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Preserved bevacizumab (Avastin®) eye drops for application in multidose containers – an in-vitro characterisation

BMC Veterinary Research volume 21, Article number: 129 (2025) Cite this article

Abstract

Purpose

Monoclonal antibodies have made an immense contribution to the treatment of various human diseases. We aimed at investigating an affordable treatment option for veterinary patients with corneal neovascularization by adding the preservative benzalkonium chloride (BAC) to bevacizumab (Avastin®) for usage in multidose containers. A comprehensive analytical similarity assessment of preserved and unpreserved bevacizumab after dilution and storage was carried out.

Methods

Diluted and preserved bevacizumab was analysed at different time points for a 4-week period and compared with unpreserved bevacizumab at the same concentrations at each time point. Native-PAGE, immunoblotting and HP-SEC were used to observe aggregation and degradation. DLS provided information about particle size and dispersity. Bevacizumab quantified by ELISA was conducted to determine its biological activity. Dose response curves and cell migration assays were performed to detect possible toxic effects and determine biological activity and efficacy of the drug using HUVECs.

Results

Native-PAGE, immunoblotting and HP-SEC analysis did not show any changes or degradation products in the presence of BAC and after storage compared to unpreserved bevacizumab. The overlapping intensity-based particle size distribution obtained from DLS showed similarity in all tested groups and homogeneity was maintained. ELISA accurately detected bevacizumab at different concentrations. HUVECs incubated with preserved or unpreserved bevacizumab showed a comparable effect on cell migration. No decrease in cell viability was detected.

Conclusion

Equivalence tests demonstrated that bevacizumab is stable after dilution, storage and preservation with BAC. Our study shows that preserved bevacizumab applied in mutidose containers can be considered as a cost-effective alternative to the otherwise single-dose treatments.

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Introduction

Corneal neovascularization (CNV) significantly contributes to ocular disorders, such as superficial pigmentary keratitis, chronic superficial keratitis and consequently rejection of corneal transplantations [1, 2]. Inflammation and other pathological insults disrupt the corneal angiogenic privilege and promote angiogenesis in the physiologically avascular cornea [3]. Although CNV is initially needed after injuries to clear infections, initiate wound healing, and prevent stromal melts, it comes with several disadvantages. Corneal neovascularization leads to tissue scarring, lipid deposition, edema, and potentially sustains inflammation that may impede visual acuity [4, 5]. Furthermore, it's noteworthy that CNV not only affects human patients, leading to a 12% decrease in visual acuity, but it also plays a substantial role in veterinary ophthalmology, where the cornea is involved in approximately 48% of reported lesions in dogs [6, 7]. Of these lesions, 30.8% are inflammatory keratopathies like ulcerative keratitis, pigmentary keratitis and chronic superficial keratitis (CSK) which are also associated with CNV [7]. Moreover, chronic inflammation of the ocular surface also contributes significantly to corneal neoplasm development, as evidenced by the occurrence of corneal vascular neoplasia in canine and feline patients with a history of chronic ocular disease, as well as the emergence of corneal B-cell lymphoma in horses with chronic immune-mediated keratitis [8,9,10,11].

In addition, CNV accompanies common causes of corneal blindness such as keratoconjunctivitis sicca in conjunction with pigmentary keratitis [12].

Typical treatment options include topically or systemically administered corticosteroids, cyclosporine and non-steroidal anti-inflammatory drugs, alongside with different surgical approaches [13,14,15]. However, these drugs show variable success in the clinics and myriad adverse effects. Therefore, proper medication options are still elusive and their development is fundamental to treat CNV and maintain corneal clarity, avascularity and vision [16].

A balance of antiangiogenic and angiogenic factors maintain the avascular state of the cornea [17]. If angiogenic factors predominate, the balance is lost and corneal vascularization is initiated. One key aspect is the action of vascular endothelial growth factor A (VEGF-A) on its receptors (mainly VEGFR-1 and VEGFR-2), as shown in experimental models of CNV [18]. In line with these observations, VEGFR-1 and VEGFR-2 are expressed in canine healthy and diseased eyes and pathological conditions induce their upregulation [19].

