FormalPara Key Points

Storage of diluted SB12 for infusion in saline solution for extended period does not compromise its quality or activity.

Extended storage of SB12 may reduce treatment delays, drug waste, and cost of treatment.

1 Introduction

Eculizumab (Soliris, Alexion Europe SAS, Alexion Pharmaceuticals, Inc.) is a recombinant humanized IgG2/4k monoclonal antibody that acts as complement inhibitor. It works by binding with high affinity to complement protein C5, blocking the protein’s cleavage to C5a and C5b and downstream formation of the terminal complement complex C5b-9. This inhibits the terminal effects mediated by C5b-9, such as intravascular hemolysis and thrombotic microangiopathy (TMA). Eculizumab is administered by intravenous (IV) infusion and is currently approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS), as well as adult patients with generalized myasthenia gravis (gMG) who are positive for anti-acetylcholine receptor (AchR) antibody and with neuromyelitis optica spectrum disorder (NMOSD) who are positive for anti-aquaporin-4 (AQP4) antibody [1, 2].

SB12 (Samsung Bioepis Ltd.) is a biosimilar referencing Soliris (eculizumab) that has obtained European Commission approval [3] based on a totality of evidence including analytical, non-clinical data, and clinical data. The clinical equivalence between SB12 and its reference eculizumab product has been demonstrated in a randomized, double-blind, multicenter, cross-over phase 3 study in 50 adult patients with PNH (NCT04058158) [4]. In this study, there were no clinically meaningful differences between SB12 and its reference eculizumab product in terms of clinical efficacy, safety, pharmacokinetics, pharmacodynamics, and immunogenicity.

The reference eculizumab product is available as a sterile, clear, colorless, preservative-free concentrated solution for IV infusion. Prior to administration, the product should be diluted to a final concentration of 5 mg/mL in 0.9% NaCl, 0.45% NaCl, or 5% dextrose in water. The unopened reference product should be stored at 2–8 °C and protected from light in the original carton, and at room temperature (≤ 25 °C) for a single period of up to 72 h. While both labels state that the product should be used immediately after dilution, the summary of product characteristics adds that the diluted product is stable when stored at 2–8 °C or room temperature for 24 h [1, 2].

When performed under controlled conditions that warrant the drug’s quality and efficacy, the preparation of antibodies for infusion in advance can be highly beneficial for patients and clinical staff. This may help to avoid unnecessary treatment delays, decrease the workload of nurses and pharmacists, and reduce costs [5]. Moreover, temperature excursions outside the conditions outlined in the reference product’s label can occur in a clinical setting and may result in unnecessary drug waste [5]. There are no extended in-use (after dilution) stability data for any eculizumab product, including biosimilar SB12. To address this knowledge gap, we evaluated a set of physicochemical and biological parameters that determine the stability of in-use SB12 upon extended storage outside of what is outlined in the reference product’s US and EU labels. The study was conducted on two batches of SB12 (including at least one batch at the end of proposed shelf-life), using the diluents indicated in the reference product’s label (0.9% NaCl, 0.45% NaCl, and 5% dextrose in water) and three different containers [polyolefin (PO) bags, glass bottles, and syringes]. The protocol followed International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) and EMA requirements for stability evaluation of biological products [6,7,8].

2 Methods

This paper does not contain any studies with human or animal participants. Study acceptance criteria were established on the basis of SB12 product stability specifications.

2.1 Sample Preparation and Study Design

In-use stability of SB12 was evaluated in two batches of SB12, one of which was toward the end of proposed shelf-life (batch 2, aged 34 months). First, the SB12 samples were removed from refrigerated storage (5 ± 3 °C) and kept in the upright position at room temperature (30 ± 2 °C with a RH of 65 ± 5%) and protected from light for 72 h. The samples were then diluted in 240 mL of 0.9% NaCl, 0.45% NaCl, or 5% dextrose in water to yield a final concentration of 5 mg/mL in accordance with the reference product’s label [1, 2] and clinical trial protocol. The diluted samples were then stored in PO bags or glass bottles at 5 ± 3 °C for up to 3 months, then at 30 ± 2 °C/65 ± 5% RH for 72 h. Samples were analyzed at the initial time point and then after 1 month, 2 months, 3 months, and 3 months plus 72 h. At month 3, 60 mL of diluted SB12 samples (0.9% NaCl in PO bags, 0.45% NaCl in PO bags, and 5% dextrose in glass bottles) were also transferred to Plastipak syringes (BD), kept at 30 ± 2 °C/65 ± 5% RH for 72 h and analyzed. Individual containers aligned with the number of testing time points were prepared for each study condition. The protein concentrations of the different containers were very similar, demonstrating a high level of homogeneity between preparations. The number of replicates varied on the basis of each test’s standard operating procedure (SOP) that was validated according to the ICH Q2(R1) guideline for validation of analytical procedure.

