Physicochemical and biological comparison of the first Brazilian biosimilar filgrastim with its reference product

Figures & data

Table 1 Analytical methods and batches used to compare Eurofarma’s Fiprima^® and Hoffman-La Roche Ltd Granulokine^®

Parameter	Analytical method	Number of batches compared
		Eurofarma	Hoffman-La Roche Ltd
Advanced analysis
Primary structure	N-terminal sequencing	5	2
	Peptide mapping – mass spectrometry	3	3
Secondary and tertiary structure	Far-UV CD	7	5
	Near-UV CD	5	5
	Intrinsic fluorescence	5	4
	Extrinsic fluorescence	3	3
	Thermostability by CD-far	4	3
	X-ray crystallography	2	In silica
Identity, charge, and purity	Bidimensional electrophoresis	8	5
Size	Mass spectrometry	6	5
Standard analysis
Identity and purity	SE-HPLC	15	7
Content, hydrophobicity, and purity	RP-HPLC	20	13
Size and purity	Reducing SDS-PAGE	15	5
Size and purity	Nonreducing SDS-PAGE	15	4
Identity, charge, and purity	PI	15	8
Purity (HCP and contaminant DNA)	ELISA	15	6
	Real-time PCR	9	7
Identity	Western blot	15	8
Receptor binding and potency (in vitro)	Cell bioassay	20	5

Abbreviations: UV, ultraviolet; CD, circular dichroism; SE-HPLC, size-exclusion high-performance liquid chromatography; RP-HPLC, reverse-phase high-performance liquid chromatography; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PI, isoelectric point; ELISA, enzyme-linked immunosorbent assay; HCP, host cell protein; PCR, polymerase chain reaction.

Figure 1 Total ion current chromatogram of mass spectromery (400–1,500 m/z) comparing Fiprima^® (upper spectrum batch P037) and Granulokine^® (lower pectrum batch B2070B05).

Figure 2 Comparison of far-UV CD spectra from Fiprima^® and Granulokine^®.

Notes: Overlay of the far-UV CD spectra of three batches of Fiprima (P037, P038, P040) and three batches of Granulokine (B2030B01, B2070B05, B2073B05).

Abbreviations: CD, circular dichroism; UV, ultraviolet.

Table 2 Deconvolution using CDSSTR database

Sample	% α helix	% β sheet	% Turns	% Nonordinated
Eurofarma P037	51	23	8	19
Eurofarma P038	52	21	8	19
Eurofarma P040	51	24	7	18
Hoffman-La Roche Ltd B2030B01	52	22	7	19
Hoffman-La Roche Ltd B2070B05	52	22	8	18
Hoffman-La Roche Ltd B2073B05	50	23	8	19

Note: Data from Sreerama et al.⁹

Figure 3 Far-UV CD spectra of samples submitted to different temperatures.

Notes: The three samples at the left are Eurofarma’s batches (A – P037; B – P038; C – P040) and samples at the right are Hoffman-La Roche Ltd (A – B2030B01; B – B2070B05; C – B2073B05).

Abbreviations: CD, circular dichroism; UV, ultraviolet.

Figure 4 Near-UV CD nonnormalized spectra of samples of Eurofarma’s (P037, P038, P040) and Hoffman-La Roche Ltd batches (B2070B05, B2073B05).

Note: Arrow indicates the most negative peak.

Abbreviations: CD, circular dichroism; UV, ultraviolet.

Figure 5 Comparison of fluorescence spectra of integral and denatured filgrastim.

Notes: Spectra superposition of three of Eurofarma’s batches (P037, P038, P040) and three of Hoffman-La Roche Ltd batches (B2030B01, B2070B05, B2073B05). Urea was the denaturing agent.

Figure 6 Comparison of tertiary structure of filgrastim.

Notes: Overlay of tertiary structure of Eurofarma’s filgrastim Fiprima^® (blue) and other human filgrastims deposited in Protein Data Bank (1CD9 in green, 1PGR in pink, and Angem’s 1RHG in yellow).

Figure 7 Electrophoresis (reduced SDS-PAGE) silver stained comparing Eurofarma batch P003 and Hoffman-La Roche Ltd batch B2010B02.

Notes: From left to right: Molecular weight standard; P003 at 100 µg/mL; P003 at 50 µg/mL; P003 at 10 µg/mL; P003 at 2 µg/mL; P003 at 1 µg/mL; CRS at 100 µg/mL; CRS at 2 µg/mL; Granulokine^® B2010B02 at 100 µg/mL; Granulokine B2010B02 at 2 µg/mL.

Abbreviations: SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; CRS, Chemical Reference Substances.

Figure 8 Comparative bidimensional electrophoresis.

Notes: Bidimensional electrophoresis of the Granulokine^® batch at the left (B1037B02) and Eurofarma’s Fiprima^® at the right (TP104). 1= Granulokine; 2= Fiprima

Figure 9 Spectra obtained of impurity peak (above) and the main filgrastim peak (below).

Note: The spectra is the result of mass spectrometry of the impurity peak and the main filgrastim peak.

Figure 10 Comparative RP-HPLC.

Notes: Superposed RP-HPLC chromatograms of Eurofarma’s Fiprima^® and Hoffman-La Roche Ltd Granulokine^®. Left: Fiprima’s batches 056/10 (blue), 057/10 (green), and 058/10 (pink) and Granulokine batch B2008B01 (red); right: Eurofarma filgrastim batches P008 (blue), P037 (light blue), P038 (green), P040 (gray), and P044 (orange) and two Granulokine batches B2030B01 (red) and B2070B05 (brown).

