Original article
Mechanically-induced aggregation of the monoclonal antibody cetuximabAgrégation mécaniquement induite de l’anticorps monoclonal cétuximab

https://doi.org/10.1016/j.pharma.2009.05.008Get rights and content

Summary

This study focuses on the mechanically-induced aggregation of the anticancer monoclonal antibody cetuximab and its mechanism by comparing two commercially-available formulations (old formulation [OF]: 2 mg/ml in phosphate buffer; new formulation [NF]: 5 mg/ml in citrate buffer with polysorbate 80 and glycine). Cetuximab aggregation under stirring was followed during a 24 h period and several methods were used to describe the aggregation kinetics (turbidimetry, size-exclusion high performance liquid chromatography, cation-exchange chromatography, dynamic light scattering and peptide mapping). Depending on the formulation, the aggregation process followed different kinetics: biexponential for the OF and mono-exponential for the NF. The percentage of aggregation after 24 h stirring-period was about 25% for the OF but was only 2% for the NF corresponding to a ten-fold improvement, demonstrating a strong protecting effect of polysorbate and glycine. 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. This improvement of cetuximab stability by a new formulation seems linked to the stabilisation of cetuximab under dimeric form by a higher protein concentration and the presence of stabilizing excipients.

Résumé

Ce travail étudie l’agrégation induite mécaniquement de l’anticorps monoclonal cétuximab en comparant le comportement de deux formulations commerciales (ancienne formulation [AF] : 2 mg/ml dans un tampon phosphate ; nouvelle formulation [NF] : 5 mg/ml dans un tampon citrate contenant du polysorbate 80 et de la glycine). L’agrégation du cetuximab sous agitation a été suivie pendant 24 heures et plusieurs méthodes ont été utilisées pour décrire la cinétique d’agrégation ; turbidimétrie, chromatographie d’exclusion de gel, chromatographie ionique, diffraction dynamique de la lumière et carte peptidique. En fonction de la formulation, le processus d’agrégation suit des cinétiques différentes : biexponentielle pour l’ancienne formulation et mono-exponentielle pour la nouvelle. Le pourcentage d’agrégation après 24 heures était d’environ 25 % pour l’ancienne formulation et seulement de 2 % pour la nouvelle correspondant à une amélioration de la stabilité supérieure d’un facteur 10 et démontrant l’effet stabilisateur majeur de l’addition de polysorbate de glycine. Les aggrégats sont essentiellement liés à une augmentation de l’exposition de l’anticorps en solution à l’interface liquide/air et sont formés sans altération chimique de la structure. L’augmentation de la stabilité du cétuximab au stress mécanique dans la nouvelle formulation semble liée à la formation d’une forme dimérique non-covalente due à la concentration plus élevée en anticorps et à la présence d’agents stabilisants.

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.

References (30)

  • C. Balestrieri et al.

    Second derivative spectroscopy of proteins: studies on tyrosyl residues

    Anal Biochem

    (1980)
  • C. Chumsae et al.

    Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

    J Chromatogr B

    (2007)
  • E.Y. Chi et al.

    Physical stability of proteins in aqueous solution: mechanism and driving forces in non-native protein aggregation

    Pharm Res

    (2003)
  • H. Schellekens

    Bioequivalence and the immunogenicity of biopharmaceuticals

    Nat Rev Drug Discov

    (2002)
  • Y.F. Maa et al.

    Protein denaturation by combined effect of shear and air-liquid interface

    Biotechnol Bioeng

    (1997)
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