Quantitative detection of therapeutic proteins and their metabolites in serum using antibody-coupled ProteinChip Arrays and SELDI-TOF-MS.
One of the important steps in developing protein therapeutics is the determination of their preliminary PK in vivo. These data are essential to design optimal dosing in animal models prior to progressing to clinical trials in man. The quantitative detection of protein therapeutics in serum is traditionally performed by ELISA, which has the prerequisite of the availability of the appropriate monoclonal antibodies. We have developed an alternative method using polyclonal antibodies immobilized on ProteinChip Arrays and SELDI-TOF mass spectrometry. This method has an advantage over ELISA since it provides simultaneously information on the clearance rate of the protein and it's in vivo processing. We compared these two methods using a RANTES variant, [(44)AANA(47)]-RANTES as the test protein in this study. Using SELDI-TOF mass spectrometry, we were able to establish that the protein is readily oxidized in serum, and moreover is processed in vivo to produce a truncated 3-68 protein, and undergoes a further cleavage to produce the 4-68 protein. These modifications are not identified by ELISA, whilst the serum exposure profiles determined by the two methods show essentially similar protein concentration values.
Favre-Kontula
Linda
L
Serono Pharmaceutical Research Institute, 14 Chemin des Aulx, 1228 Plan-les-Ouates, Geneva, Switzerland.
Johnson
Zoë
Z
Steinhoff
Tiana
T
Frauenschuh
Achim
A
Vilbois
Francis
F
Proudfoot
Amanda E I
AE
D000818:Animals; D018946:Chemokine CCL5 / Q000097:blood* / Q000378:metabolism* / Q000493:pharmacokinetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D040081:Protein Array Analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018946:Chemokine CCL5