Pharmacological inhibition of VEGF‐A has revolutionized human patients' care with diseases driven by pathological neovascularization [20,21,22,23,24,25,26,27]. One of the most potent VEGF inhibitors is bevacizumab (Avastin®, Roche). Bevacizumab is a recombinant humanized anti-VEGF immunoglobulin G1 (IgG 1) antibody and was recently administered safe and efficacious to treat dogs with CNV [28]. It was administered via a single-dose vial of 0.5 mL for each treatment time point to address concerns of stability and safety. Of note—despite its efficacy—this treatment regime is not suitable for veterinary practice because of the increased consumption and high costs when the medication is applicated in single dose vials [29]. Approximately two to three mL are needed per month to treat one eye according to the published dosages in single-dose vials [30,31,32]. Hence, the aim of this study was to overcome this problem by manufacturing bevacizumab eye drops suitable for multidose containers.

The addition of an antimicrobial preservative, most frequently the quaternary ammonium benzalkonium chloride (BAC), is necessary to prevent bacterial contamination of the multidose eye drops [33, 34]. Few studies already used preserved bevacizumab eye drops although the compatibility of the solutions is unknown [1, 30, 31]. We hypothesized that bevacizumab preserved with BAC is a safe and cost-effective alternative formulation to single-dose treatments. The aim of this study was to perform a comprehensive analytical similarity assessment. We investigated the structural, physicochemical and functional similarities of preserved and unpreserved bevacizumab after dilution and storage to assess potential differences, which could impact the clinical performance.

Results

General properties

Avastin® (25 mg/mL) showed a pH value of 6.14. When diluted with sterile 0.9% saline solution the pH value slightly dropped to 6.05. However, the values remained stable when diluted with sterile 0.9% saline solution and preserved with BAC 0.01% (pH 6.05 ± 0.00). No particles were visible macroscopically (Data not shown).

Biochemical properties by native PAGE, SDS PAGE and ELISA

Native PAGE is a quick diagnostic tool for detection of antibody aggregation. It was performed to detect any possible gross degradation or aggregation by separation of proteins and protein complexes in their native state [35]. Only one band was observed, indicating the presence of monomeric mAbs, when 5 µg of preserved or unpreserved bevacizumab were loaded per lane. Similar banding patterns and no indication of aggregation or degeneration was detected in all groups (Fig. 1a). Density obtained of the B-T0 protein band was similar to preserved (BBAC-T0), preserved and stored (BBAC-T4) and diluted and stored (B-T4) protein bands (Fig. 1b). Similarly, we did not detect aggregation in Coomassie blue-stained gels at different contrast settings. Our results were reproduced with different amounts (0.1 µg and 1 µg) loaded (Suppl. Figure 1) as well. Native gels are presented in Suppl. Figure 2.

Fig. 1
figure 1

Native PAGE migration of B-T0, BBAC-T0, B-T4, BBAC-T4 and A. a Representative native PAGE gel shows the electrophoretic pattern of the native proteins stained with Coomassie brilliant blue (5 µg loaded per lane). b Densitometry was performed on a representative gel loaded with 5 µg per lane. The density was normalized to B-T0

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Immunoblotting (SDS PAGE) was used to separate the proteins based on their mass. This allows a qualitative analysis and detection of any possible changes that may affect light or heavy chains of the samples [36,37,38,39]. The samples resolved into heavy (~ 50 kD) and light IgG chains (~ 25 kD) in all samples when either 10 ng, 25 ng or 50 ng were loaded per lane (Fig. 2a). Again, we did not observe any signs of aggregation. Densitometric analysis of the heavy (Fig. 2b) and light chain (Fig. 2c) revealed comparable mean intensities of the bands in all groups. Native blots are presented in Suppl. Figure 3.

Fig. 2
figure 2

Immunoblotting of B-T0, BBAC-T0, B-T4, BBAC-T4 and A. a Representative example of bevacizumab detected after immunoblotting on a PVDF membrane. 10 ng (1), 25 ng (2) or 50 ng (3) were loaded per lane. Two distinct bands of 25 and 55 kDa were detected, corresponding to the light and heavy chains of the IgG molecule. (b/c) Densitometry analysis of the heavy (b) and light (c) chain (n = 2)

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ELISA quantification was performed to determine the binding activity to VEGF and thus biological activity of bevacizumab after dilution and storage with or without preservative [40]. A standard curve was established using a fresh vial of Avastin® between 0.625 ng/mL and 40 ng/mL. The detected range resulted in a linear relationship (Fig. 3a). The different bevacizumab samples were diluted to a concentration of 5 ng/ml. The calculated concentrations were comparable in all groups (Fig. 3b).

