FormalPara Key Points

Sandoz rituximab biosimilar quality remains stable after prolonged exposure of product diluted in 0.9% NaCl infusion bags to temperatures up to 30 ± 2 °C, while protected from light exposure.

The in-use stability of the product was confirmed even in the worst-case scenario, whereby the starting drug product, in vials, was nearing the end of its shelf-life and was exposed to out-of-fridge conditions, simulating transport deviations (up to 25 ± 2 °C), as well as deviations at the hospital (up to 30 ± 2 °C).

1 Introduction

Regular stability studies often fail to foresee and factor in all potential events that may occur during the transport and storage of liquid in vials (LIVI) prior to dilution and administration of biologics [1]. Thus, this paper presents the data of cumulative long-term storage, subsequent out-of-fridge (OOF, in other publications also sometimes referred to as out-of-refrigeration) storage, and subsequent in-use (dilution in infusion bag) for the Sandoz rituximab (RTX) biosimilar (SDZ-RTX; GP2013; Rixathon®/Riximyo®).

Rituximab is a chimeric, mouse anti-human monoclonal antibody targeting the cluster of differentiation 20 antigen [2], and is approved for the treatment of several B-cell hematologic malignancies and autoimmune diseases [3,4,5]. Rituximab was the first anti-cluster of differentiation 20 monoclonal antibody approved by the US Food and Drug Administration for relapsed or refractory, cluster of differentiation 20-positive, B-cell, low-grade, or follicular lymphoma in 1997 [6,7,8]. Rituximab coordinates anti-tumor effects through a variety of mechanisms, including direct induction of apoptosis, antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis [9, 10].

Currently, there are several RTX biosimilars available. Here, we discuss the SDZ-RTX, which is available as a concentrate solution for infusion in a single-dose vial (100-mg or 500-mg active ingredient) at a concentration of 10 mg/mL. Approved storage conditions include storage in a refrigerator at 2–8 °C protected from light exposure [11]. Storage requirements and considerations also define that the product should always be handled according to the most current Product Information Leaflet (PIL) or Summary of Product Characteristics [11,12,13]. For example, in the European Union, the SDZ-RTX Summary of Product Characteristics states that the manufacturer does not permit the storage of the final diluted product in 0.9% NaCl for more than 24 h outside of the intended storage conditions of 2–8 °C [11]. Thus, in cases where storage of the diluted product at room temperature exceeds the permitted 24-h period, such a product must be discarded [11], despite the possibility that it is of acceptable physicochemical and biological quality.

Permitting temperature excursions that do not impact the quality and stability of the biosimilar product should be considered. Regional differences in humidity and temperature may also impact the purity and potency of SDZ-RTX during temperature excursions, so it is important to ensure stability is assessed across a range of temperatures and humidities [14, 15], for example, recapitulating the climate in regions located in climatic zones I, II, III, IVa, and IVb as defined for stability studies [16, 17].

The physicochemical and biological in-use stability of SDZ-RTX has been demonstrated upon preparation in 0.9% NaCl for an intravenous transfusion with prolonged storage of up to 30 days at 5 ± 3 °C [15]. The stability of unopened SDZ-RTX vials has also been demonstrated in OOF conditions for 21 days at temperatures up to 30 ± 2 °C and 65 ± 5% relative humidity (RH) [14].

Because of the lack of data on prolonged storage of finally diluted SDZ-RTX at room temperature conditions, this study (termed the in-use stability study) was performed to assess the impact of prolonged elevated temperature conditions, as seen in climatic zone IVb (30 ± 2 °C/75 ± 5% RH for 14 days) [16], on the in-use stability of SDZ-RTX diluted in a 0.9% NaCl infusion bag.

2 Materials and Methods

The in-use stability study was performed in the full Good Manufacturing Practices environment using Good Manufacturing Practices-validated equipment and stability chambers and product-specific validated methods, and following Good Manufacturing Practices standards.

2.1 Study Design

The in-use stability study was performed with two batches of expired, but otherwise representative, SDZ-RTX 500 mg (50 mL, LIVI; Table 1). The use of aged (and of acceptable microbial quality, i.e. sterile) material is a realistic worst-case approach for simulating real-life use of SDZ-RTX, as a product nearing the end of its shelf-life can be used in clinical settings. Batch selection for the study met European Medicines Agency guidelines (CPMP/QWP/2934/99, issued for multi-dose products), requiring the testing of a minimum of two pilot scale batches, including at least one batch nearing the end of its shelf-life [18].

