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1 Gel filtration 凝胶过滤层析技术 Content 内容 Content 内容 What is gel filtration 凝胶过滤定义? Gel filtration (GF) is a simple and reliable chromatographic method for separating molecules according to size and shape. 凝胶过滤层析是根据分子大小和形状的差异进行分离的一种简单可靠的层析技术 Gel structure 填料结构 AGAROSE 琼脂糖凝胶 A good gel for gel filtration contains about 95% water 凝胶过滤分离原理 : 空间排阻 1. Spherical particles packed into a column. 凝胶过滤填料的 95% 为水 2. Sample applied. 3. Buffer (mobile phase) and sample move through column. Molecules diffuse in and out of matrix 4. Large molecules leave the column first followed by smaller molecules in order of size. Molecules larger than the matrix pores pass straight through. - pores filled with eluent 孔内充满洗脱液 - carefully controlled size range 孔径精密控制 - chemically /physically inert (lack of adsorptive properties) 惰性 Diffusion Buffer Diffusion into the pores Buffer Diffusion out of the pores 1/

凝胶过滤层析是根据分子大小和形状的差异进行分离的一种简单可靠的层析技术 Gel structure 填料结构 AGAROSE 琼脂糖凝胶 A good gel for gel filtration contains about 95% water 凝胶过滤分离原理 : 空间排阻 1.

2 Steric exclusion leads to early elution 体积排阻程度决定洗脱位置 Molecules elute in order of size. 不同分子按照大小顺序洗出 Separation volumes 分离体积 V o V t void volume V o The largest molecules elute first; the smallest ones elute last. 大分子先洗出, 小分子后洗出 V c volume of the gel matrix V s pore volume V i V o = void volume 外水体积 = retention volume or elution volume 保留体积 / 洗脱体积 V t = total liquid volume of the bed 总液体体积 V i = inner pore volume = V c -V s V o 内孔体积 / 内水体积 V c = total geometric volume of the column 柱体积 V s = volume of gel matrix 固相凝胶的体积 The void volume V o 外水体积 V t and V c 总液体体积和柱体积 concentration 非常大的分子在外水体积洗出, V o concentration 非常小的分子的洗脱体积为内水 + 外水 V t = V +V i 柱床的几何体积, V c V o volume V o VR volume V t V c Distribution coefficient, K d 分配系数 The coefficient K av -V K d = V c - -V V s i.e., = V o + K d * V i -V = V i - : 外水体积 + 能进入的那部分内水体积 -K d : 溶质分子能够进入的内水体积 ( 内水体积 ) 占总的内水体积的比例 K d 较难测定, 常用 Kav 代替 = 保留体积 / 洗脱体积 V o = 排阻体积 / 外水体积 V c = 层析柱几何体积 V s = 固相凝胶所占的体积 V i = 孔内体积 / 内水体积 = V c -V s V o V K R -V = av V c -V - 与柱子大小和填装无关 - 可以用于比较不同的纯化结果 K av 容易得到, 也更有实际意义 2/

大分子先洗出, 小分子后洗出 V c volume of the gel matrix V s pore volume V i V o = void volume 外水体积 = retention volume or elution volume 保留体积 / 洗脱体积 V t = total liquid volume of the bed 总液体体

3 K av for very large and very small molecules 非常大的分子在 V 处洗脱 = V V K R -V V -V = = = av V c -V V c -V K av 应该在和 1 之间 concentration elution volume when K av = 1 elution with K av > 1 Adsorption has occurred. 非常小的分子在 V t 处洗脱 = V t V K = R -V = av =1 -V V c -V -V V t volume elution with K av < Serious problem! 流动相中需要加入 15mM NaCl! Calibration curves 校准曲线 Selectivity curves 选择性曲线 - 斜率越陡, 洗脱体积差异越大 - 仅可用于特定的柱子 Δ K av - 斜率越陡, 洗脱体积差异越大 - 与柱子的填装无关, 可以用于不同填料的性质比较 K av Δ log M r Δ K av Δ log M r V o K av = V c V o log M r versus the logarithm of molecular mass log M r K av versus the logarithm of molecular mass Selectivity curves 选择性曲线 K av Δ K av Δ K av - 斜率越陡, 洗脱体积差异越大 - 与柱子的填装无关, 可以用 K 于不同填料的性质比较 av 1 Exclusion limit 排阻极限不能进入填料孔的最小的分子量 Exclusion limit All molecules bigger than this elute in the void volume. 分子量大于此的分子均在外水体积洗出 Δ log M r V o K av = V c V o log M r K av versus the logarithm of molecular mass log (M r ) 3/