Fig. 3
figure 3

ELISA of B-T0, BBAC-T0, B-T4 and BBAC-T4. a An ELISA standard curve was established to detect 0.625 ng/mL to 40 ng/mL of bevacizumab. The standard curve was generated (n = 3) and data points are presented as means. The dotted line represents the best fit determined by linear curve fitting (r2 = 0.9962). b Bevacizumab samples were analyzed by ELISA at a concentration of 5 ng/ml (n = 4). From the obtained OD values, concentrations were calculated based on the standard curve and were then normalized to B-T0. Mean relative concentrations with 95% CI compared to B-T0 are depicted. OD = Optical Density

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Native PAGE, immunoblotting and ELISA indicated no changes in the biochemical properties of the drug. There is no evidence of degradation or aggregation after dilution and/or storage with or without the preservative BAC. Therefore, we conclude that bevacizumab is still biologically active and accurately detectable.

Protein profile and aggregation by High-performance size-exclusion chromatography (HP-SEC)

HP-SEC is a key tool in demonstrating biosimilarities [41]. This method allows detection of potential aggregation by quantifying the monomer levels and soluble aggregates. Protein elution profiles of the samples are illustrated in Fig. 4. All samples revealed the presence of two protein fractions, with major peaks at ~ 13 mL retention volume, corresponding to the monomeric species. In addition, a relatively minor shoulder (dimer and trimer peak) at the leading edge of the peak was observed within all samples (referred to as higher molecular weight (HMW) species). The amount of HMW was 2.59 ± 0.02%, 2.65 ± 0.22%, 2.73 ± 0.01% and 2.75 ± 0.26% for B-T0, BBAC-T0, B-T4 and BBAC-T4, respectively (Table 1). Molecular weight obtained from the major peaks centred around 151 kDa (Table 1). Isolated elution profiles of B-T0, BBAC-T0, B-T4 and BBAC-T4 is available in Supplement Fig. 4.

Fig. 4
figure 4

Overlapped high-performance size-exclusion chromatography (HP-SEC). Chromatograms of B-T0, BBAC-T0, B-T4 and BBAC-T4. Values represent means ± SD (n = 3). All samples showed the presence of two protein fractions, with a major peak at 13.17 ± 0.002 mL, 13.18 ± 0.001 mL, 13.16 ± 0.003 mL and 13.17 ± 0.003 mL retention volume for B-T0, BBAC-T0, B-T4 and BBAC-T4, respectively. Molecular weight obtained from the major peaks are 152.15 ± 0.19 kDa, 151.68 ± 0.37 kDa, 152.06 ± 0.13 kDa and 152.41 ± 0.55 kDa for B-T0, BBAC-T0, B-T4 and BBAC-T4, respectively. Mean molecular weight obtained from the minor peaks centred around 309 kDa (dimer formation) and 566 kDa (trimer formation)

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Table 1 Relative amounts of monomers and high molecular weight (HMW) species of B-T0, BBAC-T0, B-T4 and BBAC-T4
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We concluded that dilution, storage and preservation using BAC has no impact on the monodispersity of bevacizumab.

Physicochemical characterization by Dynamic Light Scattering (DLS)

DLS was conducted to analyse particle size distribution and homogeneity of the proteins (Table 2). A Polydispersity Index (PDI) < 0.3 indicates homogeneity, which is an excellent tool to identify aggregates [39]. Particle size was evaluated using the hydrodynamic diameter and was found to be similar in all groups centering around 11.65 nm (Fig. 5a) with a PDI < 0.3 (Fig. 5b). The overlapping intensity-based size distribution of all samples shows similarity in all groups (Fig. 5c). Isolated size distribution analysis of B-T0, BBAC-T0, B-T4 and BBAC-T4 is available in Supplement Fig. 5.

Table 2 Physicochemical analysis of B-T0, BBAC-T0, B-T4 and BBAC-T4
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Fig. 5
figure 5

Dynamic light scattering (DLS) analysis of B-T0, BBAC-T0, B-T4 and BBAC-T4. Values are presented as means ± SD (n = 3). a The hydrodynamic diameter was found to be 11.25 ± 0.07 nm, 11.24 ± 0.07 nm, 11.60 ± 0.49 nm and 12.51 ± 0.01 nm with PDI (b) of 0.05 ± 0.00 nm, 0.07 ± 0.02 nm, 0.07 ± 0.01 nm and 0.12 ± 0.01 nm at 25 °C for B-T0, BBAC-T0, B-T4 and BBAC-T4, respectively. Polydispersity Index (PDI) < 0.3 was used to determine homogeneity in particle size distribution. c Overlapping intensity-based distribution analysis

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When bevacizumab is diluted, stored and/or preserved parameters obtained with DLS remained unchanged compared with control (B-T0).