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
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2.2 Physicochemical Analyses

Testing for pH, protein concentration, and purity was performed as previously described [9,10,11].

2.2.1 pH

Sample pH was tested according to EP pharmacopoeia (Ph. Eur. 2.2.3) “Potentiometric Determination of pH” with a Seven Excellence pH Meter and In Lab Expert Pro-ISM (Mettler Toledo).

2.2.2 Protein Concentration

Samples were diluted with 0.9% NaCl to a concentration of 1.0 mg/mL and absorbance at a wavelength of 280 nm (A280) was measured with an ultraviolet–visible (UV–VIS) spectrophotometer (SHIMADZU); 0.9% NaCl served as the blank solution. Bovine serum albumin (BSA, Thermo Scientific, 23209) was used as an assay control after 1:1 dilution with 0.9% NaCl (Sigma, S8776) for system suitability test.

2.2.3 Size Exclusion-High-Performance Liquid Chromatography

Samples (n = 3) were injected onto a TSK gel G3000 SWXL analytical column (5 µm/7.8 mm × 300 mm; Tosoh) connected to a Waters HPLC system. A mobile phase consisting of 100 mM sodium phosphate, 300 mM NaCl, pH 6.8 was used for isocratic elution at a flow rate of 1.0 mL/min. UV detection occurred at 280 nm and data were acquired and processed by Empower®3 software (Waters).

2.2.4 Capillary Electrophoresis-Sodium Dodecyl Sulfate

Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) was performed by using a capillary electrophoresis system of PA800 plus Pharmaceutical Analysis (SCIEX). The analytical sample (n = 3) was mixed with iodoacetamide, 10 kDa internal standard, and the SDS-molecular weight sample buffer (Beckman) under non-reducing conditions. The mixture was incubated at 70 °C and loaded onto a capillary cartridge (50 µm/67 cm; Beckman coulter) for electrophoretic separation. Signals were monitored at 220 nm using a 32 Karat software (SCIEX).

2.2.5 Imaged Capillary Isoelectric Focusing

Samples of SB12 (n = 3) were mixed with pharmalyte 3–10, methyl cellulose, urea, distilled water, and pI marker. The mixtures were loaded and analyzed onto ICE3 imaged capillary isoelectric focusing (icIEF) instrument (Protein Simple). Focused protein zones were detected using a whole column UV absorption detector (280 nm).

2.3 Biological Activity and Safety

2.3.1 C5 Binding Activity

An enzyme-linked immunosorbent assay (ELISA) was conducted to evaluate C5 binding of SB12 samples (n = 2). A BSA-containing buffer was added to an ELISA plate adsorbed with C5 protein to block nonspecific binding. Serially diluted SB12 was added to bind to C5. A horseradish peroxidase (HRP)-conjugated antibody was added, followed by an appropriate chromogenic substrate. The plate was washed after each step to remove unbound substances. The optical density was read on a spectrophotometer (Molecular Device) at 450 nm. The relative C5 binding activity of SB12 was calculated by analyzing a four parameter curve plotted from data in the parallel line analysis (PLA) software (Stegmann Systems GmbH).

