FormalPara Key Points

Pfizer’s approach to formulation development for PF-06439535 drug product as a biosimilar to bevacizumab reference product (RP) was the optimization of the RP formulation.

The preferred formulation for PF-06439535 (Zirabev®, a biosimilar) is 20 mM succinate buffer at pH 5.5.

Sucrose is an effective stabilizer when PF-06439535 is stored frozen or at 5 °C.

1 Introduction

Biosimilars are biologic drugs that are highly similar to a licensed biologic (i.e., originator or reference product [RP]), with no clinically meaningful differences from the RP in terms of safety, purity, or potency [1,2,3]. Biosimilar development follows strict regulatory guidelines that require rigorous, head-to-head comparative assessments of the proposed biosimilar and the RP. These assessments include analytical, nonclinical, and clinical studies that are performed in a stepwise manner, with regulatory approval granted based on the totality of the evidence for biosimilarity that is accumulated over all stages of testing [1,2,3].

PF-06439535 (bevacizumab-bvzr; Zirabev®; Pfizer Europe MA EIGG, Brussels, Belgium, and Pfizer Inc., New York, NY, USA) is a biosimilar of bevacizumab RP (Avastin®; Genentech Inc, South San Francisco, CA, USA, and Roche Registration GmbH, Grenzach-Wyhlen, Germany) [4, 5]. PF-06439535 was approved by the European Medicines Agency and US Food and Drug Administration for use in all indications of the RP, except for hepatocellular carcinoma [4, 5]. Biosimilarity of PF-06439535 to bevacizumab RPs sourced from the European Union (bevacizumab-EU) and from the US (bevacizumab-US) was established based on results from analytical and nonclinical toxicity studies [6,7,8]. In addition, a clinical study conducted in healthy volunteers demonstrated similarity in pharmacokinetics, safety, and immunogenicity of PF-06439535 to bevacizumab RPs [9]. Furthermore, a confirmatory comparative study of PF-06439535 and bevacizumab RP in patients with non-small cell lung cancer demonstrated similarity in efficacy, with the confidence intervals for the difference between groups in objective response rate falling within the prespecified equivalence margins [10].

The work presented describes Pfizer’s approach to formulation development for PF-06439535 drug product as a biosimilar to bevacizumab RP; in particular, optimization of the RP formulation instead of the approach to utilize the same RP formulation. The goal of formulation development for PF-06439535 drug product was to identify an appropriate formulation that resulted in a product with a stability profile no worse than that of the bevacizumab RP. Similarity was defined as having no new degradation products observed for the PF-06439535 product that were not also observed in the bevacizumab RPs, and overall levels of degraded species in the PF-06439535 product that are similar to the bevacizumab RPs under the same conditions. A combination of PF-06439535 product knowledge and historical experience with the development of other monoclonal antibodies guided the selection of appropriate analytical techniques for each assessment. To accelerate the rates of degradation to be measurable on a timescale amenable to development, elevated temperature forced degradation conditions were used. The specific aim was to compare the stability of the PF-06439535 protein in multiple formulations, including the same formulation as the bevacizumab RPs.

The information and knowledge gained from drug product studies conducted during development have provided scientific understanding to support the establishment of manufacturing controls and product controls, including tests in the release and stability specification. The physicochemical and biological properties relevant to the safety, efficacy, quality, or manufacturability of the PF-06439535 drug product were investigated from early development through process validation, using a range of analytical procedures. The methods used in each development study were chosen because they are indicative of stability, and likely to generate information to support the design of a formulation and a manufacturing process that is capable of consistently delivering the intended product quality.

2 Methods

2.1 Formulation Development Strategy

2.1.1 Buffer Selection

Initial pH screening studies were performed using PF-06439535 drug substance material by formulating in acetate, histidine, and succinate buffers (at 20 mM). This assessed the impact of the buffer system and pH in the range of 5.2–6.0 on PF-06439535 in the absence of other stabilizing excipients. Additionally, development formulation stability studies compared PF-06439535 in the RP buffer system (phosphate buffer at pH 6.2 with trehalose and polysorbate 20) with buffer systems at lower pH values (pH 5.5 and 5.8).

Formulations were created by buffer exchanging the starting material (1.0 mL of PF-06439535) using 0.5–3 mL Slide-A-Lyzer cassettes (Thermo Fisher Scientific, Waltham, MA, USA) into the proper buffers/pH. After dialysis, the protein concentration was verified, followed by sterile filtration, and filled into vials to be stored in designated storage conditions.

