Evaluating Clinical Safety and Analytical Impact of Subvisible Silicone Oil Particles in Biopharmaceutical Products.

Silicone oil is a commonly used lubricant in pre-filled syringes (PFSs) and can migrate over time into solution in the form of silicone oil particles (SiOPs). The presence of these SiOPs can result in elevated subvisible particle counts in PFS drug products compared to other drug presentations such as vials or cartridges. Their presence in products presents analytical challenges as they complicate quantitation and characterization of other types of subvisible particles in solution. Previous studies have suggested that they can potentially act as adjuvant resulting in potential safety risks for patients. In this paper we present several analytical case studies describing the impact of the presence of SiOPs in biotherapeutics on the analysis of the drug as well as clinical case studies examining the effect of SiOPs on patient safety. The analytical case studies demonstrate that orthogonal techniques, especially flow imaging, can help differentiate SiOPs from other types of particulate matter. The clinical case studies showed no difference in the observed patient safety profile across multiple drugs, patient populations, and routes of administration, indicating that the presence of SiOPs does not impact patient safety.

Influence of Production Process and Scale on Quality of Polypeptide Drugs: a Case Study on GLP-1 Analogs.

PURPOSE: Manufacturing processes for polypeptide/protein drugs are designed to ensure robust quality, efficacy and safety. Process differences introduced by follow-on manufacturers may result in changes in quality and clinical outcomes. This study investigated the impact of production methods on the stability and impurities of liraglutide and semaglutide drug substances/products, and the potential impact on drug quality, efficacy and safety. METHODS: State-of-the-art analytical methods were used to compare physical and chemical stability, and impurity profiles of drug substances/products from different suppliers. Identified polypeptide-related impurities were evaluated for immunogenicity potential by in silico T cell epitope prediction. Semaglutide immunogenicity in clinical trials (SUSTAIN) was evaluated using a tiered antibody analysis. RESULTS: Manufacturing scale and process strongly impacted the physical stability of the products. Trace metals increased high-molecular-weight protein formation for liraglutide and semaglutide. Synthetic and recombinant liraglutide produced by five suppliers had distinct impurity profiles compared with the originator. In silico evaluation suggested that new impurities could be immunogenic. Immunogenicity of semaglutide in clinical trials was lower than for liraglutide. CONCLUSIONS: Differences in manufacturing processes affect chemical/physical stability and impurity profile, and may impact immunogenicity. Follow-on versions of liraglutide and semaglutide, and possibly other polypeptides, should be clinically evaluated for efficacy and safety.

Liraglutide treatment is associated with a low frequency and magnitude of antibody formation with no apparent impact on glycemic response or increased frequency of adverse events: results from the Liraglutide Effect and Action in Diabetes (LEAD) trials.

CONTEXT: Therapeutic proteins/peptides can produce immunogenic responses that may increase the risk of adverse events or reduce efficacy. OBJECTIVE: The objectives were to measure and characterize antibody formation to liraglutide, a glucagon-like peptide-1 receptor agonist, to investigate the impact on glycemic control and safety, and to compare it with exenatide, an agent in the same class. DESIGN: Antibody data were collected during six Liraglutide Effect and Action in Diabetes (LEAD) trials (26-104 wk duration). SETTING: Samples for determination of antibody formation were collected at LEAD trial sites and analyzed at central laboratories. PARTICIPANTS: Antibodies were measured in LEAD trial participants with type 2 diabetes. INTERVENTIONS: Interventions included once-daily liraglutide (1.2 or 1.8 mg) or twice-daily exenatide (10 µg). MAIN OUTCOME MEASURES: The main outcome measures included the proportion of patients positive for anti-liraglutide or anti-exenatide antibodies, a glucagon-like peptide-1 cross-reacting effect, and an in vitro liraglutide- or exenatide-neutralizing effect. Change in glycosylated hemoglobin A(1c) (HbA(1c)) by antibody status and magnitude [negative, positive (high or low level)]. RESULTS: After 26 wk, 32 of 369 (8.7%) and 49 of 587 (8.3%) patients had low-level antibodies to liraglutide 1.2 and 1.8 mg, respectively [mean 3.3% antibody-bound radioactivity out of total radioactivity (%B/T), range 1.6-10.7%B/T], which did not attenuate glycemic efficacy (HbA(1c) reductions 1.1-1.3% in antibody-positive vs. 1.2% in antibody-negative patients). In LEAD-6, 113 of 185 extension patients (61%) had anti-exenatide antibodies at wk 26 (range 2.4-60.2%B/T). High levels of anti-exenatide antibodies were correlated with significantly smaller HbA(1c) reductions (P = 0.0022). After switching from exenatide to liraglutide, anti-exenatide antibodies did not compromise a further glycemic response to liraglutide (additional 0.4% HbA(1c) reduction). CONCLUSIONS: Liraglutide was less immunogenic than exenatide; the frequency and levels of anti-liraglutide antibodies were low and did not impact glycemic efficacy or safety.

