Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

Abstract

L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

A comprehensive review on microbial l‐asparaginase: Bioprocessing, characterization, and industrial applications

“…l-Asparaginase from E. coli produces two types of enzyme, l-asparaginase I (ECI), found in the cytoplasm, and l-asparaginase II (ECII) of periplasmic origin. The latter is used for clinical applications and has a higher affinity (K m = 10-15 μM) to l-asparagine than ECI (K m = 3.5 mM) [2,8,120]. l-Asparaginases from E. chrysanthemi (Erc) and E. coli (ECII) have the similar mechanism of action against tumor cells, however their pharmacokinetics, affinity for the substrate (K m ), and immune system sensitization profile varies [120].…”

“…The downstream processing contributes to around 60-80% of the total production costs of a biotherapeutics product. Therefore, it becomes important to explore how traditional methods can be substituted with efficient and cost-effective unconventional methods for recovery and purification of biopharmaceuticals [120]. The efficiency of the protein purification process can be monitored by determining specific activity, yield, and purification fold at each step.…”

“…The efficiency of the protein purification process can be monitored by determining specific activity, yield, and purification fold at each step. Specific activity is a measure of enzyme purity and calculated by dividing total enzyme activity by total protein [117,120]. Yield or recovery (percent) is a measure of how well the enzyme activity is retained after each purification step as a percentage of the activity in the crude extract.…”

Fungal endophytes of high altitude ethnomedicinal plants as a bioresource of industrially imperative enzymes

“…Regular supply of asparagine, maintained by asparagines synthetase, is requisite for making proteins in the cell. Leukemic cells however are de cient in asparagines synthetase and depend solely on circulating blood for the supply of L-asparagine (Brumano et al 2019). The immunogenic complications associated with its present microbial sources Escherichia coli, Erwinia caratovora limits its medicinal frontier.…”

Expressão da L-asparaginase II de >i<Saccharomyces cerevisiae>/i< recombinante extracelular com glicosilação humanizada em >i<Pichia pastoris>/i<

“…A enzima nativa de E. coli, a nativa de E. coli ligada covalentemente com polietilenoglicol (ASNase peguilada) e por fim a enzima nativa de Dickeya chrysanthemi (erwinase). Também há a formulação da enzima de E. coli produzida de forma recombinante (Spectrila®, Medac) (BRUMANO et al, 2019).…”