FormalPara Key Summary Points

Why carry out this study?

Long-term stability data for unopened vials of SB11 at room temperature are limited and there is no stability information for SB11 that has been drawn into syringes for use.

The study evaluated the quality, biological activity, and presence of sub-visible particulates of SB11 in unopened vials and under in-use conditions after an extended storage period at different temperatures.

Extended storage at room temperature and advanced preparation of SB11 may benefit patients and health care providers, avoid unnecessary delays, minimize drug waste, and reduce the cost of treatment.

What was learned from the study?

Unopened SB11 vials were stable at 30 ± 2 °C/65 ± 5% RH for at least 1 month. The biological activity of SB11 and sub-visible particulates were not compromised throughout the duration of the study (2 months). The stability of in-use SB11 transferred into syringes was maintained when stored at 5 ± 3 °C for 98 days and then at 25 ± 2 °C/60 ± 5% RH for 24 h.

SB11 injections can be prepared in advance and administered to the patient without any impact on the quality and efficacy of the drug.

Introduction

Ranibizumab (Lucentis, Genentech, Novartis) is a recombinant humanized IgG1k isotype monoclonal antibody fragment that acts as an inhibitor of human vascular endothelial growth factor (VEGF)-A. By binding to bioactive isoforms of VEGF-A (including VEGF110, VEGF121 and VEGF165), ranibizumab blocks their interaction with receptors at the surfaces of endothelial cells (VEGFR1 and VEGFR2) and reduces their downstream effects [1, 2]. These include endothelial cell proliferation, neovascularization, and vascular leakage, all of which are thought to contribute to the progression of several angiogenic eye diseases [3,4,5]. Ranibizumab is currently approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for intravitreal use to treat patients with neovascular (wet) age-related macular degeneration (AMD), myopic choroidal neovascularization (mCNV), diabetic retinopathy (DR), diabetic macular edema (DME) and macular edema secondary to retinal vein occlusion (RVO) [1, 2].

SB11 (ranibizumab-nuna, Byooviz™, Samsung Bioepis, Biogen) is a ranibizumab biosimilar that has been approved by the FDA and EMA [6, 7]. The biosimilarity between SB11 and its reference product (ranibizumab) was established in a randomized, double-masked, parallel-group, multicenter phase 3 equivalence study in patients aged over 50 with neovascular AMD [8]. Both the FDA and EMA approve biosimilars based on the totality of evidence of a comprehensive comparability exercise that demonstrates that there are no clinically meaningful differences between the biosimilar and the reference product in terms of quality characteristics, biological activity, clinical safety and efficacy [8].

Both reference product (Lucentis) and SB11 are available as clear/colorless to pale yellow, preservative-free, sterile solutions for single-use intravitreal injection, to be administered under controlled aseptic conditions. Prior to use, the unopened reference product should be stored at 2–8 °C and protected from light in the original carton [1, 2]. The summary of product characteristics (SmPC) adds that the unopened product is stable at room temperature (25 °C) for up to 24 h [1]. While the storage conditions for unopened SB11 vials are the same (2–8 °C), the SmPC supports storing unopened vials in the original carton at temperatures not exceeding 30 °C for up to 1 month [6, 7].

Advanced preparation of monoclonal antibodies in conditions that do not compromise the drug’s quality and efficacy could avoid unnecessary delays, decrease the workload of clinical staff and reduce costs, benefiting patients and health care providers [9]. However, neither of the labels recommend opening the product prior to use, and there is no stability information for in-use SB11 (withdrawn into syringes). Moreover, temperature excursions outside the conditions outlined in the labels are frequent in a clinical setting and may result in unnecessary drug waste [9]. Previous research demonstrated stability in the various scenarios for Aybintio (a bevacizumab biosimilar) [10]. The present study focused on another currently approved anti-VEGF drug, SB11 (a ranibizumab biosimilar), which is used for the treatment of retinal diseases. For anti-VEGF drugs used for intravitreal administration, research confirming the in-use stability of these drugs when they are withdrawn into syringe is prevalent [11,12,13,14,15,16,17]. Additional stability information for SB11 under these scenarios is thus needed to expedite and rationalize the clinical use of this biosimilar. To address this knowledge gap, this study evaluated a set of physicochemical and biological parameters that determine the stability of SB11 (unopened vials at room temperature and the in-use product withdrawn into syringes) upon extended storage and outside of what is outlined in the US and EU labels. The study followed ICH and EMA requirements for stability evaluation of biological products [18,19,20].

