Research review paperDevelopment of reporter gene assays to determine the bioactivity of biopharmaceuticals
Introduction
Biopharmaceuticals have become the fueling engine of pharmaceutical economy, as evidenced by eight protein therapeutics in the top ten best-selling drugs. The prominent characteristics of their target specificity are correlated with their relative reliable clinical efficacy and lower side effects. Biopharmaceuticals, normally produced by living cells with large molecular weights and complicated high order structures, are quite heterogeneous in nature and highly sensitive to environmental conditions (Assenberg et al., 2013; Barnes et al., 2003; Zhu, 2012). Extensive characterization and proper quality control are essential to ensure their batch-to-batch consistency, stability, safety, and clinical efficacy (WHO, 2013). Due to their complex nature, quality control is quite different from traditional chemical drugs.
In order to ensure the quality of recombinant protein products, International Conference on Harmonization Q6B (ICH-Q6B, 1999) and other regulatory guidelines require the conduction of full characterization including physicochemical properties, biological activity, immunochemical properties (in terms of monoclonal antibody, mAbs), purity, impurities and contaminants. Regulatory expectations on biological activity remain basically unchanged given the advancement of sophisticated physicochemical techniques, due to the highly heterogeneous structure as well as complicated structure-and-function relationship of biopharmaceuticals. For complex molecules, their physicochemical information may be extensive but unable to confirm the higher-order structure which, however, can be inferred from their biological activities (ICH-Q6B, 1999).
Various biological assays are employed in the determination of biological activities for biopharmaceuticals in industry (Mire-Sluis, 2001). RGAs exploit the activation or inactivation of signal transduction, which are represented by the expression of stably transfected reporter genes under the control of appropriate regulatory elements, as the surrogates of cellular events triggered by biotherapeutics (Jiang et al., 2008). As a MOA representative, less variable and highly robust method, RGAs have become an important alternative to current bioassays in the quality control of biopharmaceuticals.
There are many good reviews on RGAs regarding to their extensive applications in basic biology research (Cevenini et al., 2016; Hill et al., 2001; Naylor, 1999; Miraglia et al., 2011), disease loci identification (Nair and Baier, 2018), noninvasive imaging (Contag and Bachmann, 2002; Roura et al., 2013), high-throughput screening for new drugs (Fan and Wood, 2007), development of natural products (Harvey and Cree, 2010; New et al., 2003), ecotoxicology (Elad and Belkin, 2017), environmental analysis (Köhler et al., 2000; Svobodova and Cajthaml, 2010) and etc. The U.S. Food and Drug Administration (FDA) has approved some biopharmaceuticals whose bioactivity determinations, as one of critical quality attributes (CQAs), are dependent on RGAs (Hofmann et al., 2016; Kronthaler et al., 2018; Lee et al., 2017), which indicates their wide acceptance both in industry and regulatory authority. Here, we focus on reviewing the applications of RGAs in the bioactivity determination of biopharmaceuticals.
Section snippets
Development of bioassays in the quality control of biopharmaceuticals
Bioassays play a key role in quality control demonstrating batch-to-batch consistency and stability of biopharmaceuticals. Bioassays to determine the potency of vaccines (Verch et al., 2018) and cell therapeutics (de Wolf et al., 2018) were well discussed elsewhere and beyond the scope of this review. Bioassays may not necessarily reflect or predict the clinical efficacy, as stated in ICH-Q6B (1999), and the correlation between the expected clinical response and the activity should be
Theoretical basis for the development of bioactivity determination methods based on RGAs
Bioactivity determination methods for biotherapeutics should be developed in accordance with the following principles: MOA-based, applicable in quality control (small variability, simple operation and transferability) and compliance with GMP (Good Manufacture Practices) validation (Rieder et al., 2010).
In order to be MOA for a biotherapeutic, RGAs should be carefully designed to monitor and correlate with the product induced events, such as signal transduction, gene expression and cellular
Current status of establishment and application of RGAs in bioactivity determination
Various therapeutic cytokines, hormones, antibody-based biotherapeutics are subject to RGAs for their bioactivity determination, and those documented are listed in Table 1.
Discussion
Although the state of the art sophisticated physicochemical assays could provide a great amount of structure information, bioassays are still irreplaceable in determining biological activity and the indication of proper high-order structures of biopharmaceuticals (ICH-Q6B, 1999). Development of biological assays for products is essential for their advancement as therapeutic products. By engineering appropriate cell lines stably transfected with plasmids consisting of luciferase gene under the
Acknowledgements
The authors thank Dr. Junxia Cao and Wenrong Yao for their help and discussion during the preparation of this manuscript, as well as Dr. Chaohong Hu for providing language editing. This work was supported by National Major Science and Technology Projects for “Major New Drugs Innovation and Development” (No. 2018ZX09736-008).
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