The excipients used in buffer preparation were analytical grade sourced from various reputable vendors in the United States (US). The formulations were stored at 40 °C for up to 12 weeks to determine the optimal buffer and pH under stressed conditions.

2.1.2 Stabilizing Excipient Selection

An important aspect of the formulation development program was to determine the stabilizing excipients for PF-06439535. Sucrose (85 mg/mL), edetate disodium dihydrate (EDTA; 0.05 mg/mL), and polysorbate 80 (PS80; 0.2 mg/mL) were evaluated for use in the PF-06439535 formulation. Analytical grade sucrose (EMD Millipore Corporation, Burlington, MA, USA), EDTA (JT Baker, Avantor, Radnor, PA, USA), and PS80 (JT Baker, Avantor, Radnor, PA, USA) were used. To determine if sucrose was an appropriate cryoprotectant for frozen storage of PF-06439535 drug substance, a formulation at the drug substance protein concentration of 100 mg/mL with all excipients (20 mM succinate at pH 5.5, 85 mg/mL sucrose, 0.05 mg/mL EDTA, 0.2 mg/mL PS80) was evaluated after 22 weeks at − 20 °C and − 40 °C. Additionally, PF-06439535 drug product at 25 mg/mL with all excipients (20 mM succinate at pH 5.5, 85 mg/mL sucrose, 0.05 mg/mL EDTA, 0.2 mg/mL PS80) was evaluated after 22 weeks at 5 °C and 25 °C and after 8 weeks at 40 °C. Finally, PF-06439535 in the succinate formulation (five lots) was compared with PF-06439535 in the RP formulation (one lot) and to bevacizumab-US and bevacizumab-EU (three lots each) after storage at 40 °C for 12 weeks.

2.2 Analytical Procedures

There are several potential routes of degradation, including changes in high molecular mass species (HMMS), charge profile, fragments, and oxidation. Therefore, testing for HMMS by size exclusion high-performance liquid chromatography (SE-HPLC), charge isoforms by cation exchange high-performance liquid chromatography (CEX-HPLC), fragments by non-reducing capillary gel electrophoresis (CGE), and methionine oxidation by limited Lys-C proteolytic mapping were performed. These analytical procedures were performed as previously described by Weiser et al. [11] and are briefly summarized below. Analytical grade excipients sourced from various reputable vendors in the US were used in all analyses.

2.2.1 Size Exclusion High-Performance Liquid Chromatography

The formation of HMMS of PF-06439535 was assessed by SE-HPLC. Isocratic elution (20 mM sodium phosphate, 400 mM sodium chloride, pH 6.0) of the samples was carried out using a YMC-Pack Diol-200 column (300 mm × 8 mm, pore size 200 Å, particle size 5 µm; YMC, Koyoto, Japan) on a Waters HPLC system (Waters Corporation, Milford, MA, USA) with ultraviolet (UV) detection and was performed at a wavelength of 280 nm.

2.2.2 Cation-Exchange Chromatography

Cation-exchange chromatography (CEX) was used to separate charge variants of PF-06439535. CEX separation was performed using a Dionex ProPac WCX-10 cation exchange column (4 mm × 250 mm, 10 micron, Thermo Fisher Scientific) on a Waters HPLC system (Waters Corporation), and the separation was monitored with UV detection at a wavelength of 214 nm.

2.2.3 Non-reducing Sodium Dodecyl Sulfate Capillary Gel Electrophoresis

Intact antibody and antibody fragments were assessed by sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE). The assay was performed under non-reducing conditions. Samples were prepared using the ProteomeLab IgG Purity/Heterogeneity Assay Kit (Beckman Coulter, Indianapolis, IN, USA) or the ProteomeLab SDS-MW Analysis Kit (Beckman Coulter). Electrophoretic separation was performed at 15 kV (normal polarity) and monitored at a wavelength of 220 nm.

2.2.4 Methionine Oxidation in the Crystallizable Fragment (Fc) Domain

Changes in the oxidation of PF-06439535 were assessed by monitoring the oxidized and non-oxidized forms of a methionine-containing peptide fragment from the crystallizable fragment (Fc) domain. PF-06439535 was digested with Lys-C and the resulting peptide fragments were analyzed by reversed-phase high-performance liquid chromatography (Waters HPLC system, Waters Corporation). Samples were injected onto the column (Zorbax 300SB, 4.6 mm × 250 mm, 5 micron; Agilent Technologies, Inc., Santa Clara, CA, USA), and the separated peptide fragments were detected using UV absorbance at 214 nm.