2.3.2 C5 Inhibition Assay

The C5 inhibitory activity of SB12 was evaluated by measuring its ability to prevent complement-induced lysis of B cells. CD20-positive B cells (Raji cells, lymphoblast-like cells, CCL-86TM, ATCC) were incubated with serially diluted SB12 (n = 2), human serum (source of complement protein), and an anti-CD20 antibody (inducer of complement dependent cytotoxicity) for 20–35 min at 37 °C/5% CO2. The cells were treated with Cytotox-GloTM following kit instructions, and the luminescence was read on a microplate reader (Perkin Elmer) at 400–700 nm. The relative C5 inhibition activity of SB12 was evaluated by analyzing a four parameter curve plotted from data in the PLA software (Stegmann Systems GmbH).

2.3.3 Particles

Particles ≥ 10 and 25 µm in size were counted according to Ph. Eur. 2.9.19 “Particulate matter in injections” with a high accuracy (HIAC) liquid particle counter.

2.3.4 DLS

Dynamic light scattering (DLS) was used to determine submicronic aggregation, and the latex standard was analyzed with samples to confirm the system suitability. A Malvern Zetasizer Nano-ZS laser light scattering system was used to obtain DLS measurements. The Zetasizer software was used for data acquisition and analysis. The size and hydrodynamic diameter distributions of the antibody were studied to check for the presence of small aggregates.

2.4 Statistical Analysis

All the generated data were analyzed by appropriate statistical methods to detect statistically significant changes from baseline in the measured parameters. Data collected from samples in PO bags (N = 5) and glass containers (N = 5) were analyzed by regression and analysis of variance (ANOVA) (α = 0.05) [12, 13]. Ranges for these data were obtained by regression tolerance interval analysis (95% confidence level and 99% population) or defined as mean ± 3 standard deviations (SD), as appropriate [14, 15]. To account for inherent assay variability, a comparative analysis of the SD obtained from the current dataset versus the SD obtained from the method validation studies (in-house data) was also conducted. If the SD from the dataset was similar to the assay variation, changes in stability were attributed to inherent assay variability and not considered relevant (even if p < 0.05).

For the samples in syringes, single data points (N = 1) were obtained for each diluent, and regression analysis was not feasible. The stability results of SB12 drawn into syringes were considered compliant when within the range calculated from the complete dataset (N = 5).

3 Results

3.1 pH and Protein Concentration

Results for pH and protein concentration over time are presented in Table 2. No meaningful changes in pH and protein concentration were observed for SB12 in PO bags and glass containers over time. The pH and protein concentrations for samples drawn into syringes were within the range of 3 SD or the range by regression tolerance interval.

Table 2 Physicochemical properties, biological activity, and subvisible particles of SB12 at baseline and 3 months + 72 h
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3.2 Purity Analyses

Purity trends over time and statistical analysis for the infusion stability study are shown in Fig. 1a–e and Table 2, respectively. Examples of chromatograms and electropherograms for SB12 samples at the initial time point and 3 months + 72 h are shown in Figs. 2, 3, and 4. For both NaCl diluents, statistical analysis showed no notable changes over time in % HMW (determined by SE-HPLC) and % total purity (determined by CE-SDS). However, there were notable changes were in %HMW for in-use SB12 diluted in 5% dextrose (Table 2). Nonetheless, the product stability specifications were met for all the diluents and containers used in the infusion stability study.

Fig. 1
figure 1

Stability trends of SB12 over time (stored at 5 ± 3 °C for 3 months, followed by 30 ± 2 °C/65 ± 5% RH for 72 h). a Percentage of HMW by SE-HPLC (n = 3); b percentage of total purity by CE-SDS (NR) (n = 3); percentage of acidic isoform (c), main isoform (d), and basic isoform (e) by icIEF (n = 3); f binding activity by C5 binding assay (n = 2); g potency by C5 inhibition assay (n = 2). C5, complement protein 5; >CE-SDS, capillary electrophoresis-sodium dodecyl sulfate; HMW, high molecular weight; icIEF, imaged capillary isoelectric focusing; RH, relative humidity; SE-HPLC, size exclusion-high-performance liquid chromatography. “n” signals the number of replicates for each experiment

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Fig. 2
figure 2

SE-HPLC chromatogram showing the total monomer and HMW impurity of SB12 at day 0 and 3 months + 72 h (stored at 5 ± 3 °C for 3 months, followed by 30 ± 2 °C/65 ± 5% RH for 72 h). Samples were diluted in a 0.9% NaCl, b 0.45% NaCl, and c 5% dextrose. HMW, high molecular weight; RH, relative humidity; SE-HPLC, size exclusion-high-performance liquid chromatography

