Focused physicochemical stability study of reconstituted trastuzumab biosimilar SB3 (Ontruzant^®) stored in the original vials over 28 days

Introduction

Trastuzumab, a humanized immunoglobulin G1 monoclonal antibody (mAb), binds to human epidermal growth factor receptor´s (HER2`s) extracellular domain thereby inhibiting growth of HER2 overexpressing tumour cells. In 2000, trastuzumab originator product Herceptin^® (Roche Registration GmbH) was approved. Since 2017, several trastuzumab biosimilars were authorized by EMA. Trastuzumab reference and biosimilar products are approved for the treatment of adult patients with metastatic or early breast cancer or metastatic gastric cancer when tumours have HER2 overexpression or HER2 gene amplification [1].

In Europe, biosimilar medicinal products are licensed according to the EMA-Guidelines which are based on comparative quality, non-clinical, and clinical studies [2]. Due to the natural variability of the biological source and to the unique manufacturing process of each manufacturer, minor differences can occur between the biosimilar and its reference product. Minor variabilities, differences in the formulation and administration devices are allowed when scientific evidence shows that it does not affect safety, efficacy, and immunogenicity of the product [3]. Because of these variabilities, physicochemical in-use stability data cannot be extrapolated and must be experimentally tested for each finished reference and biosimilar brand product [4, 5]. In-use physicochemical stability data submitted by the manufacturer are assessed during the licensing process and published in the product-specific summaries of product characteristics (SmPCs). In addition, stability studies are published in scientific journals, e.g. for the trastuzumab reference product Herceptin^® [6], [7], [8], [9], [10], [11] and the different trastuzumab biosimilar products [12], [13], [14], [15], [16], [17], [18]. In the SmPC of the trastuzumab biosimilar SB3 (Ontruzant^®) is given that the reconstituted stock solution is physicochemically stable for 7 days when stored at 2–8 °C [1]. Yun et al. reported that the reconstituted SB3 solution is physicochemically stable for 3 days when stored at 25 ± 2 °C [12]. Data on extended physicochemical stability of reconstituted Ontruzant^® solution are lacking and would facilitate efficient handling in pharmacy practice. Therefore, we designed a focused study on extended physicochemical stability of reconstituted SB3 solutions in the original vials over a period of 28 days either stored under light protection at 2–8 °C or at room temperature. For the focused study, size-exclusion high-performance liquid chromatography (SE-HPLC), ultraviolet (UV) spectroscopy, measurement of pH, and observation for visible particles and colour changes were chosen as analytical methods.

Materials and methods

Results

SE-HPLC

In the SE-HPLC chromatograms of trastuzumab Ontruzant^® test solutions a sharp main peak related to the trastuzumab monomer was detected at the retention time (R_t) of approximately 8.5 min, a small peak with R_t of about 12 min related to the excipient histidine, and a very small peak at the R_t of about 5 min, most likely related to an higher molecular weight product (HMWP) (see Figure 1). The correlation coefficient of the trastuzumab assay amounted to R²=0.9874 and proved linearity over the tested concentration range. The mean trastuzumab concentration of the intraday precision test was 1.014 mg/mL (101.4 %) ± 0.57 % relative standard deviation (RSD). The interday precision tests revealed a mean trastuzumab concentration of 1.002 mg/mL (100.2 %) ± 1.46 % RSD. Results deviated less than 2 % and proved reproducibility.

Figure 1:

Overlay of SE-HPLC chromatograms of trastuzumab SB3 solution (1.0 mg/mL) d0, d1, d7, d14, d21, d28. Main peak with high molecular weight product (HMWP) and histidine at detection wavelength 214 nm.

All monomer peaks of the test solutions stored at 2–8 °C or at 15–25 °C were inspected for changes in peak area, peak height and for the formation of additional secondary peaks in form of HMWPs and low molecular weight products (LMWPs). No substantial or systematic changes could be detected in the test solutions over the entire 28-day storage period. All main peaks and secondary peaks met the acceptance criteria (main peak deviation ≤5 %, secondary peaks maximum of 2 % in relation to the main peak). Detailed results are shown in Table 2.

Table 2:

Stability of trastuzumab SB3 reconstituted in the original vial stored at 2–8 °C and 15–25 °C over 28 days determined via SE-HPLC. Results expressed as % remaining trastuzumab concentration ± SD (n=9), initial concentration day 0 set as 100 %.

Storage temperature	SE-HPLC assay
	Initial trastuzumab monomer concentration, mg/mL ± SD (n=9)		Trastuzumab concentration remaining ± SD (n=9); measured concentration day 0=100 %
	Nominal	Measured (day 0)	d1	d7	d14	d21	d28
2–8 °C	21	20.66 (±0.29)	98.87 (±1.88)	99.22a (±1.84)	101.77 (±2.17)	102.24 (±0.99)	100.83 (±1.78)
15–25 °C	21	20.59 (±0.11)	100.29 (±0.73)	100.84 (±1.38)	101.47 (±0.70)	101.76 (±0.69)	102.22 (±0.58)

1. ^an=8. SE-HPLC, size-exclusion high-performance liquid chromatography; SD, standard deviation.

