Analysis of therapeutic proteins and peptides using multiangle light scattering coupled to ultra high performance liquid chromatography

Abstract

Analysis of the physical properties of biotherapeutic proteins is crucial throughout all the stages of their lifecycle. Herein, we used size-exclusion ultra high performance liquid chromatography coupled to multiangle light scattering and refractive index detection systems to determine the molar mass, mass-average molar mass, molar-mass dispersity and hydrodynamic radius of two monoclonal antibodies (rituximab and trastuzumab), a fusion protein (etanercept), and a synthetic copolymer (glatiramer acetate) employed as models. A customized instrument configuration was set to diminish band-broadening effects and enhance sensitivity throughout detectors. The customized configuration showed a performance improvement with respect to the high-performance liquid chromatography standard configuration, as observed by a 3 h column conditioning and a higher resolution analysis in 20 min. Analysis of the two monoclonal antibodies showed averaged values of 148.0 kDa for mass-average molar mass and 5.4 nm for hydrodynamic radius, whereas for etanercept these values were 124.2 kDa and 6.9 nm, respectively. Molar-mass dispersity was 1.000 on average for these proteins. Regarding glatiramer acetate, a molar mass range from 3 to 45 kDa and a molar-mass dispersity of 1.304 were consistent with its intrinsic peptide diversity, and its mass-average molar mass was 10.4 kDa. Overall, this method demonstrated an accurate determination of molar mass, overcoming the difficulties of size-exclusion chromatography.

Process signatures in glatiramer acetate synthesis: structural and functional relationships

“…In this regard, is mandatory to use suitable techniques to differentiate among closely related profiles against the distinctive heterogeneity profile of GA, without missing its minor populations 46 . We developed SE-UPLC 45 , SCX-UPLC and RP-UPLC methodologies 46 to evaluate molecular mass, electric charge and hydrophobicity distributions as the most reliable characteristics of the whole mixture of amino acid sequences and peptide lengths of GA through each stage of the manufacturing process (viz., polymerization, depolymerization, deprotection and purification) in order to assess critical process parameters and their impact on GA structural signatures.…”

“…All experiments were modified from the standard synthesis conditions (STD) used for GA manufacturing. GA produced by STD (GA-STD, Probioglat) has been extensively characterized in terms of its distinctive and equivalent molecular mass 45 ; electric charge and hydrophobicity 46 ; and refractive index increment and extinction coefficient 51 with respect to the reference medicinal product (Copaxone).…”

“…Samples treatment and analysis conditions for SE-UPLC were performed as previously described by Espinosa-de la Garza et al . 45 .…”

“…GA complexity may impede the complete identification of the physicochemical and biological properties utterly responsible of its efficacy and safety 35 – 43 , but it does not preclude a finding of distinctive attributes that define GA towards dissecting its identity and improve the understanding of the biological and clinical properties of glatiramer acetate 44 . For instance, GA highly disperse molecular mass distribution has been characterized, ranging from ~3 kDa to ~45 kDa with a mass-average molar mass mean (M w ) of ~10.5 kDa 45 , along its highly disperse electric charge distribution that ranges from ~1 to more than 60(e) with a mean net positive charge of ~48(e) 46 .…”

Recent applications of light scattering measurement in the biological and biopharmaceutical sciences

“…The advantages of using a SE-UHPLC column in place of a conventional SEC column include faster elution times, increased resolution, and smaller samples [49]. …”