June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
The increased stability of FpFs compared to monoclonal antibodies
Author Affiliations & Notes
  • Hanieh Khalili
    Pharmaceutics, UCL School of Pharmacy, London, United Kingdom
    NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital & UCL Institute of Ophthalmology, London, United Kingdom
  • Steve Brocchini
    Pharmaceutics, UCL School of Pharmacy, London, United Kingdom
    NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital & UCL Institute of Ophthalmology, London, United Kingdom
  • Peng Tee Khaw
    NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital & UCL Institute of Ophthalmology, London, United Kingdom
  • Ashkan Khalili
    NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital & UCL Institute of Ophthalmology, London, United Kingdom
  • Garima Sharma
    Pharmaceutics, UCL School of Pharmacy, London, United Kingdom
    NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital & UCL Institute of Ophthalmology, London, United Kingdom
  • Footnotes
    Commercial Relationships Hanieh Khalili, None; Steve Brocchini, None; Peng Khaw, None; Ashkan Khalili, None; Garima Sharma, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 377. doi:
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    • Get Citation

      Hanieh Khalili, Steve Brocchini, Peng Tee Khaw, Ashkan Khalili, Garima Sharma; The increased stability of FpFs compared to monoclonal antibodies. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):377.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Fab-PEG-Fab (FpF) molecules are new IgG mimetics that are designed to be more stable than therapeutic IgG monoclonal antibodies (MAbs). The accessible disulfides in MAbs are prone to scrambling and the hinge region is susceptible to cleavage. The FpFs have been designed to alleviate these intrinsic limitations of MAbs. The aim of this study was to evaluate FpF stability to storage and to freeze-drying.

Methods: Storage stability studies were conducted in glass vials at 37˚C with bevacizumab used in its pharmaceutical formulation and the corresponding bevacizumab derived FpF in PBS (pH 7.4) at double the bevacizumab concentration. For pre-formulation freeze-drying studies, bevacizumab was eluded over a PD-10 column to remove excipients and both the de-formulated bevacizumab and FpF were subjected to freeze-drying. Primary drying was performed at -20˚C for 12 hours at 100 μBar, followed by secondary drying at 20˚C for 2 hours. Biacore binding analyses were performed using a VEGF functionalised CM3 chip (92 RU).

Results: The FpF did not aggregate or display any light/heavy chain dissociation after 30 days storage at 37˚C as liquid (0.250 mg/mL[SB1] ). Chain dissociation and aggregation were observed for the pharmaceutical formulation of bevacizumab (0.125 mg/mL) after 7 days (37˚C). Both the FpF and deformulated bevacizumab were subjected to freeze-drying. SDS-PAGE analysis indicated there was no loss of FpF, or the occurrence of de-PEGylation or aggregation for the FpF molecule. Binding of FpF to VEGF was maintained after reconstitution. In contrast there appeared to be loss of bevacizumab as observed by decreased band intensity on SDS-PAGE. Particulates were observed when bevacizumab was reconstituted.

Conclusions: The FpF is a MAb mimetic that appears to be less prone to aggregation and precipitation than a MAb. The increased stability of FpFs indicates they have the potential to be fabricated into longer acting dosage forms than may be possible with MAbs.

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