Methods

This paper does not contain any studies with human or animal participants. The study acceptance criteria were established based on SB11 approved stability specifications as per the countries in which the drug is approved.

Sample Preparation

For the extended stability study of unopened vials, three lots of SB11 that were past the end of their shelf life (aged 48 months) were used. The samples were kept in the upright position at room-temperature conditions (30 ± 2 °C with a RH of 65 ± 5%) and protected from light. Samples were analyzed at testing intervals of 0 h (initial time point), 24 h, 72 h, 1 week, 1 month and 2 months. Testing at 24 h was only performed for one lot.

For the stability study under in-use conditions, two lots of SB11 were used, one of which was at the end of its shelf-life. The contents of the vials were withdrawn through an 18-gauge filter needle into 1-mL Luer-Lok™ (polycarbonate, PC, silicone-oil (SO)-containing) and/or Norm-ject (polypropylene, PP, SO-free) syringes. The filter needle was replaced with a 30-gauge injection needle, the air bubbles were removed, and the content was adjusted to the 0.05 mL mark, in accordance with the product label [1, 2]. The syringes were stored at 5 ± 3 °C and protected from light for 98 days and then transferred to room-temperature conditions (25 ± 2 °C/60 ± 5% RH, protected from light) for 24 h. Samples were examined at days 0 (initial time point), 21, 42, 98 and 99.

The samples were prepared under aseptic conditions and then submitted to a series of tests to evaluate their physicochemical properties and biological activities. A summary of the tests performed is detailed in Table 1.

Table 1 Testing methods and acceptance criteria
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Physicochemical Analyses

Testing for appearance (color, clarity, and visible particles), pH, and protein concentration and purity was performed as previously described [10, 21, 22].

Appearance

The manual visual inspection hood MIH DX (Eisai Machinery, USA) or a viewing station with an appropriate light source (fluorescent) and white and black backgrounds and a turbidimeter (HACH, model 2100AN) were used to inspect the color (brown standard: B1–B9 per Ph. Eur. 2.2.2) and clarity (turbidity standard in visual method: 3–80 nephelometric turbidity units (NTU) per Ph. Eur. 2.2.1) of samples and to look for visible particles (per Ph. Eur. 2.9.20).

pH

Samples were evaluated according to Ph. Eur. 2.2.3 with a Seven Excellence pH Meter and In Lab Micro Pro-ISM (a combination pH electrode and temperature probe; Mettler Toledo), which were calibrated to pH 4.01, 7.00 and 9.21 before use.

Protein Concentration

All samples were diluted to a concentration of 0.6 mg/mL, based on the expected concentration of each sample in 0.9% NaCl. Absorbance at a wavelength of 280 nm (280) was measured by a ultraviolet–visible (UV–VIS) spectrophotometer (Shimadzu or Agilent). For normalization, 0.9% NaCl served as the blank solution.

Size-Exclusion High-Performance Liquid Chromatography

Samples were injected onto a TSK gel SW2000 column (4 µm/4.6 mm × 300 mm; Tosoh) with a flow rate of 0.2 mL/min and a mobile phase that consisted of 100 mM sodium phosphate and 200 mM sodium chloride (pH 6.8). UV detection occurred at 280 nm.

Capillary Electrophoresis–Sodium Dodecyl Sulfate

Capillary electrophoresis–sodium dodecyl sulfate (CE-SDS) analyses were conducted in alkylated, non-reducing conditions with the addition of a 10 kDa internal standard using a high-performance capillary electrophoresis system (PA 800 Plus Pharmaceutical Analysis System; Sciex). The SB11 samples were electrokinetically introduced onto a capillary (50 µm/30.2 cm; Sciex). UV radiation (wavelength 220 nm) was passed through the capillary window and aperture (144712, 100 × 200 µm; Sciex) and detected by the PA 800 Plus.