Table 1 Batches used in the in-use stability study
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Prior to the in-use stability study, each undiluted SDZ-RTX LIVI batch, stored in the original folding box, was exposed to worst-case storage conditions, accounting for potential temperature transport excursions, as well as excursions at the hospital level [19]. Each SDZ-RTX LIVI batch was exposed to long-term storage for at least 36 months (shelf-life) at 5 ± 3 °C (batch 1: 41 months; batch 2: 37 months, ambient humidity). Throughout the long-term storage, SDZ-RTX LIVI batches were exposed for either 3 days (batch 1) or 2 days (batch 2) to the room temperature conditions (up to 25 ± 2 °C/60 ± 5% RH) during the manufacturing activities. After completed long-term storage, SDZ-RTX LIVI baches were exposed to the OOF condition 25 ± 2 °C/60 ± 5% RH for either 11 days (batch 1) or 12 days (batch 2), to simulate a major temperature excursion that could occur during, for example, transport of the samples to the end user. Considering both exposure to room temperature conditions and OOF conditions of 25 ± 2 °C/60 ± 5% RH, each SDZ-RTX LIVI batch was exposed to cumulative 14 days at room temperature conditions up to 25 ± 2 °C/60 ± 5% RH.

After completed OOF exposure at 25 ± 2 °C/60 ± 5% RH, both SDZ-RTX LIVI batches were exposed to 30 ± 2 °C/75 ± 5% RH for 7 days, to account for the potential temperature excursions that may occur at the hospital, as permitted according to the Summary of Product Characteristics in the European Union [11].

For the in-use stability study, the SDZ-RTX LIVI batches were diluted for an infusion. For dilution to a final concentration of SDZ-RTX of 1.0 mg/mL, an average volume based on six different 250-mL polyethylene infusion bags was determined. This approach was used as the quality of diluted SDZ-RTX in use was assessed with state-of-the-art, product-specific analytical methods requiring an accurate target concentration [14]. By following aseptic preparation practices using a unidirectional flow chamber, mimicking a hospital setting, the LIVI was diluted into a 0.9% NaCl infusion bag (250-mL polyethylene infusion bag) to a final concentration of 1.0 mg/mL. In our previous in-use study investigating the stability of SDZ-RTX at 5 ± 3 °C [15], no notable difference in the stability of SDZ-RTX diluted in 0.9% NaCl was observed between polyethylene and polypropylene infusion bags. To confirm the dilution process, sample protein concentrations of 1 mg/mL were verified by ultraviolet spectroscopy. All obtained concentrations, as measured by ultraviolet spectroscopy, were exactly 1.0 mg/mL (Fig. 1). After dilution, the bags of both SDZ-RTX batches (1 and 2) were stored protected from light exposure [11] at either 25 ± 2 °C/60 ± 5% RH for up to 30 days (condition 1) or 30 ± 2 °C/75 ± 5% RH for up to 14 days (condition 2).

Fig. 1
figure 1

Assessment of Sandoz rituximab biosimilar (SDZ-RTX) content as measured by ultraviolet (UV) spectroscopy. Sandoz rituximab biosimilar content for both batches of SDZ-RTX measured by UV spectroscopy after being diluted in 0.9% NaCl infusion bags immediately after dilution (0 days) or subjected to in-use conditions 1 (25 ± 2 °C/60 ± 5% relative humidity [RH] for 30 days) and 2 (30 ± 2 °C/75 ± 5% RH for 14 days)

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Physicochemical stability was assessed at several timepoints with one replicate (sample) per each sampling point of the sample. Samples from day 0 served as study baseline reference points. For condition 1 (25 ± 2 °C/60 ± 5% RH), the diluted products were tested on days 3, 7, 14, 21, and 30 of storage. For condition 2 (30 ± 2 °C/75 ± 5% RH), the diluted products were tested on days 3, 7, and 14. For each storage condition, the samples were transferred into the stability chamber on consecutive days in reverse order, which allowed for all to be sampled from the stability chamber on the same day. Such a study design allowed for samples, if feasible, to be analyzed simultaneously, minimizing any potential variability in the analysis [14].

