Original articleMechanically-induced aggregation of the monoclonal antibody cetuximabAgrégation mécaniquement induite de l’anticorps monoclonal cétuximab
Introduction
The stability of biotech products is a more complex issue than for other pharmacological classes. With the emerging use of therapeutic proteins such as antibodies, this problem is increasingly important and tackling it requires appropriately designed study and relevant analytical approaches. These sensitive products could undergo more complex degradation pathways during the various manipulation steps than classical drugs [1], [2]. In contrast to chemical drugs, the bioactivity of therapeutic proteins depends on more quality attributes since their large size, compositional variety and amphipathic characteristics induce specific behavior such as conformational stability, folding and unfolding, reversible or irreversible denaturation or aggregation Thus, it is difficult to assess simply the stability of biotechnology-issued anticancer drugs such as antibodies. Moreover, when no direction is given by the MAH, it may be of interest to investigate the stability profile of a given product, under specific conditions of use.
Antibody degradation pathways include both chemical and also physical alterations [1], [2], [3]. Chemical degradation implies modification of covalent bonds such as deamidation, oxidation, disulfide bridge shuffling. It is well accepted that aggregation is the primary response of monoclonal antibodies to thermal, chemical or mechanical stresses [4]. Aggregation is the main physical instability which also includes protein unfolding, adsorption or non-covalent interaction of native protein. One of the most underestimated origins of aggregation is mechanical stress which can occur during manipulations: shaking or stirring, shearing, exposure to hydrophobic gas interface (bubbling during rapid sampling by syringe or filtration) [5]. Major implications of aggregation could be the loss of efficacy and/or the appearance of toxic effects such as immunological reactions [6]. Unfortunately, these data are almost never available in manufacturer drug information files or only under very generic sentences such as “avoid shaking”.
Cetuximab is a 146 kDa chimeric IgG1κ, produced by the mammalian cell line Sp2/0 which is specifically directed against the epidermoid growth factor receptor (EGFR) over expressed in several tumor cells. It is largely used in the treatment of colorectal and head and neck cancers in association with the camphotecin derivative, irinotecan or radiotherapy. Until now, cetuximab was formulated in a 2 mg/ml solution of phosphate buffer pH 7.0 and, considering the presence of visible aggregates, it was recommended to filter the solution at 0.22 μm before use. This manipulation implied several tedious and time-consuming manipulations and could also induce possible external contamination. In order to resolve this quality problem, a new formulation has been developed and authorized for marketing. It consists of a new concentration at 5 mg/ml with modified composition including tensioactive polysorbate 80 and glycine to prevent aggregation, thus avoiding the filtration step.
The objective of this study was to assess the aggregation of cetuximab under an experimental mechanical stress and to explore its possible mechanism. Moreover, although it is usually recommended to infuse cetuximab without dilution, it also appeared interesting to test its stability in diluted solution which could be used in particular clinical situations. The appearance of visible aggregates was considered to express the response of the protein to stress; the time course of aggregation was followed by turbidimetric analysis at several wavelengths. Dynamic light scattering analysis (DLS) were also performed to explore the aggregation mechanism. Size exclusion (SE-HPLC) is used to determine the presence of soluble aggregates and to estimate the non-aggregated fraction of cetuximab. The chemical stability of non–aggregated proteins was also estimated by cation-exchange high performance liquid chromatography (CEX). Finally, the hypothesis that formed particles were non-chemically modified aggregates was tested by peptide mapping of the trypsic plus endopeptidase digests of aggregated samples.
Section snippets
Reagents and materials
Guanidine hydrochloride (GnHCl), iodoacetic acid (99% purity), sodium chloride (NaCl), acetylated trypsin, endopeptidase, dithiothreitol (DTT), ammonium bicarbonate, acetonitrile (HPLC grade) were purchased from Sigma–Aldrich (St Louis, MO, USA). Disodium hydrogen phosphate, sodium azide, sodium sulfate, potassium dihydrogenophosphate, trifluoroacetic acid (TFA; peptide grade) were acquired from Merck (Darmstadt, Germany). Formic acid (FA) was purchased from Prolabo (Fontenay-sous-Bois, France).
Turbidimetry
Both formulations of cetuximab at 2 mg/ml exhibited very different kinetic profiles of aggregation within the 24h-stirring period. Fig. 1 shows the turbidity at 350, 410 and 550 nm as a function of stirring time. The turbidity of the OF increased dramatically for the three wavelengths up to 24 h of stirring, reaching 0.809 ± 0.101 absorbance unit (AU) at 350 nm corresponding to an approximately 70-fold increase in aggregation multiplied as compared to the non-stirred sample (Table 1). After 30 min,
Discussion
The main result from this work is that an appreciable aggregation of the monoclonal antibodies cetuximab can be easily obtained by a mechanical stress which is susceptible to increase the air-liquid interfacial surface. Another result is that a modified formulation buffer including polysorbate 80, glycine and citrate buffer at pH 5.5 is able to strongly limit the physical instability of cetuximab.
The protein literature contains many references to mechanically-induced denaturation of proteins.
Conclusion
In conclusion, we have demonstrated that cetuximab can readily form aggregates in response to weak mechanical stresses such as stirring. The aggregates are mainly due to an increased exposure of antibody molecules to air/liquid interface and are formed without chemical alteration of the antibody structure. As compared to the old formulation, the recent commercially available formulation is less prone to aggregation. This improvement seems mainly due to the stabilization of cetuximab by
Conflicts of interests
None.
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