

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.

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.

Chromatography Conditions

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.

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.

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.

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.

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.

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.

Summary

Quick RecapUsing 96-well microplates for chromatography adsorbent selection and process development may expedite typical activities undertaken by process development groups.

Using such a format requires smaller sample volumes and can rapidly generate data from a number of points than might otherwise be possible in the same time frame. As a result, this method is likely to be of benefit in this important area of biopharmaceutical process development. Furthermore, using these high throughput experimental techniques increases the number of adsorbents or process conditions that can be evaluated, potentially resulting in substantial time and cost savings in process development activities.

HENRY CHARLTON, PH.D. is team leader at Prometic BioSciences Ltd., 211 Cambridge Science Park, Cambridge, CB4 0ZA, UK, +44.1223.420300, fax +44.1223.433270, henry.charlton@prometic.co.uk

BEN GALARZAben.galarza@prometic.com

BEN BEACONben.beacon@prometic.com

and KELLY LERICHEKelly.leriche@prometic.comare all research scientists,

and ROB JONESrob.jones@prometic.com is shipping and transport manager.