title: Basal buffer systems for a newly glycosylated recombinant human interferon-β with biophysical stability and DoE approaches.
abstract: The purpose of this study was to develop a basal buffer system for a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a), termed R27T, to optimize its biophysical stability. The protein was pre-screened in solution as a function of pH (2-11) using differential scanning calorimetry (DSC) and dynamic light scattering (DLS). According to the result, its experimental pI and optimal pH range were 5.8 and 3.6-4.4, respectively. Design of experiment (DoE) approach was developed as a practical tool to aid formulation studies as a function of pH (2.9-5.7), buffer (phosphate, acetate, citrate, and histidine), and buffer concentration (20 mM and 50 mM). This method employed a weight-based procedure to interpret complex data sets and to investigate critical key factors representing protein stability. The factors used were Tm, enthalpy, and relative helix contents which were obtained by DSC and Fourier Transform Infrared spectroscopy (FT-IR). Although the weights changed by three responses, objective functions from a set of experimental designs based on four buffers were highest in 20 mM acetate buffer at pH 3.6 among all 19 scenarios tested. Size exclusion chromatography (SEC) was adopted to investigate accelerated storage stability in order to optimize the pH value with susceptible stability since the low pH was not patient-compliant. Interestingly, relative helix contents and storage stability (monomer remaining) increased with pH and was the highest at pH 4.0. On the other hand, relative helix contents and thermodynamic stability decreased at pH 4.2 and 4.4, suggesting protein aggregation issues. Therefore, the optimized basal buffer system for the novel biobetter was proposed to be 20 mM acetate buffer at pH 3.8±0.2.
authors.affiliation: College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea.
authors.forename: Nam Ah
authors.initials: NA
authors.lastname: Kim
authors.affiliation: Abion Inc., Seoul, Republic of Korea.
authors.forename: Kyoung
authors.initials: K
authors.lastname: Song
authors.affiliation: College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea.
authors.forename: Dae Gon
authors.initials: DG
authors.lastname: Lim
authors.affiliation: College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea.
authors.forename: Shavron
authors.initials: S
authors.lastname: Hada
authors.affiliation: Abion Inc., Seoul, Republic of Korea; College of Pharmacy, Seoul National University, Seoul, Republic of Korea.
authors.forename: Young Kee
authors.initials: YK
authors.lastname: Shin
authors.affiliation: Department of Industrial & Management Systems Engineering, Dong-A University, Busan 604-714, Republic of Korea.
authors.forename: Sangmun
authors.initials: S
authors.lastname: Shin
authors.affiliation: College of Pharmacy, Dongguk University-Seoul, Gyeonggi 410-820, Republic of Korea. Electronic address: shjeong@dongguk.edu.
authors.forename: Seong Hoon
authors.initials: SH
authors.lastname: Jeong
mesh_terms: D002021:Buffers; D015195:Drug Design; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016899:Interferon-beta / Q000737:chemistry*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
chemical_list: D002021:Buffers; D011994:Recombinant Proteins; D016899:Interferon-beta
keywords: Biobetter; Design of experiment (DoE); Differential Scanning Calorimetry (DSC); FT-IR; Interferon; Protein formulation