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. (CZP Fab antiTNFα anti TNF α Crohns Crohn s arthritis BioSepSECS3000 BioSepSECS BioSep S3000 S BioSep-SEC-S300 (30 46 4 6 4. id i d i.d. size, , (SEC 10 pH 7.0, 70 7.0 7 0 7.0) 955, 955 95 5, (95:5 v/v, vv v/v v (DAD 21 05 0. min1. min1 1. 1 min-1 addition reversedphase reversed RPLC RP LC (RP-LC 30 C C1 150 (15 35 3 3. 8 °C 01 0.1 (v/v propanol acetonitrile (7 2 99 9 9. min1, 1, 56 5. 90 RPLC, LC, RP-LC respectively BioSepSECS300 S300 BioSep-SEC-S30 (3 i.d 7. (95: min- 15 (1 ( BioSepSECS30 S30 BioSep-SEC-S3 (95 BioSepSECS3 S3 BioSep-SEC-S (9