State of the art screening method for affinity chromatography resins

December 21, 2017

Hello and welcome to Prometic’s presentation on chromatographic screening technologies. This presentation covers an introduction into high-throughput screening and the different technologies available for chromatographic adsorbents. Following on from this, we will cover Prometic’s proprietary PuraPlate™ technology including applications, operating instructions and examples.

High-throughput screening typically involves testing many thousands of chemical drug compounds in either a 96-well or a 384-well plate format. This has now been adapted to support the rapid evaluation of chromatography adsorbents with multiple technologies available in the marketplace for use either via manual or automated platforms.

The different technologies available include PreDictor™ Plates, PhyTips®, RoboColumns® and Prometic’s PuraPlates™. These technologies can be used in different ways, for example batch mode binding with the PreDictor™ Plates, through to dynamic flow testing on automated workstations with the MediaScout® RoboColumns®. Prometic offer the PuraPlate™ which is a 96 deep-well block containing a range of different Prometic chromatography adsorbents.

The PuraPlate™ was developed by Prometic and has been successfully established during extensive screening projects over the last 15 years. The plate is used in a dynamic mode operated by gravity with no vacuum required and samples are collected via drainage outlets at the bottom of each individual column, the PuraPlate™ is user-friendly and can be operated either manually with a multi-channel pipette or with an automated workstation. Typically, the PuraPlate™ is packed with 250 microlitres of adsorbent and due to the column like format allows for scalability to conventional axial flow columns.

The PuraPlate™ has many and varied applications from high-throughput screening which allows for the evaluation of a range of adsorbents under a generic set of conditions through to process optimisation which enables rapid evaluation of a range of chromatography conditions such as load, was and elution applications to identify optimal conditions. The PuraPlate™ can also be used as a platform to evaluate a range of conditions to identify optimum cleaning strategies for adsorbent re-use and stability. Prometic has also used the platform technology to evaluate multiple batches of a single adsorbent post manufacture for performance consistency.

The PuraPlate™ is available in different formats. Option one contains a single Prometic product in each of the 96-wells to allow rapid process optimisation such as load, wash, elution and cleaning strategies. Option two contains a mixture of different adsorbents to allow for rapid evaluation of Prometic’s extensive range of off-the-shelf products to identify a potential lead candidate for your process further options include combinations of options one and two as a custom PuraPlate™.

The PuraPlate™ is a user-friendly technology in which high-throughput screening and optimisation can be carried out rapidly and with ease. Firstly, remove the upper and lower storage seals and allow the preservative to drain into a waste reservoir then equilibrate, load and elute the PuraPlate™ by applying the screening conditions as desired and collect any required fractions into suitable deep-well blocks.

Typical conditions in the PuraPlate™ mimic those used in conventional axial flow columns. Firstly, you would equilibrate the required columns, then you would load on your material. The PuraPlate™ provides a residence time of around half a minute, based on gravity flow. Next, remove any non-bound material with a post-load wash, followed by an elution step. Finally, use the specified clean-in-place strategy suitable for the adsorbents being evaluated, which standardly is 0.5 to 1 molar sodium hydroxide to remove any residual protein.

As mentioned previously, the PuraPlate™ has various applications. Option one, is using the PuraPlate™ for process optimisation by quickly evaluating products such as IEX or HIC adsorbents by varying salt or pH across the PuraPlate™ to identify the optimal conditions for binding and elution of your target protein. Another option for the PuraPlate™ is having multiple adsorbents for screening and allowing for rapid optimisation of load, wash and elution conditions. This option is employed by Prometic to rapidly screen our in-house libraries in quick screens or custom adsorbent projects.

Due to the rapid generation of a high quantity of samples from the PuraPlate™ an extensive range of analytical techniques can be utilised, such as 96-well ELISA assays, homogeneous plate assays, such as AlphaLISA or time-resolved fluorescence (HTRF), standard multi-well precast SDS-page or 96-well gels such as the E-PAGE system, automated versions of SDS analysis, such as the LabChip microfluidic system and HPLC or LCMS analysis with UPLC technology is now allowing faster run times and with 96-well plate racks supporting the high-throughput sample analysis.

The next few slides show an example of the PuraPlate™ option two, high-throughput using multiple adsorbents to identify lead candidate for lactate dehydrogenase from rabbit muscle extract, in this example the mimetic screening PuraPlate™ contains 10 different Prometic products used in two steps. The first step, using part of the plate to identify lead candidates and the second step to identify an optimum loading condition. SDS-page analysis of the elution fractions from the first step identified one of our adsorbents, Mimetic Yellow 2 A6XL, seen in lane 10, which demonstrated excellent capture and purification of the lactate dehydrogenase target protein. The second step using the remaining Mimetic Yellow 2 A6XL columns available on the same PuraPlate™ were evaluated by loading the lactate dehydrogenase at a variety of conditions, from pH 5.5 to pH 8.5. SDS-page analysis shows the optimal conditions for loading was at pH 6.

Example two demonstrates PuraPlate™ option one, high-throughput process optimisation for Prometic’s Fabsorbent™ F1P HF product, investigating a range of elution buffers at varying pH to identify an optimal strategy for selective recovery of Fab fragments bound from an e.coli lysate. SDS-page analysis shows the elution samples across the pH and buffer ranges evaluated. Lane six shows one of the promising candidates using 50 millimolar sodium acetate at pH 4.5. This slide highlights the SDS-page results for the optimal conditions identified from the PuraPlate™ investigation scaled up to a 4ml axial column using the Fabsorbent™ F1P HF adsorbent. The results show that for the elution sample for the scaled up column run in lane six has a greater than 95% purity of the Fab fragment, this purification supports the successful transfer and scale-up from PuraPlate™ to conventional column mode.

To summarise, there are a number of high-throughput screening technologies available in the market today, designed for rapid candidate selection and process optimisation of chromatography adsorbents. Prometic’s proprietary PuraPlate™ technology operates in a gravity-fed dynamic mode without the need for additional vacuum and enables scalability of chromatography adsorbents from 96-well plate format to conventional axial flow columns. The PuraPlate™ is suitable for both manual application and automated liquid handling platforms and offers a range of capabilities including lead candidate selection and process optimisation. Prometic also offers bespoke ligand discovery projects for custom-designed chromatography adsorbents to suit your needs.

Thank you for listening, and if you’d like any further information regarding the PuraPlate™ or any of our other products, please contact us using the information provided.

 

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