Analytical and Functional Similarity of Aflibercept Biosimilar ABP 938 with Aflibercept Reference Product

The similarity between ABP 938 and aflibercept RP primary structures was assessed using a number of state-of-the-art methods, including intact molecular mass by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), reduced and deglycosylated molecular mass by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), and reduced peptide mapping with liquid chromatography–mass spectrometry (LC–MS). Aflibercept is a dimeric glycoprotein with a molecular mass of 115 kilodaltons (kDa) [13]. The intact mass analysis results demonstrated that ABP 938 and aflibercept RP had similar intact molecular masses (Table 2). Reduced and deglycosylated molecular mass analyses confirmed that ABP 938 and aflibercept RP had similar molecular masses that were within ± 100 parts per million (ppm) of the theoretical mass (Table 2). In addition, the deconvoluted mass profiles were similar and there were no new species observed for the reduced deglycosylated ABP 938 as compared to aflibercept RP (Fig. 1a). The major peaks corresponded to the expected theoretical masses, which suggested that the C-terminal lysine residues were processed and the conversion of five asparagine residues to aspartic acid residues as a result of removal of the five N-glycans by peptide:N-glycosidase F (PNGase F). There were no detectable levels of N-terminal variants observed in ABP 938; however, partially processed N-terminal signal peptides were detected in aflibercept RP (Table 2). This minor difference is not expected to impact clinical performance because both ABP 938 and aflibercept RP had similar biological activities (see “Biological Activity” section). Finally, the primary amino acid sequence was determined by overlapping peptide map with tandem MS. The results confirmed that ABP 938 and aflibercept RP have the same amino acid sequence. Moreover, the peptide map profiles were visually similar, and no new peaks above the integrity limit were detected in ABP 938 as compared to aflibercept RP (Fig. 1b).

ABP 938 and aflibercept RP displayed similar extinction coefficient values as determined by amino acid analysis using ultraviolet (UV) spectroscopy, and a similar identity as evaluated by a dual anti-idiotype enzyme-linked immunosorbent assay (ELISA) (data not shown).

The HOS and biophysical properties of ABP 938 and aflibercept RP were assessed using multiple techniques. The secondary structure was characterized by Fourier transform infrared (FTIR) spectroscopy and showed strong bands in the spectra at 1639 cm⁻¹ and 1690 cm⁻¹, indicative of a β-sheet secondary structure (Fig. 2a). The tertiary structure was characterized by near-UV circular dichroism (CD) spectra which contained signals from tryptophan, tyrosine, and phenylalanine superimposed on the broad disulfide signal from 240 to 350 nm (Fig. 2b). The spectral similarity value of ABP 938, aflibercept (US), and aflibercept (EU) met the pre-defined similarity assessment criteria of ≥ 95% for both FTIR and near-UV CD spectroscopy (Table 2).

Thermostability was characterized by differential scanning calorimetry (Fig. 2c). ABP 938 and aflibercept RP displayed similar thermal melting transition temperatures at approximately 68.1 °C for T_m1 and 85.0 °C for T_m2 (Table 2). An absence of submicron particles in ABP 938 and aflibercept RP was confirmed by dynamic light scattering (Table 2). In addition, ABP 938 and aflibercept RP had similar levels of monomer, with no detectable aggregates assessed by sedimentation velocity analytical ultracentrifugation (SV-AUC) (Table 2). Finally, ABP 938 and aflibercept RP had similar molar masses for the monomer and high molecular weight (HMW) species as evaluated by size exclusion high-performance liquid chromatography (SE-HPLC) coupled with light scattering detector (Fig. 2d). ABP 938 had a lower level of HMW species compared to aflibercept RP (Fig. 2d). The theoretical masses of aflibercept RP monomer and dimer were approximately 115 kDa and 229 kDa, respectively, and the molar masses for monomer and HMW of ABP 938 and aflibercept RP were within the theoretical molar masses (Table 2). The molar masses of HMW observed in ABP 938 and aflibercept RP were consistent with that of dimers.

