William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio, USA.
Biotechnol Prog. 2020 Sep;36(5):e3010. doi: 10.1002/btpr.3010. Epub 2020 May 13.
Haptoglobin (Hp) is a plasma glycoprotein that scavenges cell-free hemoglobin (Hb). Hp has various potential therapeutic applications, but it has been mainly studied for treatment of acute hemolytic conditions that can arise from situations such as massive blood transfusion, infusion of stored red blood cells, severe burns, trauma, sepsis, radiation injury, and others. Therefore, Hp may also be beneficial during chronic hemolytic disease states such as hereditary spherocytosis, nocturnal hemoglobinuria, sickle-cell anemia, and malaria. Various methods have been developed to purify Hp from plasma or plasma fractions. However, none of these methods have exploited the large molecular weight (MW) range distribution of Hp polymers to easily isolate Hp from other plasma proteins. The present study used tangential flow filtration (TFF) to isolate polymeric Hp from plasma proteins using human Fraction IV (FIV) as the starting material. After removal of insoluble material from a suspension of FIV paste, the protein mixture was clarified on a 0.2 μm hollow fiber (HF) TFF filter. The clarified protein solution was then bracketed based on protein MW using HF filters with MW cut-offs (MWCOs) of 750, 500, and 100 kDa. Using untreated FIV, the Hp purity of the main bracket was ~75% with a total Hb binding capacity (HbBC) yield of 1.2 g starting from 500 g of FIV paste. However, pretreatment of FIV with fumed silica to remove lipoproteins increased Hp purity to >95% with a HbBC yield of 1.7 g per 500 g of FIV. Taken together this study provides a novel and scalable method to purify Hp from plasma or plasma fractions.
触珠蛋白（Hp）是一种血浆糖蛋白，可清除游离血红蛋白（Hb）。Hp 具有多种潜在的治疗应用，但主要研究用于治疗可能由大量输血、储存的红细胞输注、严重烧伤、创伤、败血症、辐射损伤等引起的急性溶血性情况。因此，Hp 在遗传性球形红细胞增多症、夜间血红蛋白尿、镰状细胞贫血和疟疾等慢性溶血性疾病状态下也可能有益。已经开发了各种方法从血浆或血浆级分中纯化 Hp。然而，这些方法都没有利用 Hp 聚合物的大分子量（MW）范围分布来从其他血浆蛋白中轻松分离 Hp。本研究使用切向流过滤（TFF）从血浆蛋白中分离聚合 Hp，用人 Fraction IV（FIV）作为起始材料。从 FIV 糊剂的悬浮液中除去不溶性物质后，在 0.2 μm 中空纤维（HF）TFF 过滤器上澄清蛋白质混合物。然后根据蛋白质 MW 使用 MW 截止值（MWCO）为 750、500 和 100 kDa 的 HF 过滤器对澄清的蛋白质溶液进行分级。使用未经处理的 FIV，主支架的 Hp 纯度约为 75%，总 Hb 结合能力（HbBC）产量为 1.2 g，起始于 500 g 的 FIV 糊剂。然而，用气相二氧化硅预处理 FIV 以去除脂蛋白可将 Hp 纯度提高到>95%，每 500 g FIV 产生 1.7 g HbBC。总的来说，这项研究提供了一种从血浆或血浆级分中纯化 Hp 的新颖且可扩展的方法。