Calibration curves 校准曲线 Selectivity curves 选择性曲线 - 斜率越陡, 洗脱体积差异越大 - 仅可用于特定的柱子 Δ K av - 斜率越陡, 洗脱体积差异越大 - 与柱子的填装无关, 可以用于不同填料的性质比较 K av Δ log M r Δ

4 Fractionation range 分级范围 Content 内容 K av K av =.1 to K av =.7 的范围内, 选择性曲线为直线 fractionation range 填料分级范围 log (M r ) Resolution factor, R s 分辨率 Rs =.6 Rs = 1 Resolution: depends on 分辨率取决于 selectivity 选择性 and efficiency 柱效 high selectivity lower selectivity Selectivity 选择性 : 峰分离的量度高选择性可以达到基线分离高选择性可以弥补低的柱效 - 峰体积有可能增加 low selectivity R s = Rs = 4 V 2 -V 1 (W 1 + W 2 ) / 2 两峰相对于其峰宽而言, 分开的有多远 high selectivity high efficiency low efficiency Efficiency 柱效 : 峰宽的量度柱效越高峰越窄高柱效需要良好的柱填装技术高柱效有时可弥补选择性不足 high efficiency low efficiency 选择最佳的凝胶 (high selectivity) Fractionation Range 分级范围 1k 1k 1k 1k K av , 1, 1,, 1,, Superdex Peptide 2. Superdex Superdex 2 4. Sephacryl S-1 HR 5. Sephacryl S-2 HR 6. Sephacryl S-3 HR 7. Sephacryl S-4 HR 4/

体积有可能增加 low selectivity R s = Rs = 4 V 2 -V 1 (W 1 + W 2 ) / 2 两峰相对于其峰宽而言, 分开的有多远 high selectivity high efficiency low efficiency Efficiency 柱效 : 峰宽的量度柱效越高峰越窄高柱效需

5 Results depend on selectivity 分离结果与选择性有关 Shape effects 形状的影响排阻极限增加 AU 28 Sephacryl S-3 Sephacryl S-2 BSA Cyt C b-l IgG Sephacryl S-1 Cytidine 1k-15k 适合较大分子 5k-25k 最佳 1k-1k 适合较小分子 K av denatured proteins 变性蛋白 native proteins 天然蛋白 log (M r ) A gel has different fractionation ranges for native, globular proteins and denatured proteins in random coil. 对于形状不同的分子, 某填料的分级范围不同 ml Shape effects 形状的影响 Efficiency 柱效 K av 1 linear polysaccharides 线性多糖 K av 1 Molecules with different shapes have different selectivity curves. 对于形状不同的分子, 有不同的选择性曲线 Efficiency depends on 取决于 : flow rate 流速 particle size of matrix 粒径大小 particle size distribution of matrix 粒径分布 globular proteins 球蛋白 log (M r ) packing quality of the column 层析柱的装填 sample volume and viscosity 样品体积和黏度 Peak width depends on particle size 粒径影响柱效 Superdex Peptide µm.14 Superdex Peptide µm retention time min Superdex 3 prep grade µm retention time min Resolution depends on sample volume 进样体积影响柱效 Superdex Peptide High Performance column 1/3_24ml µl A µl Retention volume (ml) µl Retention volume (ml) 2 µl.8.1% Vt.8% Vt 4 µl Retention volume (ml) 4 µl <2% Vt 5/

对于形状不同的分子, 某填料的分级范围不同 4 8 12 ml Shape effects 形状的影响 Efficiency 柱效 K av 1 linear polysaccharides 线性多糖 K av 1 Molecules with different shapes have different selectivity curves.