ABP 938 and aflibercept RP were evaluated for product-related substances and impurities. Size and charge variants are indicative of the stability of the products, which may change over time at the recommended storage conditions. ABP 938 and aflibercept RP had visually similar HMW and main peak profiles with no new or missing peaks as assessed by size exclusion ultrahigh-performance liquid chromatography (SE-UHPLC) (Fig. 3a). The stability data demonstrated that under the recommended storage conditions, ABP 938 maintained a lower level of HMW species compared to aflibercept RP for up to 36 months (Fig. 3a inset).

Size variants were assessed by capillary electrophoresis–sodium dodecyl sulfate (CE-SDS) in reducing and denaturing conditions. This method is suitable for quantifying fragments and the purity of the samples. ABP 938 and aflibercept RP had visually similar total pre-peak (fragmentation) and main peak (purity) profiles, with no new or missing peaks as assessed by reduced CE-SDS (rCE-SDS) (Fig. 3b). The stability data demonstrated that the level of rCE-SDS main peaks for ABP 938 remained higher than aflibercept RP for up to 36 months (Fig. 3b inset). Size variants assessed by non-reduced CE-SDS (nrCE-SDS) showed that ABP 938 and aflibercept RP had visually similar profiles with no new or missing peaks (Fig. 3c). Size variants included fragments and partial molecules; however, partial molecule species were not observed in either ABP 938 or aflibercept RP samples. The pre-peaks observed are fragmented species, which were present at low levels. The stability data demonstrated that the levels of main peaks were similar for ABP 938 and aflibercept RP (Fig. 3c inset).

Aflibercept RP is supplied in a single-dose vial and a single-dose pre-filled syringe (PFS) designed to deliver 0.05 mL at 40 mg/mL [16]. ABP 938 and aflibercept RP demonstrated similar product strength as assessed by protein concentration and volume in both vial and PFS presentations (Table 2).

Aflibercept RP acts as a soluble decoy receptor that binds VEGF-A and PlGF, thereby inhibiting the binding and activation of its cognate VEGF receptors [10, 16]. The VEGFR domain-mediated binding and cell-based functional studies demonstrated that ABP 938 was similar to aflibercept RP with respect to inhibition of VEGF-A₁₆₅-mediated proliferation in human umbilical vein endothelial cells (HUVEC), VEGF-A₁₆₅/VEGFR-2 signaling inhibition in HUVEC, VEGF-A₁₆₅/VEGFR-2 signaling inhibition in HEK293 cells, and VEGF-A₁₆₅/VEGFR-2 inhibition of binding (Fig. 4a–d). Additionally, the data suggested that ABP 938 and aflibercept RP were similar in PlGF-1/VEGFR-1 dimerization inhibition in human osteosarcoma U2OS cells, PlGF-2/VEGFR-1 dimerization inhibition in U2OS cells, PlGF-1 and PlGF-2 binding, and PlGF-1/VEGFR-1 inhibition of binding (Fig. 5a–e).

Aflibercept RP also binds to galectin-1 and VEGF-B but not to VEGF-C and VEGF-D [17, 18]. ABP 938 was similar to aflibercept RP with respect to galectin-1 and VEGF-B binding activity (Fig. 6a, b), and the absence of VEGF-C and VEGF-D binding activity (Table 3). Similar to aflibercept RP, ABP 938 showed a lack of antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC) activities (Table 3).

The studies evaluating Fc domain-mediated binding activity demonstrated that ABP 938 and aflibercept RP had similar levels of neonatal fragment crystallizable receptor (FcRn) and Fc-gamma receptor 1 (FcγRI) binding. While ABP 938 had lower levels of complement component 1q (C1q) and other FcγR binding compared to aflibercept RP (Table 3), the differences are not considered critical for clinical performance because both ABP 938 and aflibercept lacked ADCC, ADCP, and CDC activities (Table 3).