RESUMEN
Nimotuzumab, an IgG that recognizes the epidermal growth factor receptor (EGF-R) overexpressed in some tumors, is used in the treatment of advanced head and neck cancer. For the quantification of this protein in cell culture supernatants, protein G-HPLC affinity chromatography is used due to its high affinity and specificity for antibodies of this class. The technique relies on the comparison of the area under the curve of the elution peak of the samples to be evaluated versus to a calibration curve of well-known concentrations and was validated by assessment of its robustness, specificity, repeatability, intermediate precision, accuracy, linearity, limit of detection, limit of quantification, and range. According to results of the study all validation parameters fulfilled the preestablished acceptance criteria and demonstrated the feasibility of the assay for the analysis of samples of cell culture supernatant as well as drug product.
RESUMEN
Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study reports the full characterization of the mAb N-glycosylation and the consistency observed in several production batches from a perfusion mode culturing system that lasted between 68 and 150 days. It confirms that the N-glycan structures of Nimotuzumab expressed in the NS0 murine myeloma cell line are of the murine type. They consist mainly of fucosylated G0, G1 and G2 oligosaccharides, which are normally found in the CH2 region of IgG. Other minor species found were high mannose and sialylated structures. A small portion of the glycans were sialylated (â¼12%) and the only type of sialic acid detected was N-glycolyl-sialic acid, α2,6-linked to Gal. No Galα1-3Gal moieties were detected.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Oligosacáridos/química , Conformación de Carbohidratos , Cromatografía Líquida de Alta Presión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.