6 Resolution depends on sample viscosity 黏度影响柱效样品黏度过高, 分离度下降. Resolution depends on column length 柱高影响分辨率 Increasing column length increases resolution 柱高加长, 分辨率提高 relative viscosity: Haemoglobin and NaCl, viscosity changed by adding dextran x Superdex Peptide High Performance column 1x3 mm i.d x bed height x Superdex Peptide High Performance column 1x3 mm i.d x bed height relative viscosity: relative viscosity: retention time min retention time min Content 内容 Main purification tasks 主要应用 Group separations 组分离 desalting, buffer exchange, removing reagents 脱盐 / 换缓冲液 Fractionation of proteins and peptides 精细分离 complex samples, monomer/dimer 聚集体去除 Estimation size 测定分子量 Characterisation: size homogeneity 蛋白鉴定 : 均一性 Group separations 组分离 Desalting proteins 蛋白质快速脱盐 Fractionation 精细分离 Separating dimer and oligomers from monomer 去除蛋白聚集体 16% - 44% CV Column: Hitrap desalting 5ml Sample: HSA, 2 mg/ml, Load:.8, 1.3, 1.7, 2.2 ml ( 16-26% Vt ) Buffer: NaCl.5M dimer monomer Column: Superdex 75 1/3 GL Tricorn Sample: reccys-prot., protein solution containing a mixture of dimers and monomers Sample volume: 2 µl (.8% Vt ) Elution buffer: 5mM TrisHCl, 1mM EDTA,.15M NaCl ph 8.4 filtered through.22 µm sterile filter Flow rate:.5 ml/min (38cm/h) System: ÄKTAexplorer 1 Detection: A 28 nm ml V c 组分离 : 柱高 < 6cm, 进样体积较大 (< 25% CV) 精细分离 : 柱高 3-1cm, 进样体积较小 ( 一般.5-5 % CV) 6/

8.7.6.5.4 2 x Superdex Peptide High Performance column 1x3 mm i.d x bed height.8.7.6.5.4 1 x Superdex Peptide High Performance column 1x3 mm i.d x bed height relative viscosity: 4.2 4.2.3.2.3.2.1.1 relative viscosity: 11.

7 Estimating molecular size 测定分子量 Measure elution position Calibration Curve 校准曲线 Characterisation: size homogeneity 蛋白鉴定 : 均一程度 Log(M) log (M r ) 计算分子量大小 ~ Log (M r ) Superdex 2 HR 1/3 Main products 主要产品 Superdex : 首选的分子筛填料 Superdex : 首选的分子筛凝胶 Highest selectivity 选择性高, high speed 快速, scale-up 可放大 Sephacryl HR complex samples, monomer/dimes 聚集体去除, 小规模 Superose Sepharose Virus, pdna, polysaccharides, 病毒 /DNA/ 多糖 etc Sephadex Fast desalting, buffer exchange, removing reagents 快速脱盐, 置换缓冲液, 去除试剂 - 葡聚糖和琼脂糖交联而成 - 高选择性, 高分辨率 - 速度快, 吸附最低, 收率高 - 化学性质稳定 - 流速快, 耐压性能好常用于对分辨率要求比较高的精细纯化! Gel structure 填料结构 A hypothetical structure for Superdex cross-linked agarose dextran Superdex Superdex Peptide M r 1-7 Superdex 75 M r 3-7 分离分子量大小 ( 球状蛋白质 ) Superdex 2 M r 1-6 cross-section from Superdex particle Cross-linked agarose 交联琼脂糖 strong support 支撑 Dextran chain 葡聚糖链 controlled pore size for separation 控制孔径 Tricorn Superdex 1/3GL_24ml Hiload Superdex 16/26 12ml /32ml 13um 34um 7/

模 Superose Sepharose Virus, pdna, polysaccharides, 病毒 /DNA/ 多糖 etc Sephadex Fast desalting, buffer exchange, removing reagents 快速脱盐, 置换缓冲液, 去除试剂 - 葡聚糖和琼脂糖交联而成 - 高选择性, 高分辨率