Different diabetogenic potential of autoaggressive CD8+ clones associated with IFN-gamma-inducible protein 10 (CXC chemokine ligand 10) production but not cytokine expression, cytolytic activity, or homing characteristics.

Type 1 diabetes mellitus is an autoimmune disease characterized by T cell-mediated destruction of the insulin-producing beta cells in the islets of Langerhans. From studies in animal models, CD8(+) T cells recognizing autoantigens such as islet-specific glucose-6-phosphatase catalytic subunit-related protein, insulin, or glutamic acid decarboxylase (GAD) are believed to play important roles in both the early and late phases of beta cell destruction. In this study, we investigated the factors governing the diabetogenic potential of autoreactive CD8(+) clones isolated from spleens of NOD mice that had been immunized with GAD65(515-524) or insulin B-chain(15-23) peptides. Although these two clones were identical in most phenotypic and functional aspects, for example cytokine production and killing of autologous beta cells, they differed in the expression of IFN-gamma-inducible protein-10, which was only produced at high levels by the insulin-specific clone, but not by the GAD65-specific clone, and other autoantigen-specific nonpathogenic CD8 T cell clones. Interestingly, upon i.p. injection into neonatal mice, only the insulin B-chain(15-23)-reactive CD8(+) T clone accelerated diabetes in all recipients after 4 wk, although both insulin- and GAD-reactive clones homed to pancreas and pancreatic lymph nodes with similar kinetics. Diabetes was associated with increased pancreatic T cell infiltration and, in particular, recruitment of macrophages. Thus, secretion of IFN-gamma-inducible protein-10 by autoaggressive CD8(+) lymphocytes might determine their diabetogenic capacity by affecting recruitment of cells to the insulitic lesion.

An islet-homing NOD CD8+ cytotoxic T cell clone recognizes GAD65 and causes insulitis.

T cells play a central role in the development of diabetes both in man and in the non-obese diabetic (NOD) mouse. Both the CD4(+) and CD8(+) subsets of T cells are required for the normal development of IDDM in NOD mice. Islet reactive CD4(+) T cells play a clear pathogenic role as evidenced from the isolation of diabetogenic CD4(+) T cell clones. CD8(+) T cells seem to be involved in the initiation of diabetes as lack of these cells leads to protection from diabetes. We have isolated a GAD(65) reactive, cytotoxic CD8(+) T cell clone R1 that produces large quantities of IFNgamma and accelerates the onset of insulitis. This clone proliferates and produces IFNgamma in response to GAD(65) presenting APCs and kills GAD(65) presenting targets. Furthermore, it expresses TNFalpha, CD25, CD28, CD44, CD45 and LFA1, but not CD95L This is the first example of a GAD(65)specific CD8(+) T cell clone that accelerates the onset of the insulitis, although it does not appear to accelerate the onset of diabetes.

PDX-1 mediates glucose responsiveness of GAD(67), but not GAD(65), gene transcription in islets of Langerhans.

Glucose responsiveness is a fundamental metabolic feature of pancreatic beta-cells. Glucose-regulated transcription of the insulin gene is in part mediated via the homeobox transcription factor PDX-1. Another islet protein and diabetes autoantigen, glutamic acid decarboxylase (GAD), has been shown to be subject to regulation by glycemia. We have studied the mRNA level of two isoforms of GAD, GAD(65) and GAD(67), and found that GAD(67) but not GAD(65) mRNA steady-state level is regulated by glucose. By transfection of a rat GAD(67) promoter-driven luciferase reporter gene into primary rat islet cells, we demonstrate glucose-regulated expression of the reporter gene. We show that PDX-1 is able to bind to two TAAT-boxes in the GAD(67) promoter and that functional disruption of these two PDX-1 binding elements has an additive effect in severely impairing glucose responsiveness of the GAD(67) promoter. These data strongly suggest that PDX-1 is involved in glucose-regulated expression of GAD(67).