Manufacturing history of etanercept (Enbrel^®): Consistency of product quality through major process revisions

Abstract

Etanercept (ETN) (Enbrel®) is a soluble protein that binds to, and specifically inhibits, tumor necrosis factor (TNF), a proinflammatory cytokine. ETN is synthesized in Chinese hamster ovary cells by recombinant DNA technology as a fusion protein, with a fully human TNFRII ectodomain linked to the Fc portion of human IgG1. Successful manufacture of biologics, such as ETN, requires sophisticated process and product understanding, as well as meticulous control of operations to maintain product consistency. The objective of this evaluation was to show that the product profile of ETN drug substance (DS) has been consistent over the course of production. Multiple orthogonal biochemical analyses, which included evaluation of attributes indicative of product purity, potency, and quality, were assessed on >2,000 batches of ETN from three sites of DS manufacture, during the period 1998-2015. Based on the key quality attributes of product purity (assessed by hydrophobic interaction chromatography HPLC), binding activity (to TNF by ELISA), potency (inhibition of TNF-induced apoptosis by cell-based bioassay) and quality (N-linked oligosaccharide map), we show that the integrity of ETN DS has remained consistent over time. This consistency was maintained through three major enhancements to the initial process of manufacturing that were supported by detailed comparability assessments, and approved by the European Medicines Agency. Examination of results for all major quality attributes for ETN DS indicates a highly consistent process for over 18 years and throughout changes to the manufacturing process, without affecting safety and efficacy, as demonstrated across a wide range of clinical trials of ETN in multiple inflammatory diseases.

Using Titer and Titer Normalized to Confluence Are Complementary Strategies for Obtaining Chinese Hamster Ovary Cell Lines with High Volumetric Productivity of Etanercept

“…In order to investigate the performance of titer and proxy of q p as early reporters for volumetric productivity, we generated a polyclonal cell line by transfecting CHO‐S cells with a plasmid co‐expressing glutamine synthetase (GS) and etanercept and then applied MSX‐selection. Etanercept is a homodimeric Fc‐fusion therapeutic protein that is considered a potentially difficult‐to‐express protein due to its highly O‐glycosylated regions . We then developed a screening platform that combines enrichment of high q p by surface labeling‐based fluorescence activated cell sorting (FACS) with high‐throughput (HT) and semi‐automated confluence analysis of cells cultured statically as well as determination of titer in 96‐well plates.…”

Heterogeneity and variation of innovator biologics

“…The following mean and median ranges compare standard (Process A) to process revisions (Process B, C, D) based on several physiochemical characteristics: quantity of aggregated and misfolded species (18-20%, 15%, 12-14%, 15-16%), purity/impurity (30-34 ppm, 10-12 ppm, 4-5 ppm, 5-6 ppm), binding to TNF [107-108 binding units (BU), 111-113 BU, 115-117 BU, 111-113 BU] and potency to inhibit TNF-based apoptosis (1.5-1.6 U/mg, 1.7-1.8 U/mg, 1.7-1.8 U/mg, 1.7-1.75 U/mg) across all batches, respectively. 6 To minimize heterogeneity and variation in batches of biologics, manufacturers heavily monitor the creation process. Yet, the numerous sources of variation in innovator biologics are difficult to control.…”

“…Approximately 26 000 patients received innovator etanercept in clinical trials without any grossly detectable changes to the efficacy, tolerability and safety profiles from the introduction of etanercept through repeated manufacturing process changes. 6 However, there is potential for minor changes to cause big effects. Epoetin-associated pure red cell aplasia developed as a consequence of changes to the manufacturing process of the stabilizer medium.…”

LBEC0101, an etanercept biosimilar for the treatment of rheumatoid arthritis

“…Approved TNF inhibitors include etanercept, infliximab, adalimumab, certolizumab pegol, and golimumab [11]. Etanercept was the first TNF inhibitor approved to treat RA in the US (1998), Europe (2000), Korea (2003) and Japan (2005) [12][13][14]. However, while TNF inhibitor treatment is highly effective, the expensive medical cost exerts a financial burden on patients and healthcare systems [15][16][17].…”

“…LBEC0101 has been developed as a biosimilar to the etanercept reference product (ETN-RP) [37], which is a dimeric fusion protein that consists of the extracellular ligand binding portion of the 75 kDa TNF receptor linked to the Fc portion of human immunoglobulin G1 [12]. LBEC0101 binds competitively to TNF, which is overexpressed in RA [5].…”