8 Superdex :1_UV1_28nm Superdex :1_Inject min Superdex 75 nr 171:1_UV1_28nm Superdex 75 nr 171:1_Inject min min Peptide Purification 多肽纯化 Aggregates Removal 聚集体去除 SDS-PAGE Superdex Peptide - 分离范围 1-7 Da - 高化学稳定性, ph1-14, 兼容有机溶剂 ( 乙腈 +TFA) - 和 RPC 衔接, 用于 peptide 的高效分离纯化 Requirement : Monomer > 95% Superdex 2 1/3GL 比较 Superdex 75 5/15 GL & Superdex 75 1/3 GL Rapid purity check of r-protein 重组蛋白纯度快速检测 Superdex 75 1/3 GL Column volume 24 ml Flow rate.6 ml/min Sample volume 1 μl Analysis time 47 min Recombinant protein ~17 kda. Truncated form ~1 kda. Recombinant protein Truncated protein 快速得到好的分辨率! Superdex 75 5/15 GL Column volume 3 ml Flow rate.3 ml/min Sample volume 12 μl Analysis time 12 min 47 min Column: Superdex 75 5/15 GL Buffer: PBS ph 7.4 Flow rate:.3 ml/min Sample volume: 4 µl Analysis time: 12 min/run System: Ettan LC 样品纯度检测非常快可以粗略估测纯度精确的定量分析请选择 1/3 GL 12 min! 12 min! Sephacryl HR - 由葡聚糖交联 N,N- 亚甲基二乙酰胺形成 - 理化性质稳定 - 系列全, 成本低类病毒颗粒的纯化 (VLP) HiPrep 16/6 Sephacryl S-5 HR Collaboration with Novavax Inc., US 8/

9 Act FF 11:1_UV3_26nm FF 11:1_Fractions FF 11:1_Inject FF FF 11:1_EditedBaseline ml W aste Act Manual run 1:1_UV1_26nm Manual run 1:1_UV3_28nm Manual run 1:1_Cond Manual run 1:1_Fractions Manual run 1:1_Inject Manual run 1:1_Fractions@11,HIST2 Manual run 1:1_Fractions@11,HIST1 Manual run 1:1_Fractions@15,BASEM Manual run 1:1_Logbook Waste Waste Waste ml M anual run 1:1_UV1_26nm Ma nual run 1:1_U V3_28nm Manual run 1:1_Fractions M anual run 1:1_Inject % Waste Waste min Superose Sepharose - 高度交联的琼脂糖 - 分离范围广 - 理化性质稳定 - 交联的琼脂糖 - 理化性质稳定 - Fast Flow 填料流速快 :25-3cm/h Sepharose 4FF: 6kD~2MD Sepharose 6FF: 1kD~4MD - 常用于疫苗领域 Influ.& Rabies Vaccine Production 流感 & 狂犬等病毒类疫苗的生产 Cell culture supernatant 多糖疫苗 : 两步层析代替酚抽提 75k ultrafiltration SEC: Sepharose 4FF AEX: Q Sepharose FF 阴离子交换 Capto adhere Sepharose 4FF 杂蛋白比例 ultra-/diafiltration purified human influenza virus 终产品蛋白残留 < 1% 系列 1 管号多糖疫苗的检定药典 CP25: 用琼脂糖 -4B 或 CL-4B 凝胶过滤法测定, K D 值应.4 ( 即 retention2 处的 Kd2 值.4 ) K D 值表示相对分子量, 多糖抗原分子量要大于 1kd 才具有免疫原性超螺旋质粒 pdna 纯化 Clarified alkaline lysate 裂解液 HiTrap 26/1 Sepharose 6 FF 26 mm x 1 mm Vt: 53 ml RNA-removal Kd.4 HiTrap PlasmidSelect Xtra 16 mm x 25 mm Vt: 5 ml Supercoiled pdna capture Kd =.5 Purified supercoiled pdna 纯化的 pdna HiTrap SOURCE 3Q 16 mm x 25 mm Vt: 5 ml Supercoiled pdna polishing PlasmidSelect Xtra Starter Kit 9/

1 15 2 ml 134.62 14.84 147.12 1 2 3 4 5 6 7 8 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 16.85 51 53 55 57 59 61 63 65 67 69 71 73 7577 W aste Act 3.5 3. 2.5 2. 1.5 1..5. 4 3 2 1

