|
Choice of the adequate quantification method for recombinant human GM-CSF produced in different host systems Mariela Bollati
Fogolín Marcos Oggero
Eberhardt Ricardo Kratje Marina Etcheverrigaray* * Corresponding author Finanacial support:
This work was supported by
a grant from the Agencia Nacional de Promoción Científica
y Tecnológica (Argentina), BID 802, PID Nr. PMT-SID 187. Abbreviations: rhGM-CSF: recombinant human granulocyte-macrophage colony stimulating factor; ELISA: enzyme-linked-immunosorbent assays; MAb: monoclonal antibody; RIA: radioimmnunoassay; PAb: polyclonal antibody; FCS: foetal calf serum; PBS: phosphate buffered saline; LB: Luria-Bertani medium; HRP: horseradish peroxidase; OPD: o-phenylenediamine; CV: coefficients of variation; CHO: Chinese hamster ovary; SEM: standard error of the mean.
The human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor which mediates the differentiation and proliferation of granulocyte and macrophage colonies in the bone marrow. There is a lot of clinical interest in this protein due to its ability to accelerate the granulocyte and macrophage recuperation after current therapeutic regimens for cancer and bone marrow transplantation and also its potential in treating myeloid leukaemia. The hGM-CSF has been introduced into clinical treatment with promising results. The recombinant form of this hematopoietic factor (rhGM-CSF) has been produced and purified from yeast, mammalian cells and bacteria, resulting in proteins that vary in composition, glycosidic content and structure. During the production process of this factor, specific quantitative assays are required. Here, we report the development and comparison of different assays carried out in order to quantify the cytokine during a production process. Three enzyme-linked-immunosorbent assays (ELISA) were developed to quantify the hGM-CSF derived either from bacteria or mammalian cells and compared with a bioassay based on the proliferation of hGM-CSF-responsive TF-1 cells. The immunoassays were carried out with the aim of devising quick and sensitive tests without the use of radioisotopes and to avoid the use of laborious, lengthy and subjected to inherent inaccuracies bioassays. The assays were developed using non-glycosylated rhGM-CSF (E. coli-derived protein) as a standard of quantitation, glycosylated rhGM-CSF expressed in mammalian cells, two monoclonal antibodies (MAbs M7E10 and M1B8) and polyclonal antibodies against the E. coli-derived rhGM-CSF. The following quantification assays were developed: a sandwich ELISA with two MAbs, a sandwich ELISA with MAbs and polyclonal antibodies, a competitive ELISA and a cell proliferation assay. All the methods were sensitive enough for the detection of the non-glycosylated rhGM-CSF in the upstream and the downstream processes. Different samples -consisting of non-glycosylated rhGM-CSF diluted in different media- were evaluate in order to analyse the influence of the protein’s environment in its quantification. Among the ELISAs developed in this work, the sandwich ELISA with two MAbs was the least influenced by the protein environment. This method shows also the highest intra and inter-assay reproducibility becoming the most appropriate assay to quantify non-glycosylated rhGM-CSF. The sandwich ELISA with monoclonal and polyclonal antibodies had the broadest linear range. All antibodies used in the development of these assays recognised both the non-glycosylated and the glycosylated cytokine. However, when the three ELISAs were used to quantify the glycosylated rhGM-CSF, they provided different concentrations of the protein and consequently, different values of specific activity were estimated. Only the competitive ELISA with MAb M7E10 provided a value of specific activity comparable with those earlier reported. A previous work demonstrated that MAb M7E10 has a similar affinity for glycosylated and non-glycosylated rhGM-CSF, mapping a region not involved in the glycosylation site. Therefore, the competitive ELISA was the only assay useful to quantify glycosylated rhGM-CSF comprising the use this particular MAb a more accurate way to determine the glycosylated molecule. The measurement of the biological activity of this hormone may be done in vitro by proliferation assays that use hGM-CSF responsive cell lines. The specific activity of different recombinant preparations of hGM-CSF differ and are most likely to be different from the specific activity of naturally occurring materials. In this case, there is no international standard for the glycosylated-derived rhGM-CSF and consequently, it cannot be quantified by bioassay. Finally, the developed
competitive ELISA becomes the method of choice for the quantification
of the glycosylated form of this cytokine during its production process,
from cell supernatants to the purified end product. |
|
Home | Mail to Editor | Search | Archive |
||