Table 1.
Criticality level of attributes analyzed.
Fig 1.
Comparison of chromatographic profiles.
(A) Reduced peptide mapping by UV detection, (B) Reduced peptide mapping by MS analysis (with identified peptides annotated) (C) Non-reduced peptide mapping by UV detection (D) Deconvoluted mass spectra for intact mass and polydispersity analysis (Representative images).
Fig 2.
Comparison of spectral profiles.
(A) Far UV CD spectra, (B) Near UV-CD spectra, (C) Intrinsic fluorescence spectra at 280 nm and (D) 295 nm. 2D UV-visible spectra of (E) Neulasta® and (F) Lupin’s Pegfilgrastim (Representative images).
Table 2.
Comparison of secondary structure analyzed by Far UV CD.
Table 3.
Comparison of disulfide bond mapping.
Fig 3.
Comparison of higher order structures.
(A) Thermal denaturation profiles, (B) 1D 1H NMR spectra with a full intensity scale (left panel) and with an expanded intensity scale (right panel), (C) 2D 1H-13C HSQC NMR spectra at natural abundance of Lupin’s Pegfilgrastim; V0200039 (left panel) and Neulasta® 1095928 (right panel). Cross peaks represent fingerprint C-Hn resonances from the polypeptide and PEG.
Fig 4.
Comparison of functional attributes.
Dose response curve of (A) Neulasta® and (B) Lupin’s Pegfilgrastim. (C) Scatter plot of relative potency. (D) Equivalence testing of relative potency. Binding kinetics of pegfilgrastim with G-CSF receptor by SPR (E) G-CSF receptor sensorgrams, (F) KD Kinetics scatter plot, (G) Equivalence testing of receptor binding. The circles and squares represent samples analyzed at two different campaigns.
Fig 5.
Comparison of product-related impurities.
(A) SE-HPLC chromatograms analyzing size variants, (B) SEC-MALS chromatograms analyzing size variants, (C) CEX-HPLC chromatograms analyzing charge variants, (D) RP-HPLC chromatograms analyzing impurities.
Fig 6.
Scatter plots for product-related impurities.
(A) Aggregates, (B) HMW A+B, (C) Main peak, and (D) Total HMW measured by SE-HPLC. (E) Aggregates measured by AUC. (F) Aggregates, (G) HMW A+B, and (H) Main form measured by SEC-MALS. (I) Pre-peaks and (J) Main peak measured by CEX-HPLC. (K) Pre-peaks, (L) Main peak, (M) Post-peaks, (N) Dimer measured by RP-HPLC. (O) Total oxidation measured by LC-MS. Observed mass of (P) Pegylated variants measured by SE-UPLC. (Q) Free m-PEG measured by RP-UPLC-CAD. (R) Protein concentration measured by UV absorbance. The circles and squares represent samples analyzed at two different campaigns.
Table 4.
Comparison of product-related variants.
Table 5.
Comparison of total sub-visible particles obtained by MFI.
Table 6.
Summary of forced-degradation studies.
Table 7.
Results of similarity assessment including details of attributes, analytical methods and the number of batches tested.