Quality attributes of recombinant therapeutic proteins: An assessment of impact on safety and efficacy as part of a quality by design development approach

Abstract

Quality by Design (QbD) is a new approach to the development of recombinant therapeutic protein products that promotes a better understanding of the product and its manufacturing process. The first step in the QbD approach consists in identifying the critical quality attributes (CQA), i.e., those quality attributes of the product that have an impact on its clinical efficacy or safety. CQAs are identified through a science-based risk assessment taking into consideration a combination of clinical and nonclinical data obtained with the molecule or other similar molecules or platform products, as well as the published literature. The purpose of this article is to perform a comprehensive review of the published literature, supporting an assessment of the impact on safety and efficacy of the quality attributes commonly encountered in recombinant therapeutic proteins, more specifically those produced in mammalian cell expression systems. Quality attributes generally observed in biopharmaceutical proteins including product-related impurities and substances, process-related impurities, product attributes, and contaminants are evaluated one by one for their impact on biological activity, pharmacokinetics and pharmacodynamics, immunogenicity, and overall safety/toxicity.

Tailoring recombinant protein quality by rational media design

“…A large number of reports describe various impacts of glycosylation on the quality attributes of biologics including in vivo efficacy [62][63][64] , pharmacokinetics (PK) 11 , antibody-dependent cellular cytotoxicity (ADCC) 63,65 and complementdependent cytotoxicity (CDC) activities 66 , stability and overall structure of the molecule 63 , clearance and half-life in vivo 10 as well as immunogenicity 10,67 . Non-fucosylated therapeutic antibodies exhibit 50 to 1,000-fold higher efficacy than their fucosylated counterparts 65 due, in most cases, to enhanced ADCC activity 68 . Given the abundance of research efforts in this area, it is no surprise that many have concluded that glycosylation is one of the main areas requiring development 69 to improve efficacy 70 and safety 10,70 of next generation therapeutics 61 .…”

“…Ammonium levels not only inhibit galactosylation, but also have an impact on the abundance of sialylated species, due to the sparsity of galactosylated glycoforms and limited expression levels of α-2,3-sialyltransferase (SiaT) 126 . In contrast to human IgG, sialic acids are attached to the terminal galactose residues of antibodies expressed in CHO cells via an α-2,3 linkage exclusively 68 . It was reported that by reducing glutamine concentrations to 0 mM in CHO-K1 batch and perfusion cultures, sialylation was inhibited, thus leading to more abundant neutral N-linked glycans 117 .…”

“…Asparagine (Asn) residues with glycine (Gly) on their C-terminus are the most prone to deamidation 149 . Both negative and absence of impact of deamidation on potency and immunogenicity have been reported on therapeutic proteins, whereas the influence on pharmacokinetics remains to be elucidated 68 . Glutamine (Gln) deamidation may also appear at an exceedingly slower rate than for Asn, and it is reported to be not of concern for biopharmaceuticals 68 .…”

“…Met oxidation has been reported to adversely affect both mAb structure and stability 39 . Various impacts of oxidation on biological activity, stability, and half-life of the therapeutic protein have been identified, and oxidationinduced aggregation may provoke immunogenicity reactions 68 . A recent study reported the use of oxidation reagents including hydrogen peroxide and tert-butyl hydroperoxide in combination with free tryptophan resulted in selective oxidation of methionine of IgG1 formulated at 25 mg/mL in 51 mM sodium phosphate, 6% trehalose, and 0.04% polysorbate 20 (pH 6.2) 156 .…”

“…Minimal or negligible impact of C-terminal Pro amidation on potency and pharmacokinetic properties was demonstrated 44 . Even though C-and N-terminal modifications have not been reported to adversely affect safety 41,68 , potential correlations between glycosylation pattern and terminal modifications have been detected 66 , and hence highlight the importance to control the level of charge variants.…”