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.

Keywords: L-asparaginase; PEGylation; acute lymphoblastic leukemia; biobetters; nanobiotechnology; protein engineering; quality-by-design; site-directed mutagenesis.

Figure 1

Comparison between biological reference drug, biosimilar and biobetter in terms of development time, overall cost of production, patent protection, and commercial value.

Figure 2

In the traditional “Quality-by-Testing” (QbT) paradigm (A), the prospective drug product is first identified and a manufacturing process is proposed and adjusted until the finished product meets quality specifications. Afterwards, the operating parameters are locked, validated and filed with the regulatory agency. The process is then operated within narrow ranges around the set points, which (ideally) guarantees product consistency. In the “Quality-by-Design” (QbD) paradigm (B), the first step is the definition of the “Quality Target Product Profile” (QTPP) of the prospective pharmaceutical. Afterwards, using risk assessment tools, the “Critical Quality Attributes” (CQA) of the product are identified and, based on them, “Critical Process Parameters” (CPP) and “Critical Material Attributes” (CMA) are found using “Failure Mode Effects and Criticality Analysis” (FMECA), “Sensitivity Analysis” (SA), among other tools. Then, using such statistical tools as “Design of Experiments” (DoE) and “Multivariate Analysis” (MVA), the impact of the CMA and CPP on the CQA are studied, thus allowing process redesign and the removal of quality bottlenecks. Using “Process Analytical Technology” (PAT) a control strategy can then be proposed. Since, within the QbD paradigm, the whole research process is filed with the regulatory agency, the manufacturing process can more easily be improved upon (Rathore, 2014).

Figure 3

Influence of PEGylation as an engineering technique for biobetter drug development. Example: native ASNase and the respective biobetter–PEGylated ASNase (9 PEG chains of 10 KDa).

Figure 4

Pharmaceutical issues about L-Asparaginase development as a biopharmaceutical.