Biosimilarity under stress: A forced degradation study of Remicade® and Remsima™

Figures & data

Figure 1. Schematic of stress study design. Humidity/thermal stress of infliximab samples were performed by incubating the drug powders at 40°C at different %RH for 0–4 weeks, followed by reconstitution in WFI and analysis.

Figure 2. Characterization of protein aggregation in stressed samples. A. Representative SEC chromatograms of infliximab following 97% RH/40°C incubation for 0, 1, 2 and 4 weeks. B. SDS PAGE of Remicade® and Remsima™ samples stressed for 4 weeks at 0 (dry), 50, 75 and 97% RH. C. Kinetics of monomer loss at various humidity for Remicade® (solid) and Remsima™ (dashed) as detected by SEC (n = 4 ± SEM). D. Reducing SDS PAGE gel of Remicade® and Remsima™ humidity stressed samples for 4 weeks at 0 (dry), 50, 75 and 97% RH. Molecular weights (MW) are annotated on the ladder in kDa.

Table 1. Fitted rates of monomer loss for Remicade® and Remsima™ samples after incubation at 40°C and various relative humidity levels.

	Monomer Loss Rate (day^−1) ± SD (R^2)
	50% RH	75% RH	97% RH
Remsima	−0.19 ± 0.01 (0.99)	−0.23 ± 0.06 (0.86)	−0.42 ± 0.01 (0.99)
Remicade	−0.20 ± 0.01 (0.99)	−0.18 ± 0.03 (0.92)	−0.44 ± 0.01 (0.99)

Figure 3. Nanoparticle tracking analysis of unstressed (light) and incubated at 97% RH/40°C for 4 weeks (dark) samples of Remicade® (A) and Remsima™ (B). DSC thermal melts of unstressed (dashed) and incubated at 97% RH/40°C for 4 weeks (solid) samples of Remicade® (C) and Remsima™ (D).

Table 2. Impurity profile of Remicade® and Remsima™ before and after 4-week incubation at 40°C and 97% relative humidity.

		Remicade®			Remsima™
Method	Attribute	0 days	4 weeks	Statistics	0 days	4 weeks	Statistics
SEC	Dimer, %	0.1 ± 0.0	12.7 ± 1.1	P < 0.005	0.4 ± 0.0	12.4 ± 0.5	P < 0.005
NTA	Number of subvisible particulates 50–350 nm, 10*^7	2.99 ± 0.49	7.43 ± 1.96	NS	21.1 ± 1.60	33.9 ± 3.59	P < 0.005
IM MS	Fragment 50 kDa, %	—	19.7 ± 2.7	N/A	—	26.5 ± 14.2	N/A
	Fragment 100 kDa, %	—	6.0 ± 1.1	N/A	—	9.1 ± 3.4	N/A
	Dimer, %	0.8 ± 0.2	3.3 ± 0.6	P < 0.1	2.0 ± 2.5	2.9 ± 1.2	NS
	Trimer, %	—	0.2 ± 0.1	N/A	—	0.2 ± 0.1	N/A
SDS	Aggregate, %	24.1	37.5	N/A	23.8	40.8	N/A
	Fragment, %	7.0	14.1	N/A	0.0	0.4	N/A
LC-MS	Deamidation LC-N158, %	0.6 ± 0.1	7.5 ± 0.7	P < 0.05	0.5 ± 0.4	7.5 ± 0.5	P < 0.05
	Deamidation HC-N57, %	1.7 ± 0.2	17.6 ± 1.9	P < 0.05	2.2 ± 0.1	18.9 ± 0.8	P < 0.05
	Deamidation HC-N392, %	1.3 ± 0.1	10.3 ± 1.8	NS	0.5 ± 0.1	11.2 ± 1.5	P < 0.05
Activity	TNF binding, %	100.0 ± 3.0	81.8 ± 4.4	NS	111.7 ± 3.1	77.2 ± 5.9	P < 0.01
	FcγRIIIa binding, KD (nM)	173 ± 56	545 ± 117	P < 0.1	368 ± 160	680 ± 22	NS

Data shown are percentages (n = 2 lots ± SD), N/A – not applicable, NS – not significant.

Figure 4. Ion mobility spectra and corresponding mass to charge spectrograms of Remicade® (A) and Remsima™ (B) before and after 4 weeks incubation at 97% RH/40°C, Remicade® (C) and Remsima™ (D). Fragment, monomer, dimer and trimer species annotated in ion mobility spectra.

Figure 5. Biophysical characterization of humidity stressed samples of Remicade® (A, C, E, solid) and Remsima™ (B, D, F, dashed) incubated at 97% RH/40°C for 0 (blue), 2 (red) and 4 (green) weeks. Intrinsic fluorescence spectra ((A)and B), circular dichroism far UV spectra ((C)and D) and near UV spectra ((E)and F).

Figure 6. Chemical modifications of humidity stressed (97% RH) samples. Deamidation (A), oxidation (B) and N-glycosylation (C) of Remicade® (blue) and Remsima™ (orange) for native proteins and following 97% RH/40°C stress for 2 and 4 weeks as assayed by LC-MS (n = 2 lots ± SEM).

Figure 7. Binding of Remicade® and Remsima™ samples after stressing at 97% RH/40°C. A. TNF binding as measured by ELISA. B. FcγRIIIa binding as measured by BLITZ (n = 2 lots ± SEM; * P< 0.05 ** P<0.01 compared with unstressed).