2.2.5 Visible Appearance, Subvisible Particle Characteristics, and pH

The presence of visible particulates in solution was evaluated by visual inspection of samples: 4 mL of each drug product formulation was filled into 5 mL glass Type I tubing vials with West 13 mm, 4432/50 FluroTec coated serum stoppers (West Pharmaceutical Services, West Whiteland Township, PA, USA). Vials were stored upright at 40 ± 2 °C (75% ± 5% relative humidity), inspected at each timepoint for visual appearance, and returned to stability until the 12-week timepoint when subvisible particle counting was performed by light obscuration using an HIAC Particle Size Analyzer (HIAC 9703+ Pharmaceutical Particle Counter and HRLD150 Sensor; Beckman Coulter) to assess for particulate matter. Samples were evaluated using a pH electrode and meter that was calibrated before use [11].

2.2.6 Protein Concentration

UV spectroscopy (SoloVPE variable path length instrument; Repligen Corporation, Bridgewater, NJ, USA; Cary 50/60 UV spectrophotometer; Agilent Technologies) was performed for protein concentration [11].

3 Results

3.1 Formulation Development Strategy

3.1.1 Buffer Selection

PF-06439535 demonstrated optimal stability when stored at 40 °C for up to 13 days in either histidine or succinate buffer systems (pH range of 5.2–5.8) (Fig. 1).

Fig. 1
figure 1

Effect of pH and buffer on the HMMS level in PF-06439535 in biosimilar formulations (acetate, histidine, and succinate) stored at 40 °C. HMMS high molecular mass species

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PF-06439535 was more stable in the succinate formulation (pH 5.5) than in the RP formulation (pH 6.2), under both real-time and accelerated stability conditions. SE-HPLC, CEX-HPLC, and limited Lys-C proteolytic mapping results for storage of PF-06439535 at 40 °C at 0 and 8 weeks are shown in Table 1. Results demonstrated a greater increase from week 0 in HMMS with phosphate buffer at pH 6.2 compared with other formulations at lower pH values (Table 1 and Fig. 2).

Table 1 Effect of pH and buffer on the HMMS level, acidic species, and methionine oxidation in PF-06439535 drug product formulations
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Fig. 2
figure 2

Effect of pH and buffer on the HMMS level in PF-06439535 in the RP formulation (phosphate) versus biosimilar formulations (succinate or histidine) stored at 40 °C. HMMS high molecular mass species, RP reference product

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All formulations demonstrated a significant increase in acidic species from week 0 (Table 1). The level of increase (between weeks 2 and 8) in methionine oxidation was similar between the phosphate and histidine buffers, with less of an increase observed for the succinate buffer (Table 1).

Results of the pH and buffer accelerated stability studies demonstrated that succinate buffer at a concentration of 20 mM with a target pH of 5.5 was the preferred choice for the PF-06439535 formulation.

3.1.2 Stabilizing Excipient Selection

No significant changes in the product quality attributes of frozen PF-06439535 drug substance were observed after 22 weeks at − 20 °C and − 40 °C (Table 2).

Table 2 Stability of frozen PF-06439535 drug substance at 100 mg/mL with excipients
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Likewise, no changes in the product quality attributes of PF-06439535 drug product were observed after 22 weeks at 5 °C, which is the recommended storage temperature. Changes were observed at 25 °C or after 8 weeks at 40 °C as expected (Table 3). No new degraded species were observed in the biosimilar succinate formulation compared with the RP formulation.

Table 3 Stability of PF-06439535 drug product at 25 mg/mL with excipients
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The stability studies of frozen and liquid PF-06439535 provide supporting data that sucrose is an effective cryoprotectant for processing and frozen storage of PF-06439535 drug substance, as well as an effective stabilizing excipient for liquid storage of PF-06439535 drug product.

A comparison between the bevacizumab RP and the PF-06439535 drug product formulations is shown in Table 4, where the RP formulation was verified through published information available (i.e., prescribing information in the US [5] and European Public Assessment Report [4]). The selected excipients for PF-06439535 are summarized in Table 5.

Table 4 Comparison of PF-06439535 drug product and bevacizumab RPs
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Table 5 Amounts and functions of PF-06439535 excipients
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As the selected succinate formulation is different from the bevacizumab RP formulation, PF-06439535 in both formulations was directly compared. At elevated temperatures, PF-06439535 was more stable in the succinate formulation compared with the RP formulation (Figs. 3, 4, 5, 6, 7).

