Evaluation of protocols for purification of mouse monoclonal antibodies. Yield and purity in two-dimensional gel electrophoresis.
Protocols for purification of mouse monoclonal antibodies (MAbs) from nude mice ascites were investigated in order to assess the yield and to compare the purified products in two-dimensional gel electrophoresis (2DGE). Three MAbs (one IgG2 and two IgG1), selected for their differing behaviours towards protein A, were purified by ammonium sulphate precipitation and/or gel filtration, anion exchange (DEAE), hydroxylapatite and affinity (protein A) chromatography, or by a combination of these methods. Protein A constantly provided the highest purity whatever the IgG subclass. The best results in terms of yields and purity were a function of the optimization of the protein A protocol. In our study, they were obtained in a 3 h protocol (IgG2), a 16 h protocol with discontinuous pH gradient method (IgG1 with sufficiently high affinity for protein A) or a multi-step protocol involving DEAE and protein A (IgG1 with low affinity for protein A). DEAE chromatography alone provided a slightly better yield, but only moderate purity. Hydroxylapatite chromatography appeared to be less potent in terms of yield, purity and day-to-day reproducibility. Salt precipitation and gel filtration enabled only relative enrichment of the MAb solution. Some degradation products of both heavy and light chains clearly appeared in the 2DGE patterns of antibodies purified by different protocols, and seem to be partly related to the elution pH and to the duration of the purification procedure. Finally, this work highlights considerable heterogeneity not only between two different MAbs of the IgG1 subclass but also within a monoclonal population of immunoglobulins.
Manil
L
L
Motté
P
P
Pernas
P
P
Troalen
F
F
Bohuon
C
C
Bellet
D
D
D000645:Ammonium Sulfate; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification*; D000915:Antibody Affinity; D001202:Ascitic Fluid / Q000276:immunology; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D008819:Mice, Nude / Q000276:immunology; D013205:Staphylococcal Protein A
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D013205:Staphylococcal Protein A; D000645:Ammonium Sulfate