Quantitation of certolizumab pegol by validated liquid chromatography methods

Cardoso Júnior, Clóvis Dervil Appratto; Xavier, Bruna; Dumoncel, Rafaela Ferreira Perobelli; Silva, Francielle Santos da; Dalmora, Sérgio Luiz

doi:10.1590/s2175-97902023e23020

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Brazilian Journal of Pharmaceutical Sciences

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Article • Braz. J. Pharm. Sci. 59 • 2023 • https://doi.org/10.1590/s2175-97902023e23020 copy

Quantitation of certolizumab pegol by validated liquid chromatography methods

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Certolizumab pegol (CZP) is a Fab’ fragment of the humanized antibody with anti-TNF-α activity that is indicated as therapy for Crohn’s disease and rheumatoid arthritis. Using a BioSep-SEC-S3000 column (300 x 4.6 mm i.d., 5 µm particle size), a size exclusion liquid chromatography (SEC) method was developed. Mobile phase A consisted of 100 mM monobasic sodium phosphate and 200 mM sodium chloride (pH 7.0), while mobile phase B was ethanol (95:5, v/v), and the analysis was performed using a diode array detector (DAD) set to 214 nm and a flow rate of 0.5 ml min-1. In addition, a reversed-phase liquid chromatography (RP-LC) method based on gradient elution was developed on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d., 3.5 µm particle size) kept at 80 °C. Mobile phase A was 0.1% (v/v) TFA in ultrapure water, and mobile phase B was a mixture of propanol, acetonitrile, ultrapure water and TFA (70 + 20 + 9.9 + 0.1, v/v) operated at a flow rate of 1.0 ml min-1, and DAD was applied at 214 nm. CZP elution was achieved with retention times of 5.6 min and 9.0 min for SEC and RP-LC, respectively.

Keywords:
Certolizumab pegol; Monoclonal antibody; Size exclusion liquid chromatography; Reversed-phase liquid chromatography; Validation.

Method	Nominal concentration (mg mL^-1)	Mean concentrationmeasured^a (mg mL^-1)	RSD^b(%)	Accuracy (%)	Bias^c(%)
SEC	16	16.14	0.50	100.85	0.85
	20	20.05	0.30	100.27	0.27
	24	24.09	0.07	100.39	0.39
RP-LC	16	15.87	1.85	99.18	-0.82
	20	20.06	1.46	100.31	0.31
	24	23.98	0.54	99.90	-0.10

Variable	Range investigated	CZP^a (%)	RSD^b (%)	Optimized value
Monobasic sodium phosphate concentration	90 mmol L^-1	99.11	0.21	100 mmol L^-1
	100 mmol L^-1	99.93	0.10
	110 mmol L^-1	99.37	0.45
Flow rate (mL/min)	0.4 mL min^-1	100.83	0.06	0.5 mL min^-1
	0.5 mL min^-1	99.30	0.20
	0.6 mL min^-1	100.49	0.48
Mobile phase pH	6.8	100.16	0.26	7.0
	7.0	99.93	0.10
	7.2	99.85	0.14
Ethanol (%)	4	99.69	0.55	5
	5	98.12	0.16
	6	98.30	0.13
Temperature (ºC)	33	100.10	0.32	35
	35	99.59	0.14
	37	99.10	0.08
Solution stability	Autosampler 24 h	101.45	0.28	-
	2 - 8 ºC 24 h	101.01	1.27	-
	2 - 8 ºC 48 h	99.18	0.55	-
Wavelength (nm)	190 - 320			214

Variable	Range investigated	CZP^a (%)	RSD^b (%)	Optimized value
Gradient: maximum percentage of mobile phase B (%B)	39 (% B)	100.61	1.45	40 (% B)
	40 (% B)	100.45	0.99
	41(% B)	100.27	1.08
Flow rate (mL/min)	0.9 mL min-1	101.16	1.33	1.0 mL min^-1
	1.0 mL min^-1	100.94	0.47
	1.1 mL min^-1	101.72	0.96
TFA (%)	0.09	100.12	1.33	0.10
	0.10	100.85	0.44
	0.11	101.08	0.62
Temperature (ºC)	79	101.03	1.34	80
	80	100.76	0.87
	81	100.84	1.68
Solution stability	Autosampler 24 h	99.83	1.13	-
	2 - 8 ºC 24 h	99.12	0.41	-
	2 - 8 ºC 48 h	98.64	0.96	-
Wavelength (nm)	190 - 320			214

Method	Factor	Optimal	Low level	High level
SEC	Mobile phase pH	7.0	6.8	7.2
	Flow rate (mL min⁻¹)	0.5	0.4	0.6
	Column temperature (°C)	35	33	37
	Wavelength (nm)	214	212	216
RP-LC	Flow rate (mL min⁻¹)	1.0	0.9	1.1
	Column temperature (°C)	80	79	81
	Gradient: mobile phase B (%)	40	39	41
	Wavelength (nm)	214	212	216

Sample	Label claim (mg mL^-1)	SEC		RP-LC
Sample	Label claim (mg mL^-1)	HMW^a (%)	Content^a (%)	Fragments^a (%)	Main Peak^a (%)
1	200	1.12	99.18	0.94	98.93
2	200	0.77	100.23	1.02	99.25
3	200	1.83	99.17	0.87	98.72
4	200	0.84	101.47	1.33	100.24
5	200	0.92	99.22	1.51	99.09
Mean	-	1.10	100.03	1.13	99.25
SD ^b	-	0.43	1.08	0.27	0.59

Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br

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[1] *Correspondence: S. L. Dalmora. Departmento de Indústria Farmacêutica. Universidade Federal de Santa Maria. Av. Roraima nº 1000. 97105-900 - Santa Maria, Rio Grande do Sul, Brasil. Phone: +55 55 3220 8952. E-mail: sdalmora@terra.com.br.