Imaged Capillary Isoelectric Focusing

Samples of SB11 were mixed with carrier ampholytes injected into a transparent column (capillary), retained in a microscope-slide cartridge and separated by isoelectric focusing. Focused protein zones were detected using a whole-column UV absorption detector (wavelength 280 nm).

Using a capillary cartridge at 4 °C, the mixture was loaded onto the imaged capillary isoelectric focusing (icIEF) instrument. The anolyte was 0.08 M H3PO4 and the catholyte was 0.1 M NaOH.

Biological Activity and Safety

Testing for biological activity (VEGF binding activity, VEGF neutralization) and safety (sub-visible particulates) was performed as previously described [10].

VEGF Binding Assay

An enzyme-linked immunosorbent assay (ELISA) was conducted to evaluate the VEGF binding activity of SB11 samples. Bovine serum albumin (BSA)-containing buffer was added to an ELISA plate with VEGF-A 165 adsorbed to block nonspecific binding. Serially diluted SB11 was added to bind to the VEGF-A 165. A horseradish peroxidase (HRP)-conjugated anti-hIgG (Fc specific) antibody was added, followed by an appropriate chromogenic substrate. The plate was washed after each step to remove unbound substances. The optical density at 450 nm was read on a spectrophotometer (SpectraMax M5e). The relative VEGF-A 165 binding activity of SB11 was calculated by analyzing a four-parameter curve plotted from data in parallel line analysis (PLA) software.

VEGF Neutralization Assay

The VEGF-A 165 neutralization potency of SB11 was determined in KDR 293 cells using a VEGF receptor 2 (VEGFR2)-dependent reporter gene system. KDR 293 cells were genetically modified to contain the nuclear factor of activated T-cell (NFAT) binding sequence in the promoter region of the luciferase reporter gene. Bound VEGF to VEGFR2 activates the VEGFR2-dependent signal pathway and the expression of the luciferase reporter gene, which is measured by luminescence. The luminescence data was read by using the microplate reader (Envision or M5e). The neutralization potency of VEGF-A 165 by SB11 was calculated based on dose-dependent changes in luminescence levels using a four-parameter curve plotted from data in the PLA software.

Particulates

Particles ≥ 2, 5, 8, 10, 25 and 50 µm in size were counted according to Ph. Eur. 2.9.19 with HIAC 9703+ and HRLD 400 instruments (HACH).

Results

Appearance, pH and Protein Concentration

No particulate matter was detected by visual inspection, and there were no meaningful changes over time in color, clarity, pH and protein concentration (Table 2). All results met the acceptance criteria, and no notable differences were observed between in-use SB11 withdrawn in PC syringes and in-use SB11 withdrawn in PP syringes.

Table 2 Appearance, pH values and protein concentrations of unopened SB11 vials and in-use SB11 under controlled conditions
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Purity Analyses

For unopened SB11 vials at room temperature, the proportion (%) of high molecular weight (HMW) impurity (determined by SE-HPLC) and %main (determined by CE-SDS) over time met the acceptance criteria (Fig. 1). However, there were notable changes in charge distribution at month 2 of storage at room temperature, as depicted in the icIEF results (see %acidic and %main by icIEF, Fig. 1).

Fig. 1
figure 1

Stability trends of unopened SB11 vials (aged 48 months) stored at room temperature (30 ± 2 °C/65 ± 5% RH) through month 2. a Percentage of HMW by SE-HPLC. b Percentage of monomer by SE-HPLC. c Percentage of main isoform by CE-SDS (NR, Non-Reduced). Percentage of the acidic (d) and main (e) isoform by icIEF. f VEGF binding activity. g VEGF neutralization activity. CE-SDS capillary electrophoresis–sodium dodecyl sulfate, icIEF imaged capillary isoelectric focusing, RH relative humidity, SE-HPLC size-exclusion high-pressure liquid chromatography, VEGF vascular endothelial growth factor

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For in-use SB11, all results met the acceptance criteria through day 99. No notable changes were observed over time, and there was no difference between SB11 withdrawn in PC vs. PP syringes (Fig. 2). Examples of chromatograms and electropherograms for SB11 samples through day 99 are shown in Fig. 3 (HMW impurity per SE-HPLC), Fig. 4 (%main per CE-SDS) and Fig. 5 (%acidic and %main isoforms per icIEF).

