Physicochemical and Biological Examination of Two Glatiramer Acetate Products.

Herein we compared 40 mg/mL lots of the active ingredient, glatiramer acetate, manufactured by Mylan/Natco to the active ingredient, glatiramer acetate in Copaxone (Teva Pharmaceuticals, Ltd., Netanya Israel) using physicochemical (PCC) methods and biological assays. No differences were seen between the Mylan/Natco and Teva lots with some low resolution release PCC assays (amino acid analysis, molecular weight distribution, interaction with Coomassie Brilliant Blue G-250). Changes in polydispersity between Mylan/Natco and Copaxone lots were found using size exclusion chromatography and the high resolution PCC method, known as Viscotek, and suggestive of a disparity in the homogeneity of mixture, with a shift towards high molecular weight polypeptides. Using RPLC-2D MALLS, 5 out of 8 Mylan/Natco lots fell outside the Copaxone range, containing a high molecular weight and high hydrophobicity subpopulation of polypeptides not found in Copaxone lots. Cation exchange chromatography showed differences in the surface charge distribution between the Copaxone and Mylan/Natco lots. The Mylan/Natco lots were found to be within Copaxone specifications for the EAE model, monoclonal and polyclonal binding assays and the in vitro cytotoxicity assay, however higher IL-2 secretion was shown for three Mylan/Natco lots in a potency assay. These observations provide data to inform the ongoing scientific discussion about the comparability of glatiramer acetate in Copaxone and follow-on products.

Compositional differences between Copaxone and Glatopa are reflected in altered immunomodulation ex vivo in a mouse model.

Copaxone (glatiramer acetate, GA), a structurally and compositionally complex polypeptide nonbiological drug, is an effective treatment for multiple sclerosis, with a well-established favorable safety profile. The short antigenic polypeptide sequences comprising therapeutically active epitopes in GA cannot be deciphered with state-of-the-art methods; and GA has no measurable pharmacokinetic profile and no validated pharmacodynamic markers. The study reported herein describes the use of orthogonal standard and high-resolution physicochemical and biological tests to characterize GA and a U.S. Food and Drug Administration-approved generic version of GA, Glatopa (USA-FoGA). While similarities were observed with low-resolution or destructive tests, differences between GA and USA-FoGA were measured with high-resolution methods applied to an intact mixture, including variations in surface charge and a unique, high-molecular-weight, hydrophobic polypeptide population observed only in some USA-FoGA lots. Consistent with published reports that modifications in physicochemical attributes alter immune-related processes, genome-wide expression profiles of ex vivo activated splenocytes from mice immunized with either GA or USA-FoGA showed that 7-11% of modulated genes were differentially expressed and enriched for immune-related pathways. Thus, differences between USA-FoGA and GA may include variations in antigenic epitopes that differentially activate immune responses. We propose that the assays reported herein should be considered during the regulatory assessment process for nonbiological complex drugs such as GA.

Functional effects of the antigen glatiramer acetate are complex and tightly associated with its composition.

Glatiramer acetate (Copaxone®; GA) is a non-biological complex drug for multiple sclerosis. GA modulated thousands of genes in genome-wide expression studies conducted in THP-1 cells and mouse splenocytes. Comparing GA with differently-manufactured glatiramoid Polimunol (Synthon) in mice yielded hundreds of differentially expressed probesets, including biologically-relevant genes (e.g. Il18, adj p<9e-6) and pathways. In human monocytes, 700+ probesets differed between Polimunol and GA, enriching for 130+ pathways including response to lipopolysaccharide (adj. p<0.006). Key differences were confirmed by qRT-PCR (splenocytes) or proteomics (THP-1). These studies demonstrate the complexity of GA's mechanisms of action, and may help inform therapeutic equivalence assessment.

Gene expression studies of a human monocyte cell line identify dissimilarities between differently manufactured glatiramoids.

Glatiramer Acetate (GA) has provided safe and effective treatment for multiple sclerosis (MS) patients for two decades. It acts as an antigen, yet the precise mechanism of action remains to be fully elucidated, and no validated pharmacokinetic or pharmacodynamic biomarkers exist. In order to better characterize GA's biological impact, genome-wide expression studies were conducted with a human monocyte (THP-1) cell line. Consistent with previous literature, branded GA upregulated anti-inflammatory markers (e.g. IL10), and modulated multiple immune-related pathways. Despite some similarities, significant differences were observed between expression profiles induced by branded GA and Probioglat, a differently-manufactured glatiramoid purported to be a generic GA. Key results were verified using qRT-PCR. Genes (e.g. CCL5, adj. p < 4.1 × 10(-5)) critically involved in pro-inflammatory pathways (e.g. response to lipopolysaccharide, adj. p = 8.7 × 10(-4)) were significantly induced by Probioglat compared with branded GA. Key genes were also tested and confirmed at the protein level, and in primary human monocytes. These observations suggest differential biological impact by the two glatiramoids and warrant further investigation.

gamma-Glutamyl transpeptidase and glutathione biosynthesis in non-tumorigenic and tumorigenic rat liver oval cell lines.

Glutathione synthesis and growth properties were studied in the gamma-glutamyl transpeptidase(GGT)-negative, non-tumorigenic rat liver oval cell line OC/CDE22, and in its GGT-positive, tumorigenic counterpart line M22. gamma-Glutamylcysteine synthetase (GGCS) activities were comparable. Growth rates of M22 cells exceeded those of OC/CDE22 cells at non-limiting and limiting exogenous cysteine concentrations. A monoclonal antibody (Ab 5F10) that inhibits the transpeptidatic but not the hydrolytic activity of GGT did not affect the growth rates of OC/CDE22, and decreased those of M22 to the OC/CDE22 level. In GSH-depleted M22, but not in OC/CDE22 cells, the rate and extent of GSH repletion with exogenous cysteine and glutamine exceeded those obtained with exogenous cysteine and glutamate. With Ab 5F10, repletion with cysteine/glutamine was similar to that obtained with cysteine/glutamate. Repletion with exogenous GSH occurred only in M22 cells, and was abolished by the GGT inhibitor acivicin. Repletion with gamma-glutamylcysteine (GGC) in OC/CDE22 was resistant to acivicin whereas that in M22 was inhibited by acivicin. Repletion with exogenous GSH or cysteinylglycine (CG) required aminopeptidase activity and was lower than that obtained with cysteine. Unless reduced, CG disulfide did not support GSH repletion. The findings are compatible with the notions that (i) GGT-catalyzed transpeptidation was largely responsible for the growth advantage of M22 cells at limiting cysteine concentration, and for their high GSH content via the formation of GGC from a gamma-glutamyl donor (glutamine) and cyst(e)ine, and (ii) aminopeptidase/dipeptidase activity is rate-limiting in GSH repletion when GSH or CG serve as cysteine sources.