2.2 Analytical Methods

The impact of these worst-case storage conditions on the in-use stability of SDZ-RTX was evaluated using the following analytical methods: purity by size-exclusion chromatography (SEC); purity by non-reducing capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS); identity and purity by cation exchange chromatography (CEX); potency by CDC bioactivity; and particulate contamination of subvisible particles (PC-SVP) by light obscuration (LO). Details for the methods used in the SDZ-RTX in-use stability studies have been published previously [14, 15] and are shown in the Electronic Supplementary Material.

2.3 Evaluation Criteria

Changes in stability after storage were assessed by comparing the results from samples subject to prolonged in-use storage (i.e., up to 30 days under condition 1 or 14 days under condition 2) with the results from the day 0 samples that were analyzed immediately after dilution.

Moreover, the results were compared with shelf-life specification limits of SDZ-RTX LIVI for SEC, CEX, nrCE-SDS, and CDC bioactivity assessments, and pharmacopeia criteria for the PC-SVP LO assessment. Although the shelf-life specifications are applicable for SDZ-RTX LIVI only, the SEC, CEX, nrCE-SDS, and CDC bioactivity were compared with the shelf-life specification ranges also after in-use storage.

3 Results

The stability of SDZ-RTX diluted in 0.9% NaCl infusion bags was confirmed under both storage conditions (25 ± 2 °C/60 ± 5% RH for 30 days or 30 ± 2 °C/75 ± 5% RH for 14 days), as results for all assessments were within acceptance criteria for each SDZ-RTX batch. As expected, when comparing identical timepoints, larger changes in the assessed outcomes were observed after exposure to the higher temperature in condition 2 (30 ± 2 °C/75 ± 5% RH) than at the lower temperature in condition 1 (25 ± 2 °C/60 ± 5% RH). When discussing changes in the next sections, “slight changes” describe changes that do not exceed variability of the method, while “changes” or “relevant changes” describe changes over time that exceed analytical variability.

3.1 Assessment of Physicochemical Properties of SDZ-RTX

Following dilution of SDZ-RTX in 0.9% NaCl infusion bags, the protein concentration of each sample was determined by ultraviolet spectroscopy, to confirm that the dilution was accurately performed. All results demonstrated a concentration of exactly 1.0 mg/mL (Fig. 1).

3.2 Identity and Purity Assessed by CEX

Identity by CEX was confirmed for all results (data not shown). All purity results assessed by CEX were within the shelf-life specification for SDZ-RTX LIVI. The following changes in charge variants were expected and in line with results of previously conducted studies [14, 15]. During the storage of SDZ-RTX in 0.9% NaCl under conditions 1 and 2, an increase in the sum of acidic peaks was observed, whereas the sum of basic peaks showed a slight decrease (condition 2; only a very minor numerical drop was observed for condition 1), resulting in a decrease in the main peak (Fig. 2). No new or unexpected peaks were observed during comparisons between time zero and in-use conditions 1 and 2 up to 30 and 14 days, respectively (Fig. 2d).

Fig. 2
figure 2

Assessment of the identity and purity of Sandoz rituximab biosimilar (SDZ-RTX) by cation exchange chromatography (CEX). The CEX main peak (a), sum of acidic peaks (b), and sum of basic peaks (c) are shown for both SDZ-RTX batches diluted in 0.9% NaCl infusion bags and subjected to in-use conditions 1 (25 ± 2 °C/60 ± 5% relative humidity [RH] for 30 days) and 2 (30 ± 2 °C/75 ± 5% RH for 14 days). Representative chromatogram overlay (batch 1) measured by CEX (d) comparing the time zero (T0) sample (solid black line, not exposed to in-use conditions) with samples exposed to in-use conditions 1 (25 ± 2 °C/60 ± 5% RH; 3, 7, 14, 21, and 30 days) or 2 (30 ± 2 °C/75 ± 5% RH; 3, 7, and 14 days). AU arbitrary units

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3.3 Purity Assessed by SEC

Size-exclusion chromatography was used to assess the purity level and sum of high-molecular-weight species. A slight decrease in purity was detected after 30 days of storage under condition 1 and 14 days of storage under condition 2 (0.1% and 0.2% decrease, respectively) (Fig. 3). The sum of high-molecular-weight species remained virtually unchanged throughout 30 days of storage under condition 1 and throughout 14 days of storage under condition 2. All results were within the most stringent shelf-life specification limits of SDZ-RTX acceptance criteria. The observed stability trend for SEC data was in line with previous stability studies of SDZ-RTX [14, 15]. No new or unexpected peaks were observed during comparisons between time zero and in-use conditions 1 and 2 up to 30 and 14 days, respectively (Fig. 3c).

