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Technology (including: robots for production, Incompatibilities, drug production and analytics, CRS)
Proteomic approach to investigating the molecular integrity of infliximab after reconstitution and dilution in typical hospital conditions
  1. A. Salmerón-García,
  2. N. Navas Iglesias,
  3. J. Cabeza Barrera,
  4. A. Lario Simon,
  5. M.V. Longobardo Polanco,
  6. R. Ortega López,
  7. L.F. Capitán Vallvey
  1. 1Hospital Universitario San Cecilio, Pharmacy, Granada, Spain
  2. 2University of Granada (Spain), Department of Analytical Chemistry, Granada, Spain
  3. 3Instituto de Parasitología y Biomedicina ‘López-Neyra’ (CSIC), Proteomic Unit, Granada, Spain

Abstract

Background The active substance of Remicade is infliximab. It is a chimeric human-murine monoclonal antibody directed against tumour necrosis factor α (TNFα), manufactured from a recombinant cell line. Remicade is presented as powder for concentrate for solution for infusion (100 mg/phial), to be reconstituted with water for injections, diluted with saline and thereafter administered via intravenous infusion.

Purpose The purpose of this study was to investigate the suitability of a proteomic approach using MALDI-TOF mass spectrometry to test the molecular integrity of infliximab when reconstituted and diluted in the usual hospital conditions.

Materials and methods Infliximab was reconstituted in water for injection (10.0 mg/ml) and diluted with NaCl 0.9% solution (2.0 mg/ml and 0.5 mg/ml). 10 μL of the samples containing the antibody were reduced with DTT and alkylated by iodoacetamide in darkness for 30 min and they were digested by trypsin at pH 8.5 for 4 h at 37°C. The digest was loaded onto the MALDI target plate using 5 mg/ml α-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid, 50% acetonitrile as the matrix. Each digest was analysed five times by MALDI-TOF mass spectrometry using a Voyager DE-PRO (Applied Biosystems) in positive reflector mode.

Results The peptide fingerprint map (PFM) of the infliximab was obtained for the reconstituted sample and for the diluted samples right after their preparation. In this way, the molecular integrity of infliximab can be described and characterised.

Conclusions This proteomic approach for the analysis of infliximab is suitable to be used in a long stability study of the antibody reconstituted, diluted and stored refrigerated (4°C) and frozen (-20 °C) since possible changes in the antibody structure could be detected by changes in the corresponding PFM. This study stability is currently being performed by our research group.

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