A Study of the VEGF-binding Ability of Aged Bevacizumab

DILSHER S. DHOOT, MD • TREVOR J. MCFARLAND • BINOY APPUKUTTAN, PhD • J. TIMOTHY STOUT, MD, PhD

INTRODUCTION
Treatment modalities for age-related macular degeneration (AMD) have evolved considerably over the past few years. Most recently, agents that inhibit angiogenesis have shown promise in treating choroidal neovascularization (CNV) secondary to AMD.¹ Vascular endothelial growth factor (VEGF)-A is a major angiogenic factor that contributes to the neovascularization observed in AMD.^2-7 To date, 2 anti-VEGF agents have received Food and Drug Administration (FDA) approval for the treatment of neovascular AMD: pegaptanib sodium (Macugen, [OSI] Eyetech/Pfizer) and ranibizumab (Lucentis, Genentech). In phase 3 trials of pegaptanib sodium (0.3 mg) injected intravitreally vs placebo therapy, 6% of patients had significant vision improvement vs 2% in the placebo group.⁸ Phase 3 trial results for ranibizumab were encouraging, with 95% of patients maintaining their vision, and 24.8 to 33.8% having an improvement in vision up to 3 lines (15 letters) after a year of treatment.⁹
Bevacizumab (Avastin, Genentech),¹⁰ a third anti-VEGF agent, is a humanized murine monoclonal IgG1 antibody against VEGF. Bevacizumab has FDA approval for the treatment of metastatic colorectal cancer and non-small lung cancer, and trials for advanced breast and renal cancer are promising.^11-13 Both bevacizumab and ranibizumab are derived from the same murine monoclonal antibody against VEGF. Bevacizumab is a full-length antibody, with 2 binding sites for VEGF, while ranibizumab is an antigen-binding fragment with only 1 binding site for VEGF.¹⁴
Although not FDA approved for intraocular administration, bevacizumab has been used as an off-label agent in the treatment of AMD. Initial research by Michels and colleagues¹⁵ demonstrated that systemic delivery of bevacizumab 5 mg/kg improved visual acuity (VA), as well as outcomes measured by optical coherence tomography (OCT) imaging. Systemic bevacizumab is not without adverse effects, including risk of hypertension and thromboembolic events.^16,17
Local delivery of bevacizumab, such as with intravitreal injection, may decrease the risk of adverse effects compared to those occurring with systemic delivery. Several retrospective reviews have shown intravitreal bevacizumab to be potentially as efficacious as ranibizumab and well tolerated in the short term.17-19
The usual intraocular dose of bevacizumab for the treatment of AMD is 1.25 mg (0.05 mL of stock). Bevacizumab is supplied in 4-mL (100 mg) and 16-mL (400 mg) preservative-free vials at a concentration of 25 mg/mL. The 4-mL vial is most commonly used in ophthalmic practice and is formulated in 240 mg α, α trehalose dihydrate, 23.2 mg sodium phosphate (monobasic, monohydrate), 4.8 mg sodium phosphate (dibasic, anhydrous), 1.6 mg polysorbate 20, and water for injection.When stored at 4°C and protected from light, unpierced vials of bevacizumab are stable for 18 months. Once a vial is pierced, the manufacturer recommends continued storage at 4°C, protection from light, and use within 8 hours.20 Given the minute dose of each intraocular treatment relative to the supplied stock of bevacizumab, there is potential for considerable waste of this medication if practitioners adhere to the 8-hour expiration, making bevacizumab cost-prohibitive for many patients.
To date, clinicians have been either adhering to manufacturer guidelines or using bevacizumab up to 14 days after preparation for the off-label treatment of neovascular AMD.¹⁶ In this study, we test the binding of fresh, aged up to 8 weeks after initial aliquot preparation, and freeze/thawed bevacizumab to VEGF in vitro to determine whether the binding capability of this molecule diminishes with time.

METHODS
Preparation of Bevacizumab
Vials of commercially available bevacizumab (4 mL: 25 mg/mL) were obtained. Using aseptic technique under a laminar flow hood (ISO Class 5), a licensed pharmacist punctured the seal with a Mini-Spike Dispensing Pin (Braun Medical Inc., Bethlehem, Pa). The bevacizumab was then divided into aliquots of approximately 0.12 mL in multiple 1-mL Luer-Lok syringes (Becton, Dickinson & Co., Franklin Lakes, NJ). A sterile cap was used to seal each respective syringe. Syringes were labeled, placed in light-protective brown plastic bags, and refrigerated at 4°C. One vial of bevacizumab yields approximately 25 x 0.12 mL aliquots. Bevacizumab was defined as “fresh” when obtained within 8 hours of the above-outlined preparation. One week old and 8-week-old bevacizumab were prepared as above and stored at 4°C in light-protective brown plastic bags for 1 week and 8 weeks, respectively. Additional preparations of bevacizumab were stored at room temperature (25°C) for 19 days in light-protective brown bags.
The bevacizumab preparations were serially diluted to 250 ng/mL in phosphate buffer saline (PBS). Aliquots of fresh bevacizumab, 250 ng/mL, were frozen at -80°C for 20 minutes and thawed at 25°C for 30 minutes. This was done once for 1x freeze/thawed bevacizumab and three times for the 3x freeze/thawed bevacizumab. In addition, separate aliquots of fresh bevacizumab, 250 ng/mL, were denatured by boiling to 100°C. The negative control was composed of PBS alone, without any bevacizumab.
Measurement of Bevacizumab Binding to VEGF
Bevacizumab binding to VEGF was measured using a VEGF Sandwich ELISA Kit (Chemicon,Temecula, Calif ). VEGF standards of 4000, 2000, 1000, and 200 pg/mL were run with each enzyme-linked immunosorbent assay (ELISA) assay. VEGF165 protein provided in the ELISA kit was premixed for 60 minutes at 37°C in an automated shaker with the various preparations of bevacizumab: fresh, 1-week at 4°C, 8-weeks at 4°C, fresh freeze/thawed x 1, fresh freeze thawed x 3, 19-day-old at 25°C, and fresh boiled at 100°C. The respective mixtures were then loaded into ELISA wells and the standard assay protocol was followed according to the manufacturer’s instructions. Each bevacizumab preparation was run in at least duplicate. ELISA plates were read in a Victor3 multilabel reader (PerkinElmer, Shelton, Conn) at 490 nm. Statistical analysis was performed using the standard Student’s t-test.
Bacterial Culture
Fresh, 1-week old at 4°C, and 8-week-old at 4°C bevacizumab was plated on tryptic soy broth (TSB) plates and incubated at 37°C for 48 hours. Growth or lack of bacterial contamination was confirmed visually. A positive control plate with fresh sputum was also cultured for 48 hours.