Fig. 3
figure 3

Assessment of purity of Sandoz rituximab biosimilar (SDZ-RTX) by size-exclusion chromatography (SEC). The SEC purity (a) and sum of high-molecular-weight (HMW) species (b) are shown for both SDZ-RTX batches diluted in 0.9% NaCl infusion bags and subjected to in-use conditions 1 (25 ± 2 °C/60 ± 5% relative humidity [RH] for 30 days) and 2 (30 ± 2 °C/75 ± 5% RH for 14 days). Representative chromatogram overlay (c) of SDZ-RTX (batch 1) measured by SEC. The main peak and additional minor low-molecular-weight (LMW) and HMW species are indicated. AU arbitrary units, T0 time zero

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3.4 Purity Assessed by nrCE-SDS

After storage of SDZ-RTX diluted in 0.9% NaCl, a slight overall decrease in purity assessed by nrCE-SDS was observed for both SDZ-RTX batches under both storage conditions (Fig. 4a). These results are in line with findings observed during the SEC analysis, whereby slight increases of fragments were evident by both methods (in SEC, indirectly deduced by a slight reduction of purity without a change in aggregates). All nrCE-SDS results were within the most stringent shelf-life specification limits of SDZ-RTX LIVI acceptance criteria. The observed stability trend was as expected based on previous stability studies of SDZ-RTX [14, 15]. No new or unexpected peaks were observed in the electropherogram for this analysis when comparing between time zero and in-use conditions 1 and 2 up to 30 and 14 days, respectively (Fig. 4b).

Fig. 4
figure 4

Assessment of purity of Sandoz rituximab biosimilar (SDZ-RTX) by non-reducing capillary electrophoresis sodium dodecyl sulfate (nrCE-SDS). The nrCE-SDS purity (a) is shown for both SDZ-RTX batches diluted in 0.9% NaCl infusion bags and subjected to in-use conditions 1 (25 ± 2 °C/60 ± 5% relative humidity [RH] for 30 days) and 2 (30 ± 2 °C/75 ± 5% RH for 14 days). Representative electropherogram overlay (b) of SDZ-RTX (batch 1) of the nrCE-SDS. The main peak, the two heavy chains and one light chain (2HL) fragment (two heavy chains [2Hs] and one light chain [L]), and additional minor high-molecular-weight (HMW) species are indicated. AU arbitrary units, H heavy chain, HL heavy and light chain, T0 time zero

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3.5 Potency Assessed by CDC Bioactivity

The results for the cell-based CDC bioactivity assay showed inherent variability, which is higher when compared with the variability seen with high-performance liquid chromatography methods [20] such as CEX and SEC. Overall, for storage under both conditions (25 ± 2 °C/60 ± 5% RH for 30 days or 30 ± 2 °C/75 ± 5% RH for 14 days), CDC bioactivity results showed slight changes/fluctuations throughout the in-use stability study (Fig. 5). Importantly, all results were well within shelf-life acceptance criteria.

Fig. 5
figure 5

Assessment of Sandoz rituximab biosimilar (SDZ-RTX) potency measured by complement-dependent cytotoxicity (CDC) bioactivity. The CDC bioactivity is shown for both SDZ-RTX batches diluted in 0.9% NaCl infusion bags and subjected to in-use conditions 1 (25 ± 2 °C/60 ± 5% relative humidity [RH] for 30 days) and 2 (30 ± 2 °C/75 ± 5% RH for 14 days)

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3.6 Assessment of PC-SVP by LO

To confirm the quality of the batches, the presence of PC-SVP was assessed by LO. Throughout storage, fluctuations in these data were observed, which can be attributed to the variability of the method (Fig. 6). Under both conditions, results complied with pharmacopeia criteria test 1.A (solutions with nominal volume above 100 mL), European Pharmacopoeia [21] 2.9.19, US Pharmacopeia <788> [22], and Japanese Pharmacopoeia 6.07 [23]. The US Pharmacopoeia <788> guideline specifies that the preparation complies with the test if the average number of particles present in the units tested does not exceed 25 per mL equal to or greater than 10 µm and does not exceed 3 per mL equal to or greater than 25 µm, for preparations of more than 100 mL [22]. The infusion bags used in this study held 250 mL, hence these stringent limits were considered.

