Fig 1.
TIC stacked profile of INTP5 (Sample I1), US sourced Neulasta (Sample U1 and U5) and EU sourced Neulasta (Sample E1, E4 and E5).
Fig 2.
(A) Overlay of far UV CD spectra, (B) Overlay of FTIR spectra, (C) Overlay of fluorescence spectra, (D) overlay of 1D NMR spectra and (E) DSC thermogram of INTP5 and Neulasta.
Fig 3.
Shifted overlay of 1H-13C HSQC spectrum for INTP5 (Sample I1, Sample I2) and EU sourced Neulasta (Sample E3).
Table 1.
List of methods used for assessment of primary structure, higher order structure and functional characteristics of INTP5, EU sourced Neulasta and US sourced Neulasta.
Fig 4.
(A) Dose response profile of in vitro cell proliferation assay; (B) Dose response profile of receptor binding assay by flow cytometry.
Fig 5.
(A) Overlay of 1H-13C HSQC and HMBC spectra for INTP5 (Sample I1), N-terminal peptide; (B) Shifted overlay of 1H-13C HSQC spectrum for INTP5 (Sample I1, Sample I2) and EU sourced Neulasta (Sample E3).
Fig 6.
(A) Overlay of CEx chromatographic profile of INTP5, US Neulasta and EU Neulasta (Sample I2, U4 and E2); (B) Representative CEx-HPLC profile of INTP5 (Sample I2).
Table 2.
Quantitative purity analysis of INTP5, US Neulasta and EU Neulasta by CEx-HPLC.
Fig 7.
(A) TIC overlay of pegfilgrastim main peak and Pre-peak impurity (RRT 0.84) in peptide mapping (10–75 min) (B) TIC overlay of pegfilgrastim main peak and Pre-peak impurity (RRT 0.84) in peptide mapping (75–145 min).
Fig 8.
Representative CEx-HPLC profile of POP Sample I4 showing Pre-peak impurity 1 (RRT 0.60), Pre-peak impurity 2 (RRT 0.73) and Pre-peak impurity 3 (RRT 0.84).
Fig 9.
SDS-PAGE analysis of Sample I4-CEx impurity peaks (pegylation mixture output).
(Solution 1, Solution 4, Solution 5, Solution 6 and Solution 7 are the internal Reference Standard solutions).
Fig 10.
(A) TIC overlay of pegfilgrastim main peak and Pre-peak impurity 1 (RRT 0.60) in peptide mapping (10–75 min) (B) TIC overlay of pegfilgrastim main peak and Pre-peak impurity 1 (RRT- 0.60), TIC of peptide mapping (75–145 min).
Table 3.
Summary of characterization of Pre-peak Impurities 1, 2, 3 and pegfilgrastim main peak from the pegylation reaction output.
Fig 11.
(A) Overlay of RP chromatographic profile of INTP5, US Neulasta and EU Neulasta (Sample I2, U4 and E2) (B) Representative RP-HPLC profile of INTP5 zoomed at the baseline to reveal the maximum possible impurities.
Table 4.
Quantitative purity analysis of INTP5, US Neulasta and EU Neulasta by RP-HPLC.
Fig 12.
(A) Representative RP-HPLC profile of oxidized (24 hours) INTP5 Sample I2 (B) Overlay of control sample and oxidized impurity 1 (RRT About 0.45) TIC of peptide mapping (10–54 min).
Table 5.
Characterization of oxidized species (Under forced oxidation stress) RRTs are based on RP-HPLC retention times relative to the main peak corresponding to the Native protein.
Fig 13.
(A) Representative RP-HPLC profile of stressed deamidated INTP5 (Sample I2) for peak collection. (High loading- 500 μg) (B) Overlay of pegfilgrastim main peak and Post-peak Impurity (RRT about 1.06), TIC of peptide mapping (85–145 min) for peptide containing Q91.
Table 6.
Summary of characterization of peaks observed by RP-HPLC.
Fig 14.
(A) Overlay of SEC-HPLC chromatographic profile of INTP5, US Neulasta and EU Neulasta (Sample I2, U4 and E2) (B) SEC-HPLC profile of spiking Low Molecular weight (Filgrastim, F1) (green) and High Molecular Weight (purified dipegylated filgrastim) (Red) impurities in INTP5.
Table 7.
Quantitative purity analysis of INTP5, US Neulasta and EU Neulasta using SEC-HPLC.
Fig 15.
Light scattering profile of vortexed INTP5 (molar mass vs. time).
Table 8.
Determination of Molecular Weight by SEC-MALS.
Fig 16.
Representative profile for in vitro bioassay of INTP5 control (microfuge tube) and vortexed (stressed) samples.
Table 9.
In vitro bioassay and SEC-HPLC data of forced aggregated samples.