September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Optimal Buffer for Storage of Ranibizumab at Low Concentrations
Author Affiliations & Notes
  • Hui Yee Chua
    Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore
  • Rupesh Vijay Agrawal
    National Healthcare Group Eye Institute, Tan Tock Seng Hospital, Singapore, Singapore
  • Tina T Wong
    Singapore Eye Research Institute, Ocular Drug Delivery Group Singapore National Eye Centre, Singapore, Singapore
  • Peter Preiser
    Biological Sciences, Nanyang Technological University , Singapore, Singapore
  • Subbu Venkatraman
    Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore
  • Footnotes
    Commercial Relationships   Hui Yee Chua, None; Rupesh Agrawal, None; Tina Wong, None; Peter Preiser, None; Subbu Venkatraman, None
  • Footnotes
    Support  NHG Thematic Grant/13008
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 3330. doi:
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    • Get Citation

      Hui Yee Chua, Rupesh Vijay Agrawal, Tina T Wong, Peter Preiser, Subbu Venkatraman; Optimal Buffer for Storage of Ranibizumab at Low Concentrations. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3330.

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

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Purpose : Choroidal neovascularization is one of the contributing causes to vision loss for Age-related Macular Degeneration (AMD). Ranibizumab is an antigen-binding fragment (Fab) of a monoclonal antibody that binds to Vascular Endothelial Growth Factor (VEGF), thereby reducing neovascularization. The aim of this work is to identify the optimal storage buffer for ranibizumab at the 50% inhibition (IC50) concentrations. Ranibizumab was evaluated by quantifying the VEGF binding ability after storage in various buffers. The loss of activity due to storage in inappropriate buffers would lead to erroneous interpretation of the results.

Methods : Ranibizumab was stored for a period up to 7 days at 37oC in microtubes at concentrations between 6.25 to 50 ng/ml. The storage buffers used included: Phosphate buffer saline (PBS)- a physiologically simulating buffer; Intrinsic buffer- a cocktail containing trehalose, Tween 20 and histidine used for storing the commercial ranibizumab; Bovine Serum Albumin (BSA) buffer- BSA dissolved in PBS; Serum buffer- a cocktail reported by Genentech for diluting ranibizumab in a pharmacokinetic study consisting of BSA, Tween 20 and EDTA diluted in PBS. Ranibizumab activity in the different samples was subsequently evaluated by testing its activity to bind to VEGF using Enzyme Linked Immunosorbent Assay (ELISA) and comparing it to freshly diluted drug.

Results : Storage of ranibizumab in both PBS and BSA buffer resulted in a rapid loss of activity as measured by ELISA. Even at the highest concentration of 50ng/ml no activity could be detected in PBS (P= 0.0001) after one day of storage. In BSA buffer a similar rapid loss was observed and only 80% of activity was measurable after storage for one day. In contrast serum buffer showed no loss of activity after one day storage even at concentrations of ranibizumab as low as 6.25 ng/ml. In intrinsic buffer, no significant difference in the ranibizumab activity was detected after 7 days at concentrations as low 6.25 ng/ml.

Conclusions : In this study, ranibizumab was shown to lose its VEGF binding ability when stored at low concentrations in PBS and with BSA buffer. Both of these buffers are frequently reported to be used for ranibizumab dilution. However, VEGF binding was maintained when ranibizumab was stored in intrinsic and serum buffer. These findings will be helpful when working with ranibizumab at low nanogram concentrations during in-vitro drug release or transport.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.


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