Fig. 2
figure 2

Stability trends of in-use SB11 vials withdrawn into PC or PP syringes through day 99 (5 ± 3 °C for 98 days, then 25 ± 2 °C/60 ± 5% RH for 24 h). a Percentage of HMW by SE-HPLC. b Percentage of monomer by SE-HPLC. c Percentage of main isoform by CE-SDS (NR, Non-Reduced). Percentage of acidic (d) and main (e) isoform by icIEF. f VEGF binding activity. g VEGF neutralization activity. CE-SDS capillary electrophoresis–sodium dodecyl sulfate, icIEF imaged capillary isoelectric focusing, PC polycarbonate, PP polypropylene, RH relative humidity, SE-HPLC size-exclusion high-pressure liquid chromatography, VEGF vascular endothelial growth factor

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Fig. 3
figure 3

SE-HPLC chromatogram showing total monomer and HMW impurity of in-use SB11 withdrawn into PP and PC syringes at day 0 and day 99 (5 ± 3 °C for 98 days, then 25 ± 2 °C/60 ± 5% RH for 24 h). HMW high molecular weight, PC polycarbonate, PP polypropylene, RH relative humidity, SE-HPLC size-exclusion high-pressure liquid chromatography

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Fig. 4
figure 4

CE-SDS (non-reducing) electropherogram showing total purity of in-use SB11 withdrawn into PP and PC syringes at day 0 and day 99 (5 ± 3 °C for 98 days, then 25 ± 2 °C/60 ± 5% RH for 24 h). CE-SDS capillary electrophoresis–sodium dodecyl sulfate, PC polycarbonate, PP polypropylene, RH relative humidity, SE-HPLC size-exclusion high-pressure liquid chromatography

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Fig. 5
figure 5

icIEF electropherogram showing charge variants of in-use SB11 withdrawn into PP and PC syringes at day 0 and day 99 (5 ± 3 °C for 98 days, then 25 ± 2 °C/60 ± 5% RH for 24 h). icIEF imaged capillary isoelectric focusing, PC polycarbonate, PP polypropylene, RH relative humidity, SE-HPLC size-exclusion high-pressure liquid chromatography

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Biological Activity

The results from VEGF binding and neutralization assays show no notable changes over time in the biological activity of SB11 taken from unopened vials at room temperature (Fig. 1) and in-use SB11 withdrawn into PC or PP syringes (Fig. 2). All results met the acceptance criteria.

Safety Analyses

Sub-visible particulates (≥ 10, 25 and 50 µm) detected by a HIAC liquid particle counter did not exceed the acceptance criteria, and no meaningful trend was detected over time for both unopened SB11 vials and in-use SB11. Sub-visible particulates (≥ 2, 5 and 8 µm) also did not show a meaningful trend (data not shown). Examples of sub-visible particulate counts for in-use SB11 samples through day 99 are shown in Fig. 6.

Fig. 6
figure 6

Particulates of in-use SB11 withdrawn into PC or PC syringes through day 99 (5 ± 3 °C for 98 days, then 25 ± 2 °C for 24 h). *Denotes 0 particles/mL. PC polycarbonate, PP polypropylene

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Discussion

This study was designed to evaluate the stability of SB11 for an extended storage period outside the conditions in the product label. The study protocol was aligned with ICH and EMA requirements for the stability evaluation of biological products [18,19,20] and included testing for visual appearance (color, clarity, and presence of visible particles), pH, protein concentration and purity, biological activity, and sub-visible particulates. We used a combination of orthogonal and complementary methods to ensure that the results were robust and to provide reliable stability information. For instance, SE-HPLC is more adequate for detecting the main HMW impurity of SB11, while CE-SDS has higher resolution for smaller impurities and icIEF quantifies impurities based on their charge. Together, these results provide solid information about the product’s purity. The same rationale applies to biological activity, with the VEGF binding assay determining the drug’s binding activity and the VEGF neutralization assay evaluating the drug’s potency. Overall, our findings indicate that the physicochemical properties, biological activity, and sub-visible particulates of SB11 in both tested settings (unopened vials and in use) are generally maintained under the described extended storage periods.