Discussion

In-use stability data of mAb containing reference or biosimilar medicinal products need individual testing because of their complexity and heterogeneity [4, 20]. For the same reasons, physicochemical stability of (glyco)proteins such as mAbs is determined by orthogonal analytical methods.

Out of the recommended orthogonal analytical methods we focused on SE-HPLC, UV-spectroscopy, and pH measurement to determine the integrity of trastuzumab SB3. These stability-indicating methods were chosen for the extended in-use stability study as physicochemical stability of reconstituted and diluted trastuzumab SB3 was already comprehensively investigated using these methods among others [12]. SE-HPLC is well accepted to investigate the intact tertiary structure of mAbs and the absence of oligomers and soluble aggregates. We used previously the same SE-HPLC method for the stability testing of trastuzumab reference product and proved the stability indicating nature by forced degradation studies [6]. Concurrent validation of the SE-HPLC and UV assay were based on the ICH Q2 (R1) guideline [19]. Acceptance criteria for the assessment of stability, sample numbers, sampling time points and storage conditions were chosen based on the specifications of the NHS Pharmaceutical Quality Assurance Committee [4].

Many studies showed physicochemical stability of trastuzumab reference product and various biosimilars. According to the methods used in our study, reconstituted trastuzumab SB3 (Ontruzant^®) solution stored in the original glass vials revealed to be physicochemically stable over a period of 28 days regardless of the storage temperature. Impurities and degradation products were not detected. Yun et al. reported physicochemical stability data of reconstituted trastuzumab SB3 solution (3 days at 25 °C) and diluted trastuzumab SB3 infusion solution (28 days at 5 °C and 25 °C) [12]. The SE-HPLC assays of reconstituted SB3 solutions were identical in both studies during the first 3 days and did not change in our study over the extended test period of 28 days. No significant changes of the trastuzumab peak and no additional secondary peaks got obvious indicating the absence of fragmentation or aggregation. The LMWP peak at R_t 12 min was assigned to histidine which is used as a common excipient in the formulation of final medicinal mAb products [21]. pH values (6.22–6.27) remained unchanged over the extended test period and corresponded to those measured by Yun et al. in the reconstituted trastuzumab SB3 solution (pH 6.1–6.2) [12]. In addition, no change in colour, clarity, or particulate matter could be observed in both studies. Furthermore, there is evidence that higher concentrations of mAb solutions show less degradation tendencies than lower concentrations, which could be related by container absorption or the dilution of stabilising excipients [4]. The extended physicochemical stability of reconstituted, concentrated trastuzumab SB3 solution is in agreement with the previously proven stability of diluted trastuzumab SB3 infusion solution [12]. Results were independent from the chosen storage temperature (2–8 °C, room temperature) in both studies.

Limitations

Physicochemical stability of proteinaceous medicinal products should be studied by orthogonal methods. Out of the highly recommended and utilized methods [4, 7, 8, 12, 14], [15], [16], [17], [18, 20, 22] ion-exchange high-performance liquid chromatography (IE-HPLC) or imaged capillary isoelectric focusing (icIEF) were not used in this study to investigate possible changes in charge variants (acidic, main, and basic variants). Although, Yun et al. showed by icIEF that the initial isoform pattern did not change in diluted trastuzumab SB3 solutions for 28 days [12] and because the utilized UV spectroscopy method only indicates the protein content, additional IE-HPLC or icIEF are necessary to substantiate stability of reconstituted trastuzumab SB3 over 28 days. Subvisible particle measurement and submicronic aggregation were not tested in our study due to the limited available volume of trastuzumab SB3 reconstituted solution.

Finally, microbiological and physicochemical stability are to be considered concurrently when determining the stability of an individual mAb preparation. Microbiological stability of trastuzumab was tested by Karstens et al. by intended inoculation of trastuzumab diluted solution with various facultative pathogenic microorganisms [23]. Tested strains remained viable but did not show any pronounced growth supporting activity in trastuzumab solutions over the observation period. Therefore, refrigerated storage is recommended inhibiting growth of contaminating mesophilic microbes. Nevertheless, the reconstituted trastuzumab solution can still be used after a temporary interruption of refrigerated storage thus preventing waste and promoting efficiency.

Conclusions

According to the stability indicating methods used, physicochemical stability of reconstituted trastuzumab SB3 Ontruzant^® solution in original glass vials is given over a period of 28 days either stored refrigerated or at room temperature. No significant changes could be observed by the analytical methods used. However, additional experimental testing of changes in charge variants over the 28 day period should be added to substantiate the findings. From a microbiological point of view, refrigerated storage is recommended. Results promote a more efficient handling in terms of costs, environment, and practicability in everyday pharmacy practice without compromising safety and efficacy of trastuzumab SB3 preparation.