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Fig. 3
figure 3

CE-SDS (non-reducing) electropherograms showing % total purity of SB12 at day 0 and 3 months + 72 h (stored at 5 ± 3 °C for 3 months, followed by 30 ± 2 °C/65 ± 5% RH for 72 h). Samples were diluted in a 0.9% NaCl, b 0.45% NaCl, and c 5% dextrose. CE-SDS, capillary electrophoresis-sodium dodecylsulfate; RH, relative humidity

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Fig. 4
figure 4

icIEF electropherograms showing charge variants (% main, % acidic. and % basic) of SB12 at day 0 and 3 months + 72 h (stored at 5 ± 3 °C for 3 months, followed by 30 ± 2 °C/65 ± 5% RH for 72 h). Samples were diluted in a 0.9% NaCl, b 0.45% NaCl, and c 5% dextrose. icIEF, imaged capillary isoelectric focusing; pI, isoelectric point; RH, relative humidity

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No critical changes over time were observed for % acidic and % main (determined by icIEF) for SB12 diluted in 0.9% NaCl and 0.45% NaCl. However, SB12 diluted in 5% dextrose (all containers) showed notable changes in % acidic (11.4–11.8% increase) and % main (9.6–10.0% decrease) at study completion compared with the initial time point, which were statistically significant (p < 0.05) and not attributable to inherent assay variation (Table 2). The same was also observed for % basic of SB12 diluted in 5% dextrose and kept in PO bags (p = 0.017).

All the results for samples drawn into syringes were within the range of 3 SD or regression tolerance interval (Table2).

3.3 Biological Activity

No meaningful changes in C5 binding and C5 inhibition were observed over time regardless of type of diluent (0.9% NaCl, 0.45% NaCl, and 5% dextrose) and type of container (PO bag, glass bottle, and syringe) (Fig. 1f–g; Table 2).

3.4 Safety Analyses

Results for subvisible particles detected by a HIAC particle counter are presented in Table 2. There were no notable differences in subvisible particles (≥ 10 and 25 µm) for in-use SB12 across all tested diluents (0.9% NaCl, 0.45% NaCl, and 5% dextrose) and types of container (PO bag, glass bottle, and syringe). For submicronic aggregation by DLS, no increase in either the hydrodynamic diameter or the polydispersity index (PDI) was noted when stored for 2 months at 5 ± 3 °C, then 72 h at 30 ± 2 °C/65 ± 5% RH (Table 3). The inter-run assay variability during the storage period [SD 0.04–0.11 (diameter), 0.01 (PDI)] was within the range of intra-run reproducibility [SD 0.00–0.13 (diameter), 0.00–0.02 (PDI)]. In addition, regression analysis revealed that the change over the storage period was not statistically significant (p = 0.113–0.936). No populations of high hydrodynamic diameter (100–1000 nm) were present

Table 3 Hydrodynamic diameter and PDI of SB12 aggregates at 2 months + 72 h, as measured by DLS
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4 Discussion

The current study was designed to evaluate the infusion stability of in-use SB12 after an extended storage period and at higher temperatures than what is stated in the reference product’s label [1, 2]. We used different combinations of diluents (0.9% NaCl, 0.45% NaCl, and 5% dextrose in water) and storage containers (PO bags, glass bottles, and syringes) to match the conditions observed in a clinical setting. The experimental workflow was aligned with ICH and EMA requirements for stability evaluation of biological products [6,7,8] and included testing for pH, protein concentration and purity, biological activity, and subvisible particles. This orthogonal and complementary method ensures robust and reliable stability information because each method pertains to a specific aspect of stability. For instance, SE-HPLC is optimal to detect HMW impurities and CE-SDS is more adequate for smaller impurities, while icIEF recognizes different charge variant impurities. When combined, these results provide a solid evaluation of the product’s purity. The same rationale applies to biological activity, with the C5 binding assay determining the drug’s affinity and the C5 inhibition assay evaluating the drug’s potency. To assess safety, we also combined two different methods (HIAC and DLS) to look for subvisible particles and potentially immunogenic aggregates.