RESULTS
Results of ELISA assays run in quadruplicate with various bevacizumab preparations serially diluted to 250 ng/mL and premixed with VEGF protein 2000 pg/mL are shown in the Figure. Average binding, as measured by absorbance units, for the first 6 preparations of VEGF were between 0.052 and 0.056. Average binding for boiled bevacizumab (heat-inactivated control) and the PBS negative control were 0.122 and 0.125, respectively. Preparations 2 through 6 showed no statistically significant difference from the fresh bevacizumab (preparation 1) (P>.05). Preparations 1 through 6 were all significantly different from the negative control, P<.01. The boiled bevacizumab, the heat-inactivated control, was not statistically different than the PBS-negative control (P=.87). Results of ELISA assay run in duplicate with the same bevacizumab preparations serially diluted to 250 ng/mL and premixed with VEGF protein 4000 pg/mL were also statistically significant in difference, confirming the data of the first assay (data not shown). After 48 hours of incubation at 37°C, TSB plates plated with fresh, 1-week-old at 4°C, and 8-week-old at 4°C bevacizumab showed no indication of bacterial growth, indicating that our sterile aliquoting procedure was not compromised. The positive control, plated with fresh sputum, did show bacterial growth after 24 hours.

DISCUSSION
Ranibizumab has recently been granted FDA approval for intravitreal administration for the treatment of neovascular AMD. However, the cost of this medication, $1950 per intravitreal injection, is a concern for many patients and providers. Intravitreal bevacizumab has been shown by others to be an effective treatment for neovascular AMD in at least the short term.16-18With the rising costs of modern health care, it is vital to ensure widespread access to novel therapies that are effective and economical. Depending on the dose and efficiency of preparation, bevacizumab costs from $17 to $50 per intravitreal injection.²¹ Strict adherence to manufacturer guidelines would limit the use of bevacizumab to an 8-hour period once a vial of bevacizumab is punctured. This study provides objective data supporting a longer expiration time, with aseptic preparation and correct storage. These data are in concordance with a very recent report, which demonstrated up to 15.9% degradation of anti-VEGF activity of bevacizumab after withdrawal from a vial and storage at 4°C for 6 months.²² Our results indicate that there is no difference in binding between fresh and up to 8-weeks postpreparation of bevacizumab to VEGF protein as measured by sandwich ELISA. Similarly, no difference in binding was observed between fresh bevacizumab and bevacizumab that had been frozen and thawed up to 3 times.
Interestingly, bevacizumab stored at room temperature for 19 days also showed no difference in VEGF-binding from newly prepared bevacizumab.While the storage of bevacizumab at room temperature is not recommended, this evidence further demonstrates the stability of this molecule as measured by its ability to bind VEGF.
After aseptic preparation, bevacizumab prepared up to 8 weeks before culture is free of bacterial contaminants as measured by TSB culture plates. The aseptic preparation of aliquots is of vital importance and should be done by a licensed pharmacist. Single puncture of the medication vial with a device like the Mini-Spike Dispensing Pin is likely to prevent contamination during aliquot preparation.
Based on guidelines set forth by USP Chapter 797, a medication prepared in the same manner as bevacizumab (see Methods) for patient use is classified as low risk and, in the absence of passing a sterility test, can be stored for 2 weeks, if refrigerated.²³ In a retrospective review of 50 patients, bevacizumab was used up to 14 days after aseptic preparation. No serious drug-related adverse effects were identified after 3 months of follow-up.¹⁶ We have shown that bevacizumab can effectively bind VEGF up to 8 weeks after preparation. Therefore, storage for 2 weeks at 4°C with protection from light seems reasonable. When assaying for sterility, storage for longer periods of time may be a viable option. Our study adds support to the recently published paper on the storage and anti- VEGF activity of bevacizumab.²²

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All the authors of this article are affiliated with the Casey Eye Institute of Oregon Health & Science University in Portland. Dr. Stout can be reached at (503) 494-2435 or by e-mail at stoutt@ohsu.edu. The authors have no proprietary interest in these materials. This study was supported in part by an unrestricted grant to the Casey Eye Institute by Research to Prevent Blindness, Inc. (New York), and the Clayton Foundation for Research in Houston.