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Pihl Mayer posted an update 6 months, 3 weeks ago
Recombinant antibodies in single-domain format (VHHs) have been recently used for stabilizing antigens during their purification and crystallization. MK-2206 VHHs are also known for their structural stability and a significant part of them share the characteristic of remaining functionally folded also in the absence of the internal disulfide bond. Therefore, they can be expressed as intrabodies in the cell cytoplasm as well as in the bacterial periplasm. This evidence means that, in theory, VHHs can be co-expressed with their antigens independently on the redox constrains. It has also suggested the idea of using co-expression and co-purification of antigen-antibody complexes for maximizing the stabilizing effect of the antibody on its antigen during all the production steps for both cytoplasmic and periplasmic expression strategies.Aqueous two-phase systems (ATPS) have been widely and successfully used in the purification of various biological macromolecules such as proteins, nucleic acids, antibiotics, and cell components. Interfacial precipitation of the product often results in lower recovery and selectivity of ATPS. Efficient resolubilization of the interfacial precipitate offers a way to improve the recovery as well as selectivity of ATPS systems.In this protocol, we describe a method for aqueous two-phase-assisted precipitation and resolubilization of the recombinant human Granulocyte Colony Stimulating Factor (GCSF) for its selective isolation from E. coli host cell proteins as well as nucleic acids. This platform purification can be applied to other cytokines as well as most of the hydrophobic proteins that partition into the hydrophobic PEG-rich top phase. Recoveries of up to 100% of the product along with reduction of levels of E. coli host cell proteins (from 250-500 to 10-15 ppm) and of nucleic acids (from 15-20 to 5-15 ng/mL) were observed.The increasing interest of the biopharmaceutical industry to exploit plants as a commercially viable production system is demanding the development of new strategies to maximize product recovery. Aqueous two-phase systems (ATPSs) are a primary recovery technique that has shown great potential for the efficient extraction and purification of biological products, from organelles to proteins and low-molecular-weight compounds. The evaluation of different system parameters upon the partitioning behavior can provide the conditions that favor the concentration of contaminants and the desired target protein in opposite phases. The protocols described here provide the basic strategy to explore the use of ATPSs for the isolation and partial purification of native and recombinant proteins from plants and plant-derived extracts.Monoclonal antibodies (mAbs) are the fastest-growing segment in the drug market with eight of the top 20 selling drugs being mAbs and combined sales of close to 60 billion US$/year. The development of new therapeutic mAbs requires the purification of a large number of candidate molecules during initial screenings, subsequent affinity maturation campaigns, and finally the engineering of variants to improve half-life, functionality, or biophysical properties of potential lead molecules. A successful strategy to purify this ever-increasing number of mAbs in a timely manner has been the miniaturization and automation of the purification process using automatic liquid handlers (ALHs) such as Tecan’s Evo or PerkinElmer’s Janus platforms. These systems can be equipped with miniaturized columns, which are available in a wide variety of sizes and affinity matrices to cater to the need of the respective application. Various publications have described the setup of ALHs including the respective purification procedure. However, despite being very precise regarding the overall approach, most publications do not focus on the technical optimization and potential pitfalls, which can be crucial to obtain a robust process. To fill this gap, the present publication is aiming to point at some technical difficulties and suggesting potential ways to overcome these problems in order to facilitate the setup of new ALH systems for the purification of antibodies.Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. The choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices serve as a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose the best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lie in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.High-throughput process development is more and more used in chromatography. Limitations are the tools provided by the manufacturers. Here, we describe a method to select ion exchange chromatographic media using a 96-well filter microplate.