To our knowledge, this is the first publication of the extended in-use stability of an eculizumab product, including the reference product. Our findings indicate that the physicochemical properties, biological activity, and subvisible particles of SB12 diluted in 0.9% NaCl and 0.45% NaCl are generally maintained under extended storage conditions (5 ± 3 °C for 3 months, then 30 ± 2 °C/65 ± 5% RH for 72 h). There were no signs of stock instability compared with freshly prepared samples, regardless of the type of container used (PO bags and glass bottles). In addition, we show that refrigerated in-use SB12 (diluted in 0.9% or 0.45% NaCl and stored at 5 ± 3 °C for up to 3 months) is stable when withdrawn into syringes and kept at 30 ± 2 °C/65 ± 5% RH for another 72 h, allowing for administration via syringe-type pump per label instructions. These results demonstrate that in-use SB12 diluted in NaCl (0.9% and 0.45%) is stable for longer than what is claimed in the reference product’s EU and US labels, which state that diluted product should be used immediately or stored at 5 ± 3 °C or room temperature for a maximum period of 24 h [1, 2]. These data suggest that SB12 can be prepared in advance in NaCl (0.9% or 0.45%), stored under the described conditions, and administered to the patient without any impact on the quality and biological activity of the drug. The preparation of infusions in advance is a valuable tool to manage and expedite treatment cycles, while reducing the workload and increasing the efficiency of clinical staff. Furthermore, these results indicate that temperature excursions within these conditions may not warrant disposal, which is important to reduce drug waste and prevent unnecessary costs [5].

In contrast to what was observed for SB12 diluted in 0.9% and 0.45% NaCl, there were notable changes over time on acidic and main charge variants of SB12 diluted in 5% dextrose (as determined by icIEF, Fig. 1c, d; Table 2). Results showed an 11.4–11.8% increase in % acidic and 9.6–10.0% decrease in % main after 3 months and 72 h across all containers (PO bag, glass bottle, and syringe). There was also a significant change in % basic for SB12 diluted in 5% dextrose and stored in PO bags (p = 0.017, Table 2). Previous research has shown that changes in icIEF charge variants may occur as a result of dextrose-mediated protein glycation [16]. To test this hypothesis, we measured protein glycation in SB12 diluted in 5% dextrose versus 0.9% NaCl at the initial time point and end (3 months and 72 h) of the study (data not shown). We observed an increased protein glycation trend over time in both reduced and intact conditions for SB12 diluted in 5% dextrose, while no noteworthy changes occurred in SB12 diluted in 0.9% NaCl. These data suggest that the changes in icIEF charge variants observed for SB12 diluted in 5% dextrose (Fig. 1d, e) could be attributed to protein glycation. It should be noted that the observed changes in icIEF charge variants did not appear to compromise the bioactivity of in-use SB12 in 5% dextrose, which was maintained throughout the study (Fig. 1f and g; Table 2). Although there is no loss of biological activity in SB12 diluted in 5% dextrose, due to the potential instability in charge variants, the use of SB12 diluted in 5% dextrose must be restricted to the conditions defined within product label by the manufacturer.

A limitation of this study is that no evaluation of microbial properties (total viable count and sterility) for in-use SB12 was conducted. All SB12 samples were prepared and handled under aseptic conditions across all experiments, which is of critical importance to preclude the risk of microbial contamination. The stability data provided were contingent on SB12 samples being prepared under controlled aseptic conditions.

5 Conclusions

The infusion stability of SB12 after extended storage was demonstrated for longer periods and at higher temperatures (5 ± 3 °C for 3 months, then 30 ± 2 °C/65 ± 5% RH for 72 h) than what is stated on the EU and US labels of the reference product. All evaluated parameters for in-use SB12 diluted in 0.9% NaCl and 0.45% NaCl were maintained under the described conditions and for all tested containers. However, instability was observed for the diluted SB12 in 5% dextrose in water after extended storage. Overall, these data may help to reduce treatment delays while minimizing drug waste and the workload of clinical staff, without any loss in product quality and biological activity.