Cytotoxicity and efficacy by HUVEC viability and migration assay

To determine the safety and bioactivity of bevacizumab upon dilution and preservation, we studied the effect of the drug on human umbilical vein endothelial cell (HUVEC) viability and migration. HUVECs are used in this experiment since VEGF as a growth factor promotes their proliferation [42]. Therefore, when bevacizumab is present in the medium, there is an interaction between VEGF and bevacizumab, leading to an inhibitory effect on HUVEC growth. To evaluate whether bevacizumab supplemented with BAC has a toxic effect on the viability of HUVECs a dose response curve (CellTiter®-Glo assay) was performed. Comparable viability was detected in all groups (Fig. 6a) after 72 h. We assessed the functionality of preserved bevacizumab by comparing its capacity to reduce cell migration to its unpreserved counterpart (scratch assay). Percentage of scratch filling was calculated using the Adobe® Photoshop® software (Version 23.5.0, Adobe Inc., San Jose, California) (Supplement Fig. 6) modified from the analysis established by Glaß et al. [43] and Jin et al. [44]. Both unpreserved (B) and preserved bevacizumab (BBAC) decreased HUVEC cell migration compared to control (Fig. 6b). After 24 h cells treated with medium alone had already proliferated and initiated the closure of the gap; after 48 h a monolayer was formed (Fig. 6c). In contrast, cells treated with either bevacizumab (B) or bevacizumab diluted with BAC (BBAC) only partially closed the scratch wound leaving a visible gap (Fig. 6c).

Fig. 6
figure 6

Cytotoxicity and HUVEC migration assay with (B, BBAC) and without (Ctrl) treatment. Results are shown as means ± SD (n = 3). a Quantification of cell viability after treatment measured by the amount of ATP present. Relative cell viability was 92 ± 17% and 94 ± 4% for BBAC and B, respectively. b Quantification of cell migration after treatment over a 48 h period. Relative migration rates obtained were 57 ± 17% and 53 ± 17% at 24 h and 77 ± 4% and 77 ± 20% at 48 h for BBAC and B, respectively. c Representative images of preserved and unpreserved bevacizumab treated HUVECs after scratch wound. Images were taken over a 48 h period

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These data indicate that dilution and preservation of bevacizumab via BAC do not interfere with its functionality.

Discussion

Monoclonal antibodies have revolutionized the treatment of vascular driven human diseases and beyond [45,46,47,48]. It is of utmost importance to favour a widespread use in veterinary health care as well. We aimed at contributing to an affordable treatment option for veterinarian patients with CNV. We have proven here the feasibility to create stable and functional bevacizumab eye drops by adding the preservative BAC. Addition of BAC would allow for longer usage in multidose containers with prevention of bacterial contamination. This, in fact, provides a cost-effective and safe alternate formulation to single-dose treatments that require the usage of new single-dose vials, ending up in the disposal of loads of unused medication.

Alternately, to our approach, preservative-free bevacizumab eye drops could be manufactured using newly developed eye drop bottles containing a silicone membrane filter, and a one-way valve system to prevent retrograde microbial contamination. Rate of microbial contamination is around 21.7 (after two weeks), in standard multidose containers without preservatives. The usage of valve-featured bottles resulted in a slight but statistically insignificant reduction (13% of bacterial growth). Furthermore, the authors observed difficulties in administrating the eye drops using these valve-featured multidose bottles, which negatively affected owners’ compliance [49].

Benzalkonium chloride, a nitrogenous cationic surface-acting agent, is the most commonly used preservative in ophthalmic preparations at concentrations ranging from 0.004%—0.02% as seen in ketorolac (ACULAR®, Allergan Inc., California, USA) or latanoprost eyedrops (Xalatan®, Viatris Healthcare GmbH, Delaware, USA) [50]. It is an effective and safe agent, well documented experimentally in clinical trials [34]. Nevertheless, when it comes to BAC usage in veterinary patients, particularly in brachycephalic animals who already suffer from chronic ocular surface conditions due to their anatomical features, BAC could potentially worsen these susceptibilities [51,52,53,54]. Median application times of bevacizumab eye drops described in human [30, 31] and veterinary literature [28, 32] are three to four weeks. Adverse effects are most likely to occur after long-term usage like with glaucoma medications in human patients and are not expected in short-term usage [53, 54]. Due to the potential ocular toxicity of BAC, in-vivo studies are essential to evaluate its safety on the ocular surface for cases where extended exposure is required.