10 Sephadex - 由葡聚糖交联异表氯醇形成的多孔结构 - Sephadex G1: 1-7 Da 多肽纯化, oligo 脱盐, 抗生素聚集体质控分析 -Sephadex G25: 1k 5kDa MW>5kD 蛋白快速脱盐 Buffer exchange using 使用脱盐柱快速脱盐 HiPrep 26/1 Desalting Column Sample: BSA dissolved in 5 mm piperazine,.5 M sodium chloride, ph 6.2 Column: HiPrep 26/1 Desalting_53ml Buffer: 2 mm sodium phosphate,.15 M sodium chloride, ph 7. System: ÄKTAprime, 2 ml/min Content 内容 4 key questions 4 个问题 1. What is the aim of the experiment? 实验目的? - High resolution fractionation 精细分离 or 还是 - Group separation (desalting, buffer exchange)? 组分离, 缓冲液置换? High resolution fractionatio 精细分离 : Superdex, Superose, Sephacryl Group separation 组分离 : Sephadex 4 key questions 4 个问题 4 key questions 4 个问题 2. What is the molecular weight of the target protein/contaminants? 目标蛋白和杂质的分子量 Choose a gel filtration medium with the fractionation range that covers the molecular weight values of interest in your sample and that gives highest difference in size of target protein/contaminants/salt 选择分级范围能包含目标蛋白, 且使杂质和目标蛋白的分离度最大的填料 3. How much sample are they loading? 进样量是多少? Desalting 脱盐 25% CV PD-1 (gravity) loading capacity 2.5 ml HiTrap 5 ml loading capacity 1.5 ml HiPrep 53 ml loading capacity 15 ml Fractionation polishing 精细纯化 μl analytical column 分析 1%CV - 1/3 loading capacity 25 μl - 5/15 loading capacity 5 μl ml preparative column 制备 5%CV - 16/6 loading capacity 5 ml - 26/6 loading capacity 13 ml 更大规模, 可以自行填装柱子 1 /

$- High resolution fractionation 精细分离 or 还是 - Group separation (desalting, buffer exchange)? 组分离, 缓冲液置换?$

11 4 key questions 4 个问题如何得到满意的结果? 4. Will you be scaling up? 是否打算放大 Superdex - $$$ (1 st choice 首选 ) Sephadex - $ (desalting 脱盐 ) Sephacryl - $$ (large column packing is difficult 放大较困难 ) Appropriate industrial chromatography system and column available? 是否有合适的工业层析系统和层析柱? 使用选择性最高的填料! 使用预装柱! 尽量使用粒径小而均一的填料合理的进样量 / 柱高 - Group separation 组分离 : < 25% CV, 短 - Fractionation 精细分离 :.5-5% CV, 3-1cm 注意样品黏度降低流速以改善传质速度 ( 通常 5-2cm/h) 流动相中推荐使用 15mM NaCl Content 内容 Pros 优点 Simple 简单 Separates by size 根据大小分离 Fast method for buffer exchange 快速缓冲液置换 Very gentle, high yields 温和, 高收率 Works in any buffer solution buffer 没有特殊要求 Removes dimers and aggregates 去除聚集体 Cons 缺点 Limited sample volume 处理体积有限 Dilute the sample 会产生一定的稀释 Low flow rate 流速慢, 分离时间长 Scale-up 大规模放大有一定的困难 Q & A Please visit: 11 /

available? 是否有合适的工业层析系统和层析柱? 使用选择性最高的填料! 使用预装柱! 尽量使用粒径小而均一的填料合理的进样量 / 柱高 - Group separation 组分离 : < 25% CV, 短 - Fractionation 精细分离 :.

为了改进 SEC 色谱柱的分离表现 - 色谱填料 - 1. 更小的粒径更高的理论塔板数更高的反压, 更小的孔隙容量. 更大的孔隙容量 ( 校准曲线的斜率更为平缓 ) 分离系数 ; 需要被

为了改进 SEC 色谱柱的分离表现 - 色谱填料 - 1. 更小的粒径更高的理论塔板数更高的反压, 更小的孔隙容量. 更大的孔隙容量 ( 校准曲线的斜率更为平缓 ) 分离系数 ; 需要被新款适用于 UHPLC/HPLC 的 SEC 色谱柱的介绍东曹 ( 上海 ) 生物科技有限公司 Jan, 015 为了改进 SEC 色谱柱的分离表现 - 色谱填料 - 1. 更小的粒径更高的理论塔板数更高的反压, 更小的孔隙容量. 更大的孔隙容量 ( 校准曲线的斜率