Abbreviation: RP-HPLC, reverse-phase high-performance liquid chromatography.

Table 3 Retention of the peaks from RP-HPLC and their area percentage

Batches	Retention (mL)	Area/ peak area (%)	Batches	Retention (mL)	Area/ peak area (%)
Eurofarma	12.84	0.44	Eurofarma	12.84	0.25
056/10	13.19	0.08	P008	14.28	99.41
	14.31	99.48		15.20	0.34
Eurofarma	12.82	0.35	Eurofarma	12.83	0.40
057/10	14.30	99.37	P037	14.20	99.27
	15.69	0.29		15.16	0.33
Eurofarma	12.87	0.35	Eurofarma	14.33	99.24
058/10	14.32	99.43	P038	14.96	0.32
	15.73	0.23		15.12	0.44
Hoffman-La	12.79	0.22	Eurofarma	14.23	99.46
Roche Ltd	14.23	99.73	P040	15.15	0.26
B2008B01	17.82	0.05		15.25	0.28
Hoffman-La	14.31	99.42	Eurofarma	11.25	0.04
Roche Ltd	15.04	0.36	P044	12.93	0.94
B2030B01	15.38	0.22		14.31	99.02
Hoffman-La	14.20	99.62
Roche Ltd	15.18	0.18
B2070B05	15.44	0.19

Abbreviation: RP-HPLC, reverse-phase high-performance liquid chromatography.

Figure 11 Comparative SE-HPLC.

Notes: Superposition of SE-HPLC chromatograms of (left top) Fiprima^® batch 378/11 (blue) and Granulokine^® batch B2039B13 (red). Right top: Fiprima (blue) batch 365/10 and Granulokine (red) batch B2009B01; left bottom: Eurofarma’s filgrastim P006 (red) and Granulokine (blue) batch B2011B01; right bottom: Eurofarma’s filgrastim 058/10 (blue) and Granulokine batch B2010B01 (red).

Abbreviation: SE-HPLC, size-exclusion high-performance liquid chromatography.

Table 4 Retention of the peaks from SE-HPLC and their area percentage

Batches	Retention in mL (area/peak area %)	Batches	Retention in mL (area/peak area %)
Eurofarma	20.84 (99.89)	Hoffman-La	20.84 (99.97)
378/11		Roche Ltd B2039B13
Eurofarma	19.99 (99.10)	Hoffman-La	19.96 (99.10)
365/10		Roche Ltd B2009B01
Eurofarma	19.29 (99.83)	Hoffman-La	20.73 (99.25)
P006		Roche Ltd B2011B01
Eurofarma	20.57 (99.58)	Hoffman-La	20.78 (99.81)
058/10		Roche Ltd B2010B01

Abbreviation: SE-HPLC, size-exclusion high-performance liquid chromatography.

Figure 12 Comparative potency: Eurofarma’s batches in dark gray and Hoffman-La Roche Ltd batches in light gray.

Figure 13 Zoom of SE-HPLC after intense agitation of filgrastim.

Notes: Left: Eurofarma’s batch 378/11; right: Hoffman-La Roche Ltd batch B2039B13.

Abbreviation: SE-HPLC, size-exclusion high-performance liquid chromatography.

Table 5 Products behavior after being submitted to different stress conditions

Stress condition	Test	Criteria	Product	Prestress	Poststress	12 months	24 months
12 hours at 0°C	Content RP-HPLC	540–660 μg/mL	Eurofarma	571 μg/mL	546 μg/mL	568 μg/mL	557 μg/mL
			Hoffman-La Roche Ltd	580 μg/mL	543 μg/mL	583 μg/mL	560 μg/mL
	Impurity and aggregates^a	≤2.0%	Eurofarma	0.9%, 0.1%	0.9%, 0.2%	1.1%, 0.8%	0.9%, 0.2%
			Hoffman-La Roche Ltd	0.4%, 0.2%	1.3%, 0.2%	0.5%, 0.2%	0.4%, 0.1%
	Bioassay (potency)	80%–125%	Eurofarma	106.3%	92.3%	99.3%	104%
			Hoffman-La Roche Ltd	110.5%	93.0%	99.6%	104.7%
12 hours at 37°C	Content RP-HPLC	540–660 μg/mL	Eurofarma	571 μg/mL	546 μg/mL	563 μg/mL	561 μg/mL
			Hoffman-La Roche Ltd	580 μg/mL	585 μg/mL	570 μg/mL	557 μg/mL
	Impurity and aggregates^a	≤2.0%	Eurofarma	0.9%, 0.4%	0.9%, 0.5%	0.9%, 0.5%	0.9%, 0.4%
			Hoffman-La Roche Ltd	0.1%, 0.2%	0.2%, 0.1%	0.8%, 0.2%	0.3%, 0.1%
	Bioassay (potency)	80%-125%	Eurofarma	96.7%	90.5%	97.0%	99.6%
			Hoffman-La Roche Ltd	110.5%	85.2%	ND	94.1%

Notes: ^aImpurities were evaluated by RP-HPLC (the first result presented in the cell) and aggregates by SE-HPLC (the second result presented in the cell).

Abbreviations: RP-HPLC, reverse-phase high-performance liquid chromatography; SE-HPLC, size-exclusion high-performance liquid chromatography; ND, not determined.

Sreerama N, Woody RW. Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with and expanded reference set. Anal Biochem. 2000;287(2):252–260.

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