Creating and handling therapeutic proteins is challenging because of the mAbs tendency to aggregate [55, 56]. In aggregates, two or more monomeric units of mAb may bind irreversibly to one another, reducing efficacy, enhancing immunogenicity, and causing side effects in patients [57, 58]. The presence of aggregates is considered a critical quality attribute (CQA) for the U.S. Food and Drug Administration (FDA) approval [41]. Therefore, the understanding of how the presence of preservatives may affect the structural stability of bevacizumab is essential for practical applications. Considering the safety, efficacy, immunogenicity (aggregation) and biological activity for mAb quantification we used native PAGE, immunoblot, ELISA, DLS and HP-SEC.

Native PAGE was performed to separate proteins and protein complexes in their native state, immunoblotting to separate the proteins based on their mass and ELISA for analysing the biological activity of mAbs through their specific binding to VEGF. Kaja et al. [59] obtained similar results when investigating preserved bevacizumab with native PAGE, immunoblot and ELISA, although using different storage conditions and concentrations.

To the best of our knowledge, this is the first study investigating preserved bevacizumab eye drops with DLS and HP-SEC. High-performance size-exclusion chromatography was used to quantify levels of monomers and soluble aggregates of preserved and stored bevacizumab eye drops. A population of HMWS was shown in all samples (2.64 ± 0.22%), similar to reported data [60]. Kahook et al. [61] reported HMWS in diluted bevacizumab and suspected them to arise from intermolecular disulfide crosslinks forming dimers or trimers of bevacizumab IgG monomers. This would be consistent with the measured molecular weight (~ 309 and ~ 566 kDa).

These methods, although highly sensitive, require drug samples to be diluted. Dilution results in a loss of bioactivity (up to 50%) and might induce aggregation [62]. To overcome these difficulties, we further investigated the samples with DLS. It is a well-established method for the determination of aggregates of proteins [63]. In contradistinction to other techniques, no dilution of the samples is required and therefore allows an investigation of the native structure of proteins and aggregates [64]. The hydrodynamic diameter of the drugs with PDI values of ≤ 0.3 remained stable over a four-week period when measured by DLS indicating homogeneity [65]. Similar results were reported by Khalili et al. [36] and Paul et al. [66] for undiluted bevacizumab stored for up to six months. A slight increase of the hydrodynamic diameter of preserved bevacizumab after storage (BBAC-T4) could be observed (0.12 ± 0.01 nm). It is reported that pH or medium alterations and formed oligomers can lead to changes in the hydrodynamic radius [64, 67, 68]. Although the main peaks presented a uniform size distribution and no aggregates were detected, minor changes of pH values could be seen in preserved (pH 6.05) and unpreserved (pH 6.14) bevacizumab. This could possibly be an explanation for changes in the hydrodynamic diameter.

Ophthalmic solutions typically require a pH value between 4.5 and 9.0, ideally falling within the physiological range of 7.0–7.7 to prevent local irritation and enhanced lacrimation, which can lead to rapid drug clearance through the lacrimal system [69, 70]. Ophthalmic preparations with lower pH values, such as 6.05, can increase tear turnover, causing swift clearance from the ocular surface [71]. However, benzalkonium chloride acts as a penetration enhancer, possibly counteracting these effects by facilitating drug penetration through the ocular epithelia [72]. Furthermore, some ophthalmic formulations must be prepared within an acidic pH for long-term stability, as seen in the case of pilocarpine with a pH range of 4.5 to 5.5 [73]. Nonetheless, in-vivo studies are necessary in order to assess ocular compatibility.

A concentration of 2.5 mg/mL bevacizumab was used. This choice was based on findings from previous veterinary studies, which showed no significant ocular side effects in dogs, neither with nor without ocular surface diseases [28, 32]. A concentration of 0.01% BAC was chosen, as this has previously been described in the literature for the preserving of bevacizumab eye drops in human patients with chronic keratitis [1, 30, 31]. Notably, in these studies BAC and bevacizumab with concentrations as high as 10 mg/mL showed to be safe and without ocular side effects [1, 30, 31].

