Jiang W, Schwendeman S P
Division of Pharmaceutics, College of Pharmacy, The Ohio State University, 500 West 12th Avenue, Columbus, Ohio 43210, USA.
Biotechnol Bioeng. 2000 Dec 5;70(5):507-17.
A formaldehyde-mediated aggregation pathway (FMAP) is suggested as being primarily responsible for the aggregation of lyophilized tetanus toxoid (TT; a formalinized antigen) in the presence of moisture. The general occurrence of the FMAP was examined by using bovine serum albumin (BSA) and ribonuclease A (RNase) as model antigens; both protein antigens were formalinized according to a method commonly used to detoxify bacterial toxins. To clearly delineate the FMAP from other aggregation mechanisms, the aggregation kinetics and mechanism of both unmodified antigens (BSA and RNase) and formalinized antigens (f-BSA and f-RNase) were evaluated. We report that formaldehyde treatment introduces more rapid and extensive aggregation in antigens under conditions that favor the FMAP (i.e., 80% relative humidity and 37 degrees C). Consistent with formaldehyde-mediated crosslinking, f-antigen aggregates were covalent and non-disulfide-bonded, whereas BSA aggregates were disulfide-linked and RNase even did not aggregate under the same conditions. Coincorporation of amino acids (histidine and lysine), which strongly interact with formaldehyde, as well as prior antigen reduction with cyanoborohydride, significantly inhibited f-BSA aggregation, but showed no selective effect on BSA aggregation. Mechanistic analysis of f-BSA aggregates, inhibition studies, and similar reactivity of f-BSA with TT all confirmed the existence of the FMAP at moisture levels intermediate between the dry and solution state. This study demonstrates the potential for covalent reactions between formalinized protein antigens and neighboring chemical or biochemical species even after formalinization, and provides a general approach to inhibit the FMAP.
一种甲醛介导的聚集途径（FMAP）被认为是冻干破伤风类毒素（TT；一种经福尔马林处理的抗原）在有水分存在时聚集的主要原因。通过使用牛血清白蛋白（BSA）和核糖核酸酶A（RNase）作为模型抗原，研究了FMAP的普遍存在情况；这两种蛋白质抗原均按照常用于使细菌毒素解毒的方法用福尔马林处理。为了清楚地将FMAP与其他聚集机制区分开来，评估了未修饰抗原（BSA和RNase）和经福尔马林处理抗原（f-BSA和f-RNase）的聚集动力学和机制。我们报告，在有利于FMAP的条件下（即相对湿度80%和37摄氏度），甲醛处理会使抗原中出现更快、更广泛的聚集。与甲醛介导的交联一致，f-抗原聚集体是共价且非二硫键连接的，而BSA聚集体是二硫键连接的，并且在相同条件下RNase甚至不聚集。与甲醛强烈相互作用的氨基酸（组氨酸和赖氨酸）的共掺入，以及先用氰基硼氢化钠还原抗原，均显著抑制f-BSA聚集，但对BSA聚集没有选择性影响。对f-BSA聚集体的机制分析、抑制研究以及f-BSA与TT的相似反应性均证实，在干燥状态和溶液状态之间的中间水分水平下存在FMAP。本研究证明了经福尔马林处理的蛋白质抗原与相邻化学或生物化学物质之间即使在福尔马林处理后仍可能发生共价反应，并提供了一种抑制FMAP的通用方法。
