Hello, within this presentation I am going to discuss with you the new Multi-Mode Mimetic Ligand™ Library developed by Prometic Bioseparations. For both product capture of impurities and polishing applications, small synthetic ligands have become an established in downstream processes. Historically, the identification of a specific ligand suitable for target protein binding has required specialised companies and whilst usually very successful can be expensive and time-consuming. There are now a growing number of generic multi-mode ligands available on the market, incorporating both ion exchange and hydrophobic functionalities which have been successful for general polishing and host-cell protein removal applications.
Prometic have combined both a ligand discovery component and multi-modal ligands into a cost-effective and easy-to-use, diverse screening library suitable for the identification of both product capture and polishing steps. In the following slides, I will describe the Multi-Mode Mimetic Ligand™ Library as well as a couple of applications including monoclonal antibody and human plasma feedstocks.
The Multi-Mode Mimetic Ligand™ Library contains a diverse array of 96 synthetic ligands incorporating combinations of functional binding groups. The groups include Cationic, Anionic, Aliphatic and Aromatic groups as well as other specific functional groups pre-selected based on their proven ability. Figure 1, shows the layout of the Ligand Library with six zones of ligand functionalities for ease of use. The library can be used manually, as shown in figure 2, using a multi-channel pipette or via a robotic workstation. The library functions in a gravity-fed mode with no need for a vacuum or centrifugation, obtaining an approximate half minute residence time once the feedstock or buffer is loaded into each well. The non-bound and elution fractions can be analysed with a variety of high-throughput techniques to identify promising candidates. All 96 different adsorbents in the Ligand Library can then be further evaluated in a variety of formats available from Prometic.
In the first application, the Multi-Mode Mimetic Ligand™ Library was used to screen a monoclonal antibody feedstock from a CHO cell culture supernatant. Table one shows the screening conditions used, in this example the equilibration buffer was designed to match the feedstock and fifty millimolar citrate buffer at pH 3 was used for the elution.
Figure 3 shows the non-reduced SDS-PAGE results of a number of elution fractions selected from zone two of the library. Figure 4 shows the host cell protein results for the elution fractions from the entire 96-well Ligand Library. The data shows that the Multi-Mode Mimetic Ligand™ Library exhibits an extensive range of functionalities across the different zones and highlighted a number of promising candidates for both product capture and polishing applications, as shown on the next slide.
The gel in figure 5 shows a number of lead candidates for target protein capture and recovery with good host cell protein clearance. The gel in figure 6 shows a number of promising candidates for polishing applications with high host cell protein capture and low qualitative monoclonal antibody presence in the elution fractions.
This next application demonstrates the use of the Multi-Mode Mimetic Ligand™ Library with a natural feedstock, Human Source Plasma. Table 2 shows the screening conditions used. The elution buffer used is the generic Ligand Library elution condition which is 50 millimolar citrate buffer with 1 molar salt and 10% hexanediol at pH 7.5.
Figure 7 shows the non-reduced SDS-PAGE results of selected elution fractions from zones 2 to 5. Figure 8 shows a qualitative measurement scoring of three selected proteins based on the presence of each protein in the elution SDS-PAGE. Overall, the data shows a number of promising candidates for different target capture and an extensive range of performance across the Multi-Mode Mimetic Ligand™ Library.
So, to conclude the Multi-Mode Mimetic Ligand™ Library is divided into groups of ligands of similar function in 6 different zones, the applications presented successfully demonstrate the identification of several promising candidates for the capture and purification, or polishing step, of a monoclonal antibody as well as the ability to selectively bind and elute a variety of different biomolecules from a complex natural feedstock. In summary, the Multi-Mode Mimetic Ligand™ Library is a cost effective, easy-to-use, rapid screening toolbox for the identification of a ligand suitable for the purification of a wide range of target proteins from a variety of sources, both natural and recombinant.