Alteration of the formulation can be associated with a loss of bioactivity [74]. We therefore studied the effect of clinically relevant doses of preserved and unpreserved bevacizumab eye drops on endothelial cell viability (cytotoxicity) and migration (efficacy). Bevacizumab was able to decrease HUVEC migration when administered unpreserved and preserved. Forty-eight hours after treatment a slight recovery and increase in migration of the endothelial cells was detected. This could be due to the relatively short application time (10 min), which was used to mimic the contact time of ophthalmic preparations with the ocular surface in-vivo. Further studies are needed to identify appropriate application frequency of preserved bevacizumab eye drops in patients with naturally occurring CNV.

Conclusion

This study shows that bevacizumab is stable in its biochemical properties when diluted and preserved with BAC over a period of four weeks. Equivalence tests demonstrated that both preserved and unpreserved bevacizumab show similar properties. In-vivo studies are needed to indicate the use of preserved bevacizumab eye drops in patients with naturally occurring CNV.

Methods

Materials

Drug preparation

Bevacizumab 25 mg/mL (Avastin®) was obtained from Roche (Basel, Switzerland). A 2.5 mg/mL solution of bevacizumab was aseptically prepared in compliance with good manufacturing practice with sterile 0.9% saline solution as a solvent. Four groups were studied (Table 3); the solution was either preserved (BBAC) or unpreserved (B) and subsequently aliquoted to 2 mL in commercially available plastic eye drop containers. Solutions were examined at two different time points; either stored for 4 weeks protected from light at 2–4 °C (T4) or freshly prepared (T0). Benzalkonium chloride was used as a preservative. The final solution of preserved bevacizumab eye drops contained 0.01% BAC as prescribed in the European Pharmacopeia (EMA/495737/2013) [75]. As a control Avastin® (25 mg/mL, same lot number) diluted to 2.5 mg/mL with sterile 0.9% saline solution was used (B-T0). A fresh vial of undiluted Avastin® (A) (25 mg/mL, same lot number) was used as a standard.

Table 3 Sample preparation of B-T0, BBAC-T0, B-T4 and BBAC-T4
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Methods

Study design

The bevacizumab solutions (B-T4, BBAC-T0, BBAC-T4) were evaluated for their physicochemical stability after dilution and storage with or without BAC as a preservative. Over a four-week period, the solutions were analysed and compared with Avastin® (25 mg/mL, same lot number) diluted to 2.5 mg/mL with sterile 0.9% saline solution (B-T0) as recommended by the manufacturer (Avastin® prescribing information, 01.2021). Samples were prepared from different vials but the same batch. Different approaches were chosen to investigate selected critical quality attributes (CQAs) concerning safety, efficacy and/or biological activity and immunogenicity (Table 4) [76, 77].

Table 4 Summary of investigations
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Determination of pH

The pH values of the formulations were determined using a previously calibrated pH-meter (inoLab Level 3, Xylem Analytics Germany Sales GmbH & Co. KG, Weilheim, Germany). Measurements were carried out in triplicates.

Visual inspection

Solutions were visually inspected in front of a white and black panel before carrying out further experiments to confirm the absence of particles. Presence of any particles was noted.

Native polyacrylamide gel electrophoresis (Native PAGE)

Native PAGE was carried out as previously described [59]. Briefly, samples were diluted with phosphate buffered saline (PBS) in a total volume of 20 μL. The solution was electrophoresed at concentrations of 5 µg, 1 µg and 0.5 µg on a native 6% poly-acrylamide gel without SDS. Gels were stained with Coomassie Blue (SimplyBlueTM Safe Stain, Invitrogen, Carlsbad, CA) according to manufacturer’s protocol, photographed (OM-D E-M10 Mark III, Olympus, Shinjuku, Tokyo, Japan) and/or scanned. Densitometry was performed on a representative gel and data was normalized to B-T0.