Fig. 3
figure 3

SE-HPLC analysis of HMMS content in PF-06439535 in the RP formulation and biosimilar succinate formulation stored at 5 °C, 25 °C, or 40 °C. SE-HPLC size exclusion high-performance liquid chromatography, HMMS high molecular mass species, RP reference product

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Fig. 4
figure 4

Limited Lys-C proteolytic mapping analysis of methionine oxidation in PF-06439535 in the RP formulation and biosimilar succinate formulation stored at 5 °C, 25 °C, or 40 °C for 8 weeks. RP reference product

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Fig. 5
figure 5

CEX-HPLC analysis of acidic species in PF-06439535 in the RP formulation and biosimilar succinate formulation stored at 5 °C or 25 °C for 22 weeks and at 40 °C for 12 weeks. CEX-HPLC cation exchange high-performance liquid chromatography, RP reference product

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Fig. 6
figure 6

CEX-HPLC analysis of basic species in PF-06439535 in the RP formulation and biosimilar succinate formulation stored at 5 °C or 25 °C for 22 weeks and at 40 °C for 12 weeks. CEX-HPLC cation exchange high-performance liquid chromatography, RP reference product

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Fig. 7
figure 7

Non-reducing CGE analysis of fragments in PF-06439535 in the RP formulation and biosimilar succinate formulation stored at 5 °C, 25 °C, or 40 °C for 8 weeks. CGE capillary gel electrophoresis, RP reference product

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The stability of PF-06439535 in the succinate formulation versus the RP formulation was assessed at 25 mg/mL after storage at 5 °C and 25 °C for 22 weeks or at 40 °C for 12 weeks. The results demonstrated that the PF-06439535 product was more stable in the succinate formulation compared with the RP formulation in that upon storage at elevated temperatures, there was less of an increase in HMMS (Fig. 3) and slightly less of an increase in methionine oxidation (Fig. 4).

Other parameters such as levels of acidic species, basic species, and fragments (Figs. 5, 6, 7, respectively) were similar between the two formulations. Samples stored at the same temperature exhibited similar increases in basic species regardless of formulation (Fig. 6). For example, PF-06439535 formulated in the RP formulation showed a similar chromatographic profile to PF-06439535 formulated in the succinate formulation after storage at 40 °C for 12 weeks. In addition, the overall level of basic species was likely overestimated due to the challenging integration of the degraded samples.

No new degraded species were observed for samples using the succinate formulation compared with the RP formulation and the overall degradation profiles appeared similar between the two formulations. These data support the suitability of the succinate formulation for the PF-06439535 product and demonstrate it is the preferred choice for the PF-06439535 product. Additional studies such as subjecting samples to repeated freeze-thaw stress demonstrated no differences for PF-06439535 product formulated in either of the two formulations. Both formulation systems were stable with no change in the HMMS level after five freeze–thaw exposures (electronic supplementary material [ESM] Fig. S1).

Finally, PF-06439535 in the succinate formulation was more stable than PF-06439535 in the RP formulation, as less HMMS were formed when stored at an elevated temperature of 40 °C for 12 weeks (Fig. 8).

Fig. 8
figure 8

SE-HPLC analysis of HMMS in PF-06439535 in the biosimilar succinate formulation and bevacizumab-US, bevacizumab-EU, and PF-06439535 in the RP formulation stored at 40 °C for 12 weeks. SE-HPLC size exclusion high-performance liquid chromatography, HMMS high molecular mass species, RP reference product

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Furthermore, no new degraded species were observed in PF-06439535 compared with bevacizumab RP, and the overall degradation profiles were similar (ESM Figs. S2–S4).

3.1.3 Subvisible Particle Characteristics, pH, and Protein Concentration

The histidine and succinate formulations had similar levels of subvisible particles (ESM Fig. S5). PF-06439535 in the RP formulation had higher levels of subvisible particles and was more similar to the levels of subvisible particles observed in the RP (ESM Fig. S5). Levels of subvisible particles in the bevacizumab-EU product may have been slightly elevated due to the age of the product; it was used as a control in this study for overall reference but was used beyond its established expiry. There were no significant differences between formulations in pH or protein concentration data (ESM Tables S1 and S2).

