Physicochemical, biological, functional and toxicological characterization of the European follow-on glatiramer acetate product as compared with Copaxone

Abstract

For more than 20 years, Copaxone (glatiramer acetate, Teva), a non-biological complex drug, has been a safe and effective treatment option for multiple sclerosis. In 2016, a follow-on glatiramer acetate product (FOGA, Synthon) was approved in the EU. Traditional bulk-based methods and high-resolution assays were employed to evaluate the physicochemical, functional, and bio-recognition attributes, as well as the in vivo toxicity profile of the active substances in Copaxone and Synthon EU FOGA lots. These tests included quality control tests applied routinely in release of Copaxone lots, as well as additional characterization assays, gene expression studies and a rat toxicity study. Even though the Synthon FOGA was designed to copy and compete with Copaxone, the active substances were found to be similar in only 7 of the tested 14 (50%) methods (similar is defined as within approved specifications or within the inherent microheterogeneity range of tested Copaxone batches, or not showing statistically significant differences). With additional methods applied, consistent compositional differences in attributes of surface charge distribution, molecular size, and spatial arrangement were observed. These marked differences were concordantly observed with higher biological activity of some of the Synthon EU FOGA lots compared with Copaxone lots, including potency and cytotoxicity activities as well as gene expression of pathways that regulate apoptosis, IL-2, and inflammation signaling. These observations raise concerns for immunogenicity differences, particularly in (repeated) substitution settings. Another orthogonal finding demonstrated increased frequency of injection-site local toxicity observations for the Synthon EU FOGA in an in vivo daily dosing rat study, thus warranting further qualification of the link between compositional and functional differences in immunogenicity, and potential impact on long-term efficacy and safety.

Keywords: Copaxone; FOGA; Follow-on glatiramer acetate product; Glatiramer acetate; NBCD; Non biological complex drug; Substitutability.

Fig. 1

MWD, CEX, and Viscotek TDAmax Assays. A. Peak maximum molecular weight for Copaxone and Synthon FOGA product – Actual data. B. Distribution of peak maximum molecular weight (Da) for simulated Copaxone data and Synthon EU FOGA mean value. Avg = average; FOGA = follow-on glatiramer acetate; LSL = lower specification limit; USL = upper specification limit. Grey columns represent the distribution of peak maximum MW of 2500 simulated Copaxone averages of the six samples randomly drawn from the total 957 Copaxone readings. The red line represents the observed average peak maximum MW of the six available Synthon EU lots. C. CEX. Characterization of surface charge distribution of Copaxone lots and Synthon EU FOGA lots (Peak 1 = Negatively Charged Population, Peak 2 = Weak Positively Charged Population; Peak 3 = Strong Positively Charged Population). D. Viscotek TDAmax. Conformational characterization of polymer molecular weight distribution for Copaxone lots and Synthon EU FOGA lots.

Fig. 2

2D-MALLS, IMMS, and AFM Assays. A. 2D-MALLS. Molecular mass elution profiles as a function of hydrophobicity for Copaxone and Synthon EU FOGA lots. 2D-MALLS = 2 dimensional multi angle laser light scattering; EU = European Union; FOGA = follow-on glatiramer acetate Synthon EU FOGA samples are shown in red; 6 lots of Copaxone (overlay) are shown in blue. B. IMMS. The pixel-by-pixel compositional comparison of Synthon lots to a randomly selected Copaxone lot identified extensive differences (as exemplifed by the red and blue dots in the scattergraph of Clift 1503711E). IMMS = ion mobility mass spectrometry; Red = above range; Blue = below range; White = within range of COPAXONE's microheterogeneity, as tested by 6 randomly selected lots of similar expiration dates. C. AFM. Comparison of Copaxone and Synthon EU FOGAs are generally similar in morphology. EU = European Union; FOGA = follow-on glatiramer acetate.