Immunoblotting

Immunoblotting was performed as previously described [59]. Briefly, samples were diluted in PBS to a total volume of 40 μL containing 4 × loading dye. Proteins were denatured and resolved by a 10% sodium dodecyl sulphate polyacrylamide gel for 30 min at 70 V and for 90 min at 90 V at concentrations of 10 ng, 25 ng and 50 ng. Proteins were transferred to polyvinylidene difluoride membranes (PVDF, Immobilon®-P, Merck, Darmstadt, Germany) by overnight wet transfer at 4 °C. Membranes were blocked in 5% milk for 1 h, and probed with diluted sheep anti-human IgG (GE Healthcare, Piscataway, NJ) at 4 °C overnight. The next day, detection was performed by chemiluminescent visualisation using the ChemiDoc Touch™ Imaging System (BioRad Laboratories Inc., Hercules, California) according to manufacturer’s protocol. Protein expression levels were quantified using the Image Lab software (Version 6.1, BioRad Laboratories Inc., Hercules, California). All blots were performed in one experiment.

Enzyme-linked immunosorbent assay (ELISA)

ELISA was carried out as previously described [59]. Briefly, an ELISA was developed using 96-well plates coated with human IgG-specific goat IgG (Abcam, Cambridge, United Kingdom) overnight at 4 °C. Bevacizumab samples were diluted in deionized water to a concentration of 5 ng/mL. After blocking, 100 μL of the diluted samples were added to each well. Wells were then incubated with 30 ng biotinylated recombinant human VEGF-165 (antibodies-online GmbH, Aachen, Germany) for 1 h. Peroxidase-conjugated streptavidin (Extravidin-Peroxidase; Sigma, St. Louis, USA) was diluted 1:2000 in PBS and added to each well. Peroxidase activity was determined by incubation with 100 μL peroxidase substrate solution 3,3′,5,5′-tetramethylbenzidine (TMB) (Sigma). Absorbance at 450 nm was quantified in a microplate reader (FlexStation 3; Molecular Devices, Sunnyvale, CA). Absorbance at 650 nm was subtracted as a reference. The standard curve was obtained by serial dilutions of Avastin® (25 mg/mL, same lot number) in PBS. The linear range was between 0.625 ng/mL and 40 ng/mL. Experiments were carried out in triplicates.

High-performance size-exclusion chromatography (HP-SEC)

High-performance size-exclusion chromatography analysis was conducted on an OMNISEC multi-detector GPC/SEC (Malvern Panalytical, Worcestershire, UK) equipped with a refractive index, right angle light scattering (RALS), and UV/VIS diode array detector. Briefly, proteins were separated on a Superdex S200 increase 10/300 GL column (Cytiva) maintained at 25 °C, using PBS as an isocratic mobile phase at a flow rate of 0.5 mL/min. The autosample chamber was maintained at 20 °C and detectors were maintained at 25 °C. The injection volume was 60 µL. Protein concentration was measured online by using the refractive index detector. A dn/dc of 0.185 was taken. The instrument was calibrated using commercially available bovine serum albumin solution (BSA) (2 mg/mL) (Thermo Scientific™ Pierce™). Elution profiles of the samples were monitored using an ultraviolet (UV) absorbance detector. A qualitative comparison between samples was conducted using an overlay of these chromatograms. Measurements were carried out in triplicates.

Dynamic Light Scattering (DLS)

The hydrodynamic diameter and polydispersity index of bevacizumab were measured with a Malvern Zetasizer Nano ZS instrument (Malvern Instruments Ltd., UK) according to the manufacturer’s protocol. Briefly, samples (~ 700 μL) were measured using a PMMA semi-micro cuvette (Y197.1, Carl Roth, Germany) at 25.0 °C and with a detecting angle of 173 °C. Data were collected in an automatic mode, typically requiring a measurement duration of 150 s. Data were analysed using the Zetasizer software (Version 8.01.4906, Malvern Instruments Ltd., Malvern, UK). Quality reports of all the measurements were checked to ensure that the obtained data met the quality criteria. Measurements were carried out in triplicates.

Cell culture

Cells were purchased from American Type Culture Collection (ATCC®, USA, cat# PCS-100–010™). Cells were grown in Vascular Basal Medium (ATCC®, USA, cat# PCS-100–030™) supplemented with VEGF Growth kit (ATCC®, USA, cat# PCS-100–041™) containing rhEGF, rhFGF, rhVEGF, rhIGF-1, ascorbic acid, hydrocortisone, heparin, L-glutamine and 2% FBS in a 37 °C and 5% CO2 humidified incubator. Cells were seeded at 5000 cells/cm2 in T-75 flasks. After reaching 80—100% confluence the monolayer was washed with PBS (ATCC®, USA, cat# 30–2200) and trypsinized (Trypsin–EDTA for primary cells, ATCC®, USA, cat# PCS-999–003™) before being used in experiments. For experiments, a limited medium was formulated containing only rhVEGF as a growth factor according to Liu et al. [78] to produce a significant drug-specific dose-dependent inhibition. HUVECs up to passage five were used in all experiments.