4 Discussion

The goal of this formulation development study was to identify an appropriate formulation that produced a PF-06439535 drug product with a stability profile no worse than that of the bevacizumab RP. The requirements were that the formulation should provide adequate stability, safety, and compatibility for dosing; however, in line with our approach when developing formulations for other biosimilars, we chose not to replicate the formulation of the RP but rather followed an approach designed to obtain an optimal formulation in terms of these aspects while supporting similarity to the licensed RP. A screening study was conducted to determine the optimal buffer and pH for PF-06439535 under stressed conditions. The bevacizumab RP’s pH of 6.2 was not selected for use in the PF-06439535 formulation due to the observed increase in the formation of HMMS at pH 6.2 compared with lower pH values. Rather, optimal stability was demonstrated through stressed studies in the pH range of 5.2–5.8. Therefore, a target pH of 5.5 was selected for PF-06439535, allowing for ±0.3 pH units on either side of the target.

Effective buffers at pH 5.5 that were evaluated in the stress testing of PF-06439535 included histidine and succinate. The succinate formulations had slightly lower HMMS and methionine oxidation than the histidine formulations. Therefore, succinate was selected as the buffer for the PF-06439535 formulation. A concentration of 20 mM succinate was selected based on Pfizer’s prior experience with monoclonal antibody formulations. This concentration of succinate buffer has previously demonstrated sufficient buffering capacity to maintain the pH of the drug substance, drug product, and dosing solution (PF-06439535 diluted in 0.9% sodium chloride).

As a protein, PF-06439535 is stabilized by an amorphous sugar to prevent conformational changes of the antibody during freezing. Sucrose is a commonly used cryoprotectant for parenteral formulations of biologics, and prior experience has demonstrated this excipient to stabilize protein conformation during frozen storage of drug substance and liquid storage of drug product. Therefore, sucrose was selected as the cryoprotectant and tonicity modifier in the PF-06439535 formulation, based on its compatibility with frozen storage.

Platform formulation experience has previously demonstrated that EDTA protects proteins exposed to stainless steel containers during production from metal ion-induced instability. EDTA was included in the PF-06439535 formulation as a chelating agent because the drug substance and drug product will be exposed to stainless steel containers during production. EDTA was included at a level (0.05 mg/mL) expected to chelate metal ions leaching from the container and thus protect PF-06439535 from metal ion-induced instability.

As PF-06439535 is a protein, a surfactant is a commonly added excipient to prevent physical instabilities such as aggregation, precipitation, and interactions with component surfaces. PS80 is a commonly used surfactant for parenteral formulations. Based on historical experience with PS80 in monoclonal antibody formulations, it was evaluated for use in the PF-06439535 formulation. A PS80 concentration of 0.2 mg/mL was selected for the PF-06439535 formulation, to reduce the potential for HMMS formation due to interfacial shear stress. Furthermore, vegetable-derived PS80 with low peroxide content was used in the formulation to minimize peroxide-induced oxidation over the drug product shelf-life.

In summary, the developed formulation for PF-06439535 included succinate buffer at pH 5.5, with sucrose, EDTA, and PS80 as stabilizing excipients. The PF-06439535 product appeared more stable in the succinate formulation compared with the RP formulation following storage at elevated temperatures. The essential elements for Pfizer’s drug product development included the legal requirements for biosimilar approval in global jurisdictions with a regulatory framework for licensure of biosimilar products. Development of the PF-06439535 drug product formulation considered aspects relevant to both developing a pharmaceutically acceptable product and achieving similarity to the RP on a global basis.

Regulatory guidance dictates that a biosimilar must have the same strength, dosage form, and route of administration as its RP, while other features, such as the formulation, can differ from that of the licensed RP as long as they provide the same or better product quality in terms of stability, safety, compatibility for dosing, and support for similarity to the licensed RP [1,2,3]. Therefore, the priority was for the drug product presentation to match the RP for attributes that are clinically relevant to dosing, such as the amount of active ingredient per vial, protein concentration, preparation (i.e., diluents), and route of administration (i.e., intravenous, subcutaneous). Additionally, while aiming for similarity, leveraging substantial prior knowledge gained from the development of other Pfizer monoclonal antibodies, while using compendial excipients, to achieve the best quality product during formulation development resulted in the selection of a commercial formulation with the most robust stability profile and that was optimized for the PF-06439535 product.

5 Conclusions

Formulation development for PF-06439535 focused on developing a drug product similar to the RP. Liquid vial presentations of PF-06439535 have been developed (400 and 100 mg/vial; at 25 mg/mL) to be prepared and administered in the same way as the RP. The formulation for PF-06439535 does not match the RP formulation but was considered the most robust of several formulations, with enhanced quality.