The results were analyzed according to EU and US specifications, which establish different acceptance quality criteria. Unopened SB11 vials at room temperature met the acceptance criteria at month 2 by EU standards, beyond the room-temperature stability claims (1 month) in the EU label of SB11. There were noteworthy changes in the purity of unopened SB11 vials at room temperature through month 2. We observed a decrease in total purity (per %main in CE-SDS) and more so in charge distribution after 72 h (per %acidic and %main in icIEF, Fig. 1). These results were still within the US acceptance range at month 1, but assay and manufacture variations should be considered in order to minimize risk. For this reason, the shelf life of unopened vials at room temperature under US specifications is only 72 h. These results suggest that SB11 stability is maintained beyond the conditions stated in the US label (2–8 °C), which could allow unnecessary waste following temperature excursions in clinical practice to be reduced.

For in-use SB11 withdrawn into syringes (5 ± 3 °C for 98 days, then 25 ± 2 °C/60 ± 5% RH for 24 h), all results met the acceptance criteria through study completion. These results extend the pool of stability information to in-use SB11, which is absent in both US and EU labels. In-use SB11 withdrawn into syringes showed no signs of physicochemical instability at day 99 when compared to freshly prepared SB11. In addition, the biological activity determined by two different methods was maintained through day 99. These results are in accordance with a previous report on the long-term functional stability of in-use ranibizumab, in which biological activity was maintained for up to 4 weeks after transfer into plastic syringes [14]. This suggests that SB11 injections can be prepared in advance, stored under the described conditions, and administered to the patient without any impact on the quality and biological activity of the drug.

Most syringes used for intravitreal injection contain SO as lubricant, which is frequently released as droplets into the contents of the syringe [16, 23]. Previous research showed that bevacizumab and ranibizumab withdrawn into plastic syringes were contaminated with SO microdroplets [24, 25]. These microdroplets have been found in the vitreous of patients after intravitreal injection and could lead to adverse ocular reactions [16, 24, 26]. To address this safety issue, we incorporated SO-free (PP, HSW Norm-Ject) and SO-containing (PC, BD Luer-Lok) syringes in this in-use stability study. Interestingly, we did not observe differences in the proportion of HMW impurity and sub-visible particles between both types of syringes (Figs. 3 and 6), showing that the presence of SO did not increase microdroplets and protein aggregation. We also show that the physicochemical and biological properties of SB11 are not compromised in the absence of a protective SO layer, which is in line with previous research [16].

As all samples were manipulated under aseptic conditions across all experiments, no evaluation of the microbial properties (total viable count and sterility) of in-use SB11 was conducted. This is because the possibility of microbial contamination depends on the environment of use, so showing that there is no microbial contamination in a study performed under aseptic conditions cannot provide safety-related considerations. This limitation of the study represents a weakness in that there is no guarantee that there will be no safety issue, particularly in the case of poor prepared in-use SB11 under insanitary conditions. SB11 vials are supplied as sterile and preservative-free solutions, and their administration should be conducted under controlled aseptic conditions. Therefore, it is important that the sterility of in-use SB11 is maintained upon extended storage. For that reason, SB11 should be administered per label guidelines in each country in which it is approved. In the event that the in-use product can be prepared sufficiently aseptically through good manufacturing practices, safety concerns such as the risk of endophthalmitis can potentially be lowered, and our findings in terms of product quality and efficacy may support extended in-use storage [27]. However, if SB11 is withdrawn into a syringe under in-use conditions in which the sterile state is not maintained, it should be used immediately to preclude the risk of microbial contamination.

Conclusions

Overall, the results show that SB11 is stable for longer periods and at higher temperatures than what is stated in the labels of the reference product and SB11. This applies to unopened vials at room temperature and in-use SB11 withdrawn into syringes for intravitreal injection, showing that advanced preparation is feasible and there is a broader range of acceptable temperature excursions. This information can help to avoid unnecessary delays in patient treatment without any loss in quality and biological activity, lower the workload of health care providers and reduce costs associated with drug waste.