Fig. 3

Potency ex vivo cell-based assay. A. Relative potency of Copaxone and Synthon EU FOGA - actual data. B. Distribution of average relative potency (%) simulated data for Copaxone vs Synthon EU FOGA product - actual data. Avg = average; LSL = lower specification limit; USL = upper specification limit. Grey columns represent the distribution of averages of relative potency of 2500 simulated Copaxone mean values, drawn out of the total observed data from 231 lots tested. The red line represents the observed average relative potency of the 6 available Synthon EU FOGA lots. Note: Specification limits relate to the individual values not averages since the variation of mean values - standard error (SE) is smaller than the variation of individual readings - standard deviation (SD). Therefore, specification limits on chart are given for display purposes only. C. Relative potency of cytotoxicity induction Copaxone and Synthon EU FOGA product - actual data.

Fig. 4

Gene expression in THP-1 experiments. A. Number of differentially expressed probesets in each lot vs. lot contrast. Virtually no differences were found between lots of Copaxone (upper left quadrant) and lots of Synthon (lower right quadrant); however, each Copaxone vs. Synthon lot comparison exhibits differentially expressed probesets. B. Top GSEA immune pathways that significantly differ in lot vs. lot comparisons, broken down by number of contrasts with adj. p < 0.05 in the 9 Copaxone vs. Synthon contrasts (purple), 3 Synthon vs. Synthon contrasts (red), and 3 Copaxone vs. Copaxone contrasts (blue). The pathways differ by 2/3 or more of the Copaxone vs. Synthon contrasts, but rarely differ in the Synthon vs. Synthon or Copaxone vs. Copaxone contrasts. C. The volcano plots, which highlight Copaxone vs. Synthon differences in 4 hallmark pathways particularly relevant to the mechanism of action of Copaxone, show GSEAresults, with y-axis indicating significance (-log10 adj p) and x-axis indicating the GSEA enrichment score. In each plot, the number of datapoints within each category corresponds to the lot vs. lot contrasts indicated in Fig. 4A (9 Copaxone vs. Synthon, and 3 each of Synthon vs. Synthon and Copaxone vs. Copaxone). In each lot vs lot contrast, the GSEA analysis is performed on the differential expression data of the first lot versus the second one (lot1–lot2). Each dot is labeled with the specific lot contrast, abbreviated to drug (C=Copaxone, S=Synthon), and the last two (Copaxone) or three (Synthon) characters (for example Copaxone P62356 is abbreviated to C56). The abundance of significant and negative ES values (upper left corner of plot) for Copaxone vs. Synthon lot comparisons reflects lower expression of pathway genes in Copaxone relative to Synthon in each of the 4 pathways.

Fig. 4

Gene expression in THP-1 experiments. A. Number of differentially expressed probesets in each lot vs. lot contrast. Virtually no differences were found between lots of Copaxone (upper left quadrant) and lots of Synthon (lower right quadrant); however, each Copaxone vs. Synthon lot comparison exhibits differentially expressed probesets. B. Top GSEA immune pathways that significantly differ in lot vs. lot comparisons, broken down by number of contrasts with adj. p < 0.05 in the 9 Copaxone vs. Synthon contrasts (purple), 3 Synthon vs. Synthon contrasts (red), and 3 Copaxone vs. Copaxone contrasts (blue). The pathways differ by 2/3 or more of the Copaxone vs. Synthon contrasts, but rarely differ in the Synthon vs. Synthon or Copaxone vs. Copaxone contrasts. C. The volcano plots, which highlight Copaxone vs. Synthon differences in 4 hallmark pathways particularly relevant to the mechanism of action of Copaxone, show GSEAresults, with y-axis indicating significance (-log10 adj p) and x-axis indicating the GSEA enrichment score. In each plot, the number of datapoints within each category corresponds to the lot vs. lot contrasts indicated in Fig. 4A (9 Copaxone vs. Synthon, and 3 each of Synthon vs. Synthon and Copaxone vs. Copaxone). In each lot vs lot contrast, the GSEA analysis is performed on the differential expression data of the first lot versus the second one (lot1–lot2). Each dot is labeled with the specific lot contrast, abbreviated to drug (C=Copaxone, S=Synthon), and the last two (Copaxone) or three (Synthon) characters (for example Copaxone P62356 is abbreviated to C56). The abundance of significant and negative ES values (upper left corner of plot) for Copaxone vs. Synthon lot comparisons reflects lower expression of pathway genes in Copaxone relative to Synthon in each of the 4 pathways.