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. 2024 Sep 19;16(9):1222.
doi: 10.3390/pharmaceutics16091222.

Comprehensive Analysis of Cetuximab Critical Quality Attributes: Impact of Handling on Antigen-Antibody Binding

Alicia Torres-García  1   2 , Anabel Torrente-López  2   3 , Jesús Hermosilla  2   3 , Amparo Hernández  3 , Antonio Salmerón-García  2   4 , José Cabeza  2   4 , Natalia Navas  2   3
Affiliations

Comprehensive Analysis of Cetuximab Critical Quality Attributes: Impact of Handling on Antigen-Antibody Binding

Alicia Torres-García et al. Pharmaceutics. .

Abstract

Background/objectives: Cetuximab, formulated in Erbitux® (5 mg/mL), is a therapeutic monoclonal antibody (mAb) widely used in several cancer treatments. Currently, there is insufficient knowledge about the behavior of cetuximab with regard to the risk associated with its routine handling or unintentional mishandling in hospitals. Forced degradation studies can simulate these conditions and provide insights into the biophysical and biochemical properties of mAbs.

Methods: In this study, we conducted a deep physicochemical and functional characterization of the critical quality attributes of cetuximab in control samples and under controlled degraded conditions, including freeze-thaw cycles, heat, agitation, and light exposure. To achieve this purpose, we used a set of proper analytical techniques, including CD, IT-FS, DLS, SE/UHPLC-UV, UHPLC-MS/MS, and ELISA, to check functionality based on antigen-antibody binding.

Results: The results revealed that light exposure was the stress stimuli with the greatest impact on the drug product, leading to the formation of non-natural oligomers, fragmentation, and oxidation of methionine residues. Additionally, cetuximab (Erbitux®, 5 mg/mL) showed a tendency to aggregate when submitted to 60 °C for 1 h. In terms of functionality, cetuximab (Erbitux®, 5 mg/mL) samples were found to be affected when subjected to freeze-thaw cycles, 60 °C (1 h), and when exposed to light (daylight with room temperature excursion and accelerated light exposure).

Conclusions: Thus, we suggest that Erbitux® (5 mg/mL) should be shielded from these environmental conditions, as they compromise both the safety and efficacy of the drug product.

Keywords: ELISA; Erbitux® analysis; cetuximab characterization; comprehensive analysis; forced degradation; peptide mapping-RP/UHPLC-MS/MS; stability study.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 4
Figure 4
Cetuximab primary sequence. Regions highlighted in green, orange, and blue correspond to CDR1, CDR2, and CDR3, respectively. Adapted from Ayoub D et al. [34].
Figure 1
Figure 1
Far-UV CD spectra of cetuximab (5 mg/mL) stressed and control samples.
Figure 2
Figure 2
Intrinsic tryptophan fluorescence emission spectra of cetuximab (Erbitux®, 5 mg/mL) stressed and control samples.
Figure 3
Figure 3
Representative chromatograms SE/UHPLC-UV cetuximab samples. (A) Freeze-thaw cycles stress, (B) agitation stress, (C) temperature stress, and (D) light stress. The retention times indicated in the chromatograms are the means of three replicates.
Figure 5
Figure 5
Relative abundance of most important PTMs identified in cetuximab. (A) Oxidations, (B) pyroglutamination, (C) glycosylation located on H-N88 residue, and (D) glycosylation located on H-N299 residue. Error bars depict standard deviation (n = 3).
Figure 6
Figure 6
Comprehensive glycan profile of cetuximab. The various isoforms are arranged in decreasing order of relative abundance (%) based on the control sample both in (A) H-N88 residue and (B) H-N299 residue.
Figure 7
Figure 7
Calibration curve for cetuximab ELISA analysis. (A) Standard curve. (B) Logarithmic scale curve.
Figure 8
Figure 8
ELISA binding assay comparing control and stressed samples. (A) Freeze-thaw cycles stress, (B) agitation stress, (C) temperature stress, and (D) light stress. Conditions marked with asterisk (*) showed statistical differences when compared to the control.
See this image and copyright information in PMC

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