COMPARATIVE ANALYSIS OF CONFORMATIONAL STABILITY AND ENZYMATIC ACTIVITY OF L-ASPARAGINASE TYPE II BIOSIMILARS FROM Escherichia coli

INTRODUCTION: Since its introduction, multiple l-asparaginase (EcA2) formulations have been approved, yet comparative studies reveal marked pharmacokinetic and pharmacodynamic differences among biosimilars. In Brazil, the SUS imported Aginasa^® until early 2017, when a severe supply shortage forced a switch to Leuginase^®—a product later suspended amid efficacy and safety concerns. In 2018, Zenatti et al. demonstrated that Leuginase^® preparations contained higher levels of host cell protein contaminants, provoked stronger immune reactions, and exhibited three fold lower plasma activity in vivo despite matching Aginasa^® in vitro potency. That same year, Cecconello et al. detected therapeutic plasma activity in 81 % of Aginasa^® samples but in only 3 % of Leuginase^® batches, underscoring the critical need for ongoing enzymatic activity monitoring during acute lymphoblastic leukemia therapy. To investigate the molecular underpinnings of these differences, our group started a biophysical comparison study of Aginasa^® versus Leuginase^®. We observed distinct variations in molecular mass, molecular dynamics, oligomerization state, and excipient composition. Notably, excipient induced alterations in the N terminal loop—which acts as a lid over the active site—modulate enzyme conformation in solution. Mass spectrometry sequencing also revealed that Aginasa^® presents two amino acid substitutions, N64D and S252T in comparison to Leuginase^®. AIMS: Our study aims to produce these EcA2 variants identified in the biopharmaceuticals Aginasa^® and Leuginase^® and compare their structural integrity, conformational dynamics, thermal stability, and enzymatic activity. MATERIAL AND METHODS: The EcA2 variants (4M refers to Aginase^® and 2M to Leuginase^®) were expressed in the E. coli BL21 (DE3) strain and purified by hydrophobic interaction and ion exchange chromatography. Enzymatic activity was quantified by direct asparagine absorptiometry, three-dimensional structure was evaluated by NMR spectroscopy, and thermal stability was assessed by intrinsic fluorescence. RESULTS: EcA2 from both biosimilars showed similar specific activity, global conformation, and oligomeric distribution, as investigated by NMR and SEC-HPLC. However, the 2M variant showed a statistically significant difference in thermal stability when compared to the 4M variant. CONCLUSION: The difference of the in vivo activity of these EcA2 variants, might be related to their difference in conformational stability and/or to other effect related to their formulations.

This work was supported by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional pra o Desenvolvimento Cietnífico e Tecnológico (CNPq), Financiadora de Estudos e Projetos (FINEP), Fundação Maria Emília Pedreira Freire de Carvalho (FME) and by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).