Yu Chi-Ming, Chin Chim Yong, Franses Elias I, Wang Nien-Hwa Linda
School of Chemical Engineering, Purdue University, 480 Stadium Mall Drive, West Lafayette, IN 47907-2100, USA.
J Colloid Interface Sci. 2006 Jul 15;299(2):733-9. doi: 10.1016/j.jcis.2006.02.039. Epub 2006 Mar 6.
Probing protein aggregation in situ is quite important for analyzing and developing chromatographic protein purification processes. A spectroturbidimetry method with a photodiode array detector is developed and tested for probing insulin aggregation in solution and determining the aggregation number, n(m). All aggregates examined are in the Rayleigh light scattering regime, where the turbidity between 400 and 350 nm is proportional to lambda(-4). Insulin at 25 degrees C in 3.5 N acetic acid is mainly monomeric (non-aggregated). At 25 degrees C and lower acetic acid concentrations, from 0.1 to 1 N, the average insulin aggregation number n(m) ranges from 2.9 to 1.6. Aggregates, with n(m) = 2-3, are found in 2.6 N acetic acid with 20 vol% acetonitrile. In 0.8 N acetic acid with 20 vol% denatured ethanol, n(m) = 1.2. At 4 degrees C, as acetic acid concentration decreases from 3.5 to 0.1 N, n(m) decreases from 2.4 to 1.8. In 2.8 N acetic acid with 20 vol% denatured ethanol at 4 degrees C, insulin exists mainly in monomer form. In situ probing of size exclusion chromatography, SEC, effluents in 3.5 N acetic acid at 4 degrees C shows n(m) = 1.6 at the fronting portion (a mixture of monomers and dimers or other oligomers) and n(m) = 1.1 (mostly monomers) at the tailing portion of the main peak. In another example, for LysPro-insulin in reversed phase chromatography at 4 degrees C, complex elution patterns and broad peaks are due to substantial aggregation. For a linear gradient of acetonitrile from 10 to 60 vol% at 4 degrees C, n(m) ranges from 2.2 to 12, in order of elution. For a linear gradient of ethanol from 30 to 50 vol% at 4 degrees C, n(m) ranges from 14 to 27, in order of elution. Analytical HPLC results at 25 degrees C imply that the aggregates are reversible.
原位探测蛋白质聚集对于分析和开发蛋白质色谱纯化工艺非常重要。开发并测试了一种采用光电二极管阵列检测器的分光比浊法，用于探测溶液中的胰岛素聚集并确定聚集数n(m)。所有检测的聚集体都处于瑞利光散射区域，其中400至350nm之间的浊度与λ(-4)成正比。25℃下3.5N乙酸中的胰岛素主要为单体（非聚集态）。在25℃和较低乙酸浓度（0.1至1N）下，胰岛素的平均聚集数n(m)范围为2.9至1.6。在含有20体积%乙腈的2.6N乙酸中发现了n(m)=2 - 3的聚集体。在含有20体积%变性乙醇的0.8N乙酸中，n(m)=1.2。在4℃下，随着乙酸浓度从3.5N降至0.1N，n(m)从2.4降至1.8。在4℃下含有20体积%变性乙醇的2.8N乙酸中，胰岛素主要以单体形式存在。4℃下在3.5N乙酸中对尺寸排阻色谱（SEC）流出物的原位探测显示，前沿部分（单体和二聚体或其他寡聚体的混合物）的n(m)=1.6，主峰尾部部分的n(m)=1.1（主要是单体）。在另一个例子中，对于4℃下反相色谱中的赖脯胰岛素，复杂的洗脱模式和宽峰是由于大量聚集所致。在4℃下乙腈从10体积%到60体积%的线性梯度洗脱中，n(m)按洗脱顺序从2.2到12变化。在4℃下乙醇从30体积%到50体积%的线性梯度洗脱中，n(m)按洗脱顺序从14到27变化。25℃下的分析型高效液相色谱结果表明聚集体是可逆的。