Fig. 6
figure 6

Assessment of particulate contamination-subvisible particles (PC-SVP) by light obscuration (LO) throughout storage during the in-use stability study [21,22,23]. Particulate contamination-subvisible particles (a) ≥10 µm and (b) ≥25 µm detected throughout the study for both Sandoz rituximab biosimilar batches diluted in 0.9% NaCl infusion bags and subjected to in-use conditions 1 (25 ± 2 °C/60 ± 5% relative humidity [RH] for 30 days) and 2 (30 ± 2 °C/75 ± 5% RH for 14 days). JP Japanese Pharmacopoeia, Ph Eur European Pharmacopoeia, USP US Pharmacopoeia

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4 Discussion

The objective of this study was to evaluate the in-use stability of SDZ-RTX after dilution in a 0.9% NaCl infusion bag and subsequent prolonged storage at room temperature conditions. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q5C guidelines state the product shelf-life must be supported by real-time data, at the very least spanning the intended shelf-life [24]. The design of this study allowed for evaluation of the worst-case scenario, where the product in vials (LIVI) would be near the end of its shelf-life (approximately 3 years after production) and encounter a major unexpected temperature excursion (14 days at temperatures up to 25 ± 2 °C/60 ± 5% RH), followed by a temperature excursion at the hospital (7 days at 30 °C/75% RH). Following this, SDZ-RTX was diluted into a 0.9% NaCl infusion bag and exposed to realistic in-use conditions of 25 °C ± 2 °C/60% ± 5% RH for up to 30 days or 30 ± 2 °C/75 ± 5% RH for up to 14 days. The analytical methods employed are known to have stability-indicating potential for analysis of SDZ-RTX [14, 15], and all results observed were within stringent acceptance criteria set to ensure patient safety and drug efficacy. Previous methods that are known not to be stability indicating for SDZ-RTX (data not shown) were excluded from the study. Our findings show the quality of SDZ-RTX is stable even after exposure to OOF conditions and subsequent in-use studies. As such, studies such as these can help to prevent unnecessary product wastage and contribute to maintenance of a robust patient supply of SDZ-RTX.

We did not assess the stability of SDZ-RTX under non-aseptic conditions and after exposure to microbial activity; therefore, it is imperative that the preparation of SDZ-RTX takes place in aseptic conditions in the clinical setting. The microbial quality under each condition was intentionally not tested, as the microbial quality is, after the primary packaging of SDZ-RTX in vials is breached (i.e., during the dilution process), dependent solely on the dilution process. This process is unique to each time the dilution is performed, and therefore, results from any assessments of microbial activity in this study could not be applied to future dilution processes.

Overall, the results from this study suggest that the quality of SDZ-RTX diluted in infusion bags containing 0.9% NaCl remains appropriate even after being exposed to the worst-case study conditions. These data should not be used to sanction use of SDZ-RTX after the PIL-stated shelf-life (36 months), nor do they justify temperature excursions or deviations from the PIL that may occur during shipping, storage, or administration of SDZ-RTX. Sandoz rituximab biosimilar must be used as stated in the accompanying PIL. However, the results do support an extended quality window for in-use SDZ-RTX, to allow the addition of a single-time exposure to OOF conditions to the current approved storage conditions. This will prevent product waste, further reducing the financial burden in healthcare markets, and help ensure there is a robust supply of SDZ-RTX for patients [14].

5 Conclusions

These results demonstrate that SDZ-RTX batches aseptically diluted in 0.9% NaCl infusion bags remained of appropriate physicochemical and biological quality even after extended storage either at 25 ± 2 °C/60 ± 5% RH for 30 days or at 30 ± 2 °C/75 ± 5% RH for 14 days. This holds true even for a product near the end of its shelf-life previously exposed to OOF temperature excursions. Therefore, these data suggest SDZ-RTX is suitable for administration to patients after long-term storage, near the end of its shelf-life, with an additional OOF excursion (while in the original vial) and aseptic dilution into infusion bags, and finally exposure of the diluted product to realistic in-use conditions of 25 ± 2 °C/60 ± 5% RH for 30 days or 30 ± 2 °C/75 ± 5% RH for 14 days.