Polymerized hemoglobin (Hb)-based oxygen carriers (HBOCs) are a scalable and cost-effective red blood cell (RBC) substitute. However, previous generations of commercial polymerized HBOCs elicited oxidative tissue injury in vivo due to the presence of low molecular weight polymeric Hb species (<500 kDa) and cell-free Hb (64 kDa). Polymerized human Hb (PolyhHb) locked in the tense quaternary state (T-state) exhibits great promise to meet clinical needs where past polymerized HBOCs failed. This work shows that separation of T-state PolyhHb via a two-stage tangential flow filtration (TFF) purification train such that the Hb polymers are bracketed between 500 kDa and 0.2 μm creates a uniform polymer size and largely eliminates the Hb species which elicit deleterious side effects in vivo. Biophysical characterization of these materials demonstrates their potential effectiveness as an RBC substitute and verifies the low percentage of low molecular weight Hb polymers and cell-free Hb. Size exclusion chromatography confirms that T-state PolyhHb can be consistently produced in a size range between 500 kDa and 0.2 μm. Furthermore, the average molecular weight of all PolyhHb species produced is one or two orders of magnitude larger than that of the commercial polymerized HBOCs Hemolink and Oxyglobin, respectively. Haptoglobin binding kinetics confirms that two-stage TFF processing of PolyhHb reliably removes cell-free Hb and low molecular weight polymeric Hb species. T-state PolyhHbs demonstrate lower auto-oxidation rates compared to unmodified Hb and prior generations of commercial polymerized HBOCs. These results demonstrate T-state PolyhHb's feasibility as a next-generation polymerized HBOC for potential use in transfusion medicine.

中文翻译：

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大小介于 500 kDa 和 0.2 μm 之间的紧张四元态聚合人血红蛋白的生物物理特性

基于聚合血红蛋白 (Hb) 的氧载体 (HBOC) 是一种可扩展且经济高效的红细胞 (RBC) 替代品。然而，前几代商业聚合 HBOC 由于低分子量聚合 Hb 种类 (<500 kDa) 和无细胞 Hb (64 kDa) 的存在而引起体内氧化组织损伤。锁定在紧张四元态（T 态）的聚合人 Hb (PolyhHb) 在满足过去聚合 HBOC 无法满足的临床需求方面表现出巨大的前景。这项工作表明，通过两级切向流过滤 (TFF) 纯化系列分离 T 态 PolyhHb，使 Hb 聚合物介于 500 kDa 和 0.2 μm 之间，从而形成均匀的聚合物尺寸，并在很大程度上消除了引起有害物质的 Hb 物质。体内副作用。这些材料的生物物理表征证明了它们作为红细胞替代品的潜在有效性，并验证了低分子量血红蛋白聚合物和无细胞血红蛋白的低百分比。尺寸排阻色谱证实 T 态 PolyhHb 可以在 500 kDa 至 0.2 μm 之间的尺寸范围内持续产生。此外，所生产的所有 PolyhHb 物种的平均分子量分别比商业聚合 HBOC Hemolink 和 Oxyglobin 大一或两个数量级。触珠蛋白结合动力学证实，PolyhHb 的两阶段 TFF 处理可靠地去除了无细胞 Hb 和低分子量聚合 Hb 物质。与未修饰的 Hb 和前几代商业聚合 HBOC 相比，T 态 PolyhHb 表现出较低的自动氧化率。这些结果证明了 T 态 PolyhHb 作为下一代聚合 HBOC 在输血医学中的潜在用途的可行性。