Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications.

/pdf/preparative-purification-of-recombinant-proteins-current-k8j5zjxz4g.pdf

Preparative Purification of Recombinant Proteins: Current Status and Future Trends

Novel peptide ligand with high binding capacity for antibody purification.

New functional materials can in principle be created using colloids that self-assemble into a desired structure by means of a programmable recognition and binding scheme. This idea has been explored by attaching 'programmed' DNA strands to nanometre- and micrometre- sized particles and then using DNA hybridization to direct the placement of the particles in the final assembly. Here we demonstrate an alternative recognition mechanism for directing the assembly of composite structures, based on particles with complementary shapes. Our system, which uses Fischer's lock-and-key principle, employs colloidal spheres as keys and monodisperse colloidal particles with a spherical cavity as locks that bind spontaneously and reversibly via the depletion interaction. The lock-and-key binding is specific because it is controlled by how closely the size of a spherical colloidal key particle matches the radius of the spherical cavity of the lock particle. The strength of the binding can be further tuned by adjusting the solution composition or temperature. The composite assemblies have the unique feature of having flexible bonds, allowing us to produce flexible dimeric, trimeric and tetrameric colloidal molecules as well as more complex colloidal polymers. We expect that this lock-and-key recognition mechanism will find wider use as a means of programming and directing colloidal self-assembly.

http://absimage.aps.org/image/MAR09/MWS_MAR09-2008-004682.pdf

Lock and Key Colloids

Arginine is one of the most prominent residues in protein interactions, and arginine hydrochloride is widely used as an additive in protein solutions because of its exceptional effects on protein association and folding. The molecular origins of arginine effects on protein processes remain, however, controversial, and little is known about the molecular interactions between arginine cations and protein surfaces in aqueous arginine solutions. In this study, we report a unique biochemical phenomenon whereby clusters of arginine cations (Arg+) are associated with a protein surface. The formation of protein-associated Arg+ clusters is initiated by Arg+ ions that associate with specific protein surface loci through cooperative interactions with protein guanidinium and carboxyl groups. Molecular dynamics simulations indicate that protein-associated Arg+ ions subsequently attract other Arg+ ions and form dynamic cation clusters that extend further than 10 A from the protein surface. The effects of arginine on th...

Protein-Associated Cation Clusters in Aqueous Arginine Solutions and Their Effects on Protein Stability and Size

http://wolfson.huji.ac.il/purification/Course92632_2014/MixedMode/How2011.pdf

Evaluation of selectivity in multimodal anion exchange systems: a priori prediction of protein retention and examination of mobile phase modifier effects.

A majority of column chromatographies use only selected salts, e.g., ammonium sulfate, NaCl, Citrate and phosphate in hydrophobic interaction chromatography (HIC) and NaCl in ion exchange and dye affinity chromatographies. Alternatively, a pH range below or above the neutral value is often used to reduce affinity interactions, e.g., in Protein-A or dye affinity column chromatography. Although these parameters are easily manipulated, they are not necessarily the optimal conditions for high recovery and resolution of the proteins. So-called co-solvents have been used, although to a limited extent, to manipulate performance of column chromatography. Here the term co-solvent is used to indicate its relatively high concentrations required for these applications, meaning that it also serves as solvent along with water. Ethylene glycol and MgCl(2) have been used to elute specific antibodies from antigen-affinity column. Arginine has also been used for the same purpose. Arginine has much wider applications for various column chromatographies, including size exclusion chromatography (SEC), HIC and affinity chromatography. Polyethylene glycol and glycine have also been used to improve the performance of HIC and hydroxyapatite chromatography. This review summarizes these applications of co-solvents for column chromatographies.

Solvent Modulation of Column Chromatography

HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation.

https://www.mdpi.com/1999-4923/13/1/113/pdf

Pete Gagnon

Papers

Solvent Modulation of Column Chromatography

Multiple-Monitor HPLC Assays for Rapid Process Development, In-Process Monitoring, and Validation of AAV Production and Purification.

Excluded Cosolvent in Chromatography.

Aggregation detection and removal biopharmaceutical proteins.

Removal of empty capsids from adeno-associated virus preparations by multimodal metal affinity chromatography.