Analytical Development, Virdante Pharmaceuticals, Cambridge, MA 02142, United States.
J Immunol Methods. 2012 Aug 31;382(1-2):167-76. doi: 10.1016/j.jim.2012.05.022. Epub 2012 Jun 6.
Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation of IgG glycans (neutral (G0, G1, and G2), mono- and di-sialylated) using simple procedures. The method should prove useful for monitoring glycan changes in clinical settings.
典型的临床级人 IgG（静脉注射免疫球蛋白，IVIG）用于碳水化合物分析，源自数千名健康供体。本文介绍了 IgG 及其 Fc-Fab 片段的定量高分辨率聚糖谱。在变性和非变性（天然）条件下，用 Fc 特异性内切糖苷酶 S 和通用 PNGase F 消化后建立了聚糖谱。天然 PNGase F 对 IgG 的聚糖谱与内切酶 S 相似（但不完全相同）。内切酶 S 图谱不包含带有双分支 GlcNAc 的聚糖。从胃蛋白酶和 Ide S 蛋白酶消化物中分离的 Fc 片段的 PNGase F 聚糖谱相同。分离的 Fab 片段和先前去糖基化的 IVIG（天然条件）产生相同的聚糖谱。使用 2-氨基苯甲酸（2AA）标记的高分辨率 HPLC 建立了聚糖谱。该方法可准确测定唾液酸化水平。使用内部标准测定 Fc 和 Fab 的碳水化合物含量，并对蛋白和聚糖回收率进行校正。Fab 部分含有约 14%的总碳水化合物，这意味着 IVIG 中的每个分子有 2.3 个糖链，其中 2 个糖链位于 Fc 的 CH2 结构域。Fc 聚糖由中性（N）84.5%组成；单唾液酸化（S1）15%和双唾液酸化（S2）0.5%。相比之下，Fab 含有 N，21%；S1，43%和 S2，36%。在 Fab 和 Fc 中的各种聚糖（N、S1 和 S2）中发现，分支 N-乙酰葡萄糖胺和岩藻糖的分布差异很大。总 IgG 聚糖谱（Fab 加 Fc）中 N 为 78.5%；S1，17%和 S2，4.5%。在 78%的中性聚糖中，聚糖 G0、G1 和 G2（分别有 0、1 和 2 个半乳糖）的分布百分比分别为 26%、49%和 25%。来自不同制造商的各种临床级 IVIG 制剂的聚糖谱几乎相同。开发了一种快速 HPLC 分析方法，用于分离和定量 IgG 聚糖（中性（G0、G1 和 G2）、单唾液酸化和双唾液酸化），方法简单。该方法在临床环境中监测聚糖变化应该很有用。
