Antibodies aggregates

Fig.8a, b a Liposome immunocompetition assay (LIC) J l,liposome/PCB competition zone, b Liposome immunoaggregation assay (LIA) R2,liposome/antibody aggregation zone. Cl, C2 anti-biotin capture zones. Published with permission of ACS Copyright Office [209]... 

Optimal conditions for both antibody and complement dilutions have to be determined empirically, by a cytotoxicity test (see below). The complement-mediated lysis reaction proceeds between antibody-saturated target cells as one reactant, and complement as a second reactant. Therefore, when target cell surface antigens are saturated with the corresponding antibody, lysis will not increase with increasing antibody concentration. On the other hand, antibody aggregates, which can form at high concentrations (usu-... 

Miller GW, Nussenzweig V (1975) A new complement function Solubilization of antigen-antibody aggregates. Proc Natl Acad Sci USA 72 418 22. 

A. Litzen, 1. K. Walter, H. Krischollek, and K. G. Wahlund. Separation and qnantitation of monoclonal antibody aggregates by asymmetrical flow field-flow fractionation and comparison to gel permeation chromatography, A aZ. Biochem. 212 169 (1993). 

Antibodies must not be stored for extended periods at their working dilutions, unless a stabilizing protein such as BSA has been added. Storing undiluted antibody in aliquots eliminate the need for repeated freeze/thaw, which may cause antibody aggregation or denaturation. 

Over the last few years, new resins have been launched which exhibit more than one mode of interaction. The goal of the combination of hydrophobic interaction principles with ionic interactions is the generation of complementary selectivities for mixtures difficult to separate by just ionic or hydrophobic interactions (Johansson et al, 2003a, 2003b). Especially, the separation of antibody aggregates from the corresponding monomeric form can be achieved with these resins. 

A hydrophobic interaction membrane chromatography method for rapid and efficient separation and analysis of monoclonal antibodies aggregates, able to resolve Campath-IH monomer, dimer, trimer, tetramer, and pentamer, was recently reported [269]. Other applications of hydrophobic interaction membrane chromatography include the separation of model proteins [270], the purification of humanized monoclonal antibody using a stack of microporous synthetic membranes [271], and the fractionation of human plasma proteins using a 0.1 pm microporous PVDF membrane [272]. 

Wang L, R. Ghosh R. Fractionation of monoclonal antibody aggregates using membrane chromatography. J. Membr. Sci. 2008 318 311-316. 

Hawe, A., Kasper, J.C., Friess, W., Jiskoot, W. (2009) Structural properties of monocloneil antibody aggregates induced by freeze-thawing and thermal stress. Eur 7 Pharm Sci, 38 (2), 79-87. 

Liu, S., Wang, H., Hao, Z., Tracy, M., and Liu, X. (2014) Analysis of monoclonal antibodies, aggregates, and their fragments by size exclusion chromatography coupled with an orbitrap mass spectrometer. PN-64082-EN-06148, Thermo Fisher Scientific, Sunnyvale, CA, USA. 

Kukrer B, Fihpe V, van Duijn E, Kasper PT, Vreeken RJ, Heck AJR, Jiskoot W. Mass spectrometric analysis of intact human monoclonal antibody aggregates fractionated by size-exclusion chromatography. Pharm Res 2010 27 2197-204. 

Joubert MK, Luo QZ, Nashed-Samuel Y, Wypych J, Narhi LO. Classification and characterization of therapeutic antibody aggregates. J Biol Chem 2011 286 25118-25133. 

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