Data_Sheet_1_Impact of Excipients on Stability of Polymer Microparticles for Autoimmune Therapy.docx

Therapies for autoimmune diseases such as multiple sclerosis and diabetes are not curative and cause significant challenges for patients. These include frequent, continued treatments required throughout the lifetime of the patient, as well as increased vulnerability to infection due to the non-specific action of therapies. Biomaterials have enabled progress in antigen-specific immunotherapies as carriers and delivery vehicles for immunomodulatory cargo. However, most of this work is in the preclinical stage, where small dosing requirements allow for on-demand preparation of immunotherapies. For clinical translation of these potential immunotherapies, manufacturing, preservation, storage, and stability are critical parameters that require greater attention. Here, we tested the stabilizing effects of excipients on the lyophilization of polymeric microparticles (MPs) designed for autoimmune therapy; these MPs are loaded with peptide self-antigen and a small molecule immunomodulator. We synthesized and lyophilized particles with three clinically relevant excipients: mannitol, trehalose, and sucrose. The biophysical properties of the formulations were assessed as a function of excipient formulation and stage of addition, then formulations were evaluated in primary immune cell culture. From a manufacturing perspective, excipients improved caking of lyophilized product, enabled more complete resuspension, increased product recovery, and led to smaller changes in MP size and size distribution over time. Cocultures of antigen-presenting cells and self-reactive T cells revealed that MPs lyophilized with excipients maintained tolerance-inducing function, even after significant storage times without refrigeration. These data demonstrate that excipients can be selected to drive favorable manufacturing properties without impacting the immunologic properties of the tolerogenic MPs.

针对多发性硬化症（multiple sclerosis）、糖尿病（diabetes）等自身免疫性疾病（autoimmune diseases）的治疗手段无法实现根治，且会给患者带来诸多严峻挑战。这些挑战包括患者需终身接受频繁且持续的治疗，同时由于治疗的非特异性作用，患者的感染易感性显著升高。生物材料（biomaterials）作为免疫调节载荷（immunomodulatory cargo）的载体与递送系统，为抗原特异性免疫治疗（antigen-specific immunotherapies）的发展提供了助力。然而，此类研究大多仍处于临床前阶段（preclinical stage），由于给药剂量要求较低，可实现免疫治疗的按需制备。要实现这类潜在免疫治疗手段的临床转化（clinical translation），生产、保存、储存与稳定性均为亟需重点关注的关键参数。本研究中，我们测试了赋形剂（excipients）对用于自身免疫治疗的聚合物微粒（polymeric microparticles, 简称MPs）冷冻干燥（lyophilization）过程的稳定作用；此类微粒负载有肽类自身抗原（peptide self-antigen）与小分子免疫调节剂（small molecule immunomodulator）。我们采用三种临床相关赋形剂——甘露醇（mannitol）、海藻糖（trehalose）与蔗糖（sucrose）——合成并冻干了微粒制剂。我们基于赋形剂配方与添加时机，对制剂的生物物理特性进行了评估；随后在原代免疫细胞培养（primary immune cell culture）体系中对制剂开展了功能验证。从生产视角来看，赋形剂可改善冻干产品的结块现象，实现更充分的重悬，提升产品回收率，并使MPs的粒径与粒径分布随时间的变化幅度显著降低。抗原呈递细胞（antigen-presenting cells）与自身反应性T细胞（self-reactive T cells）的共培养实验结果显示，经赋形剂冻干的MPs仍可维持其诱导免疫耐受的功能，即便在未冷藏的条件下储存较长时间后亦是如此。本研究数据表明，可通过筛选赋形剂，在不影响致耐受性MPs（tolerogenic MPs）免疫特性的前提下，优化其生产相关性能。