HUVEC viability

Cells were plated at a density of 1 × 104 cells/well in 96-well plates (Falcon; Becton Dickinson Labware, Plymouth, England). After overnight incubation, cells had formed a monolayer with 80 – 100% confluency and were treated with preserved bevacizumab (BBAC) or unpreserved bevacizumab (B) for 10 min to mimic topical drug treatments [79]. After this time, solutions were removed and normal cell culture conditions were restored for 24 h. At termination of the assay, 100 μl CellTiter-Glo® 2.0 Reagent (Promega, Madison, WI) was added to each well, absorbance at 490 nm was recorded using a microplate reader (EnSpire® Multimode Plate Reader, PerkinElmer, Waltham, MA, USA) after incubation and shaking (600 rpm) for 30 min following manufacturer’s protocol. Results are expressed as a percentage of the measured absorbance of control cells. Experiments were carried out in triplicates.

HUVEC migration

Cells were inoculated onto a 12-well plate (Falcon; Becton Dickinson Labware, Plymouth, England) at a density of 1 × 105 cells/well. After a confluent monolayer had formed overnight, a scratch was introduced with a sterile 1000 μl tip. Cell debris was removed by washing with PBS. Cells were treated with either preserved bevacizumab (BBAC) or unpreserved bevacizumab (B) for 10 min. After removal of the treatments, medium was supplied. Scratch filling was documented at different time points (0 h, 24 h, 48 h and 72 h). Experiments were carried out in triplicates.

Statistics

The results of the experiments performed in triplicates are expressed as mean ± standard deviation (SD). Native PAGE and SDS-PAGE were performed in duplicates although on different gels. The results are therefore presented in a descriptive manner. A one-sided equivalence test was used to determine whether unpreserved and preserved bevacizumab are equal. Equivalence bounds were specified based on the mean ± 15% of B-T0. Equivalence between the groups was given, if the 95% CI was completely within the equivalence interval.

Data availability

The datasets used and analysed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

The authors want to thank Prof. Lea Ann Dailey of the Division of Pharmaceutical Technology and Biopharmaceutics of the University of Vienna for her generously provided knowledge and expertise. Additionally, many thanks to Josef Martinus Ellersdorfer and the staff of the Institute of Pharmacology and Toxicology of the University of Veterinary Medicine Vienna for their technical support. The research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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  1. Department of Companion Animals and Horses, University of Veterinary Medicine Vienna, Veterinaerplatz 1, Vienna, 1210, Austria

    Ulrike Lessiak & Barbara Nell

  2. Institute of Pharmacology and Toxicology, University of Veterinary Medicine Vienna, Veterinaerplatz 1, Vienna, 1210, Austria

    Tania Brandstoetter, Klara Klein, Lisa Scheiblecker & Andrea Hoelbl-Kovacic

  3. BOKU Core Facility Biomolecular & Cellular Analysis, BOKU – University of Natural Resources and Life Sciences, Muthgasse 11, Vienna, 1190, Austria

    Georg Mlynek

  4. Division of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Josef-Holaubek-Platz 2, Vienna, 1090, Austria

    Lukas Wimmer

  5. Department of Biomedical Sciences, University of Veterinary Medicine Vienna, Veterinaerplatz 1, Vienna, 1210, Austria

    Alexander Tichy

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All authors read and approved the final manuscript. UL contributed to the design of the study, performed analysis, interpreted data and wrote the manuscript. TB contributed to the design of the study, helped to perform analysis, was a major help in interpreting data, helped to write the manuscript and critically revised the manuscript. BN and AHK contributed to the study design, helped to interpret data and critically revised the manuscript. KK, GM, LW and LS performed analysis, helped to interpret the data and critically revised the manuscript. AT performed statistical data analysis, helped to interpret data and critically revised the manuscript.

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Lessiak, U., Brandstoetter, T., Nell, B. et al. Preserved bevacizumab (Avastin®) eye drops for application in multidose containers – an in-vitro characterisation. BMC Vet Res 21, 129 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12917-025-04592-4

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