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©2017 Waters Corporation 1 COMPANY CONFIDENTIAL
关于肽分析
沃特世消耗品部
王悦
Mar 27, 2018
©2017 Waters Corporation 2 COMPANY CONFIDENTIAL
Outline
肽分离常见问题与解决方案
肽分析应用推荐
如何保证分析的稳定
工具与资源
总结
©2017 Waters Corporation 3 COMPANY CONFIDENTIAL
Outline
肽分离常见问题与解决方案
肽分析应用推荐
如何保证分析的稳定
工具与资源
总结
©2017 Waters Corporation 4 COMPANY CONFIDENTIAL
肽的结构特点 肽是由氨基酸依次连接而成的化合物 -氨基酸是两性化合物,具有两性离子特性
增加肽的疏水保留 -低pH,抑制C端羧基解离 -0.1% TFA,pH=1.8-2.0
抑制碱性基团与硅醇基的离子交换 -离子对试剂,屏蔽碱性基团(同时也会增加反相保留) -严重降低质谱信号,且在液质上的残留效应明显
丙氨酸
人胰岛素
比伐卢定
不同的来源与指向: 肽药—发酵/合成/半合成。 分析目的在于质量研究与工艺纯化。 结构类似,同系物(杂质)多。 肽谱—蛋白水解片段,用于结构表征。 不同来源对分析时间要求不一样。
©2017 Waters Corporation 5 COMPANY CONFIDENTIAL
肽专用柱分离技术
www.waters.com/gotpep
130Å /300Å
• 相对硅胶基质峰形对称性更佳
• 300Å 适合 >5 000 Da肽
• 低pH (1.8) 或高 pH(10)
130Å
• TFA和FA皆有良好峰形
• 表面带正电荷,适合pH<5
100Å
• 硅胶基质色谱柱
• 对极性肽有更强的保留
• 提升键合技术改善峰形
专用柱概念
两性基团的存在,对填料要
求更苛刻
分离度高
专门挑选的填料—肽混标质控,分离性能
更高
重现性远高于常规柱
分离性能更稳定
保留时间重现性更好
方法转移(QC,或者不同site)更顺利
©2017 Waters Corporation 6 COMPANY CONFIDENTIAL
BEH—肽分析的经典选择
Bridged Ethanes In Silica Matrix
Polyethoxysilane (BPEOS)
Si
EtO
O
CH2 CH2
SiO
Si
EtO
OEt
Si O
O
OEt
O
Si
O
Si
OEt
O
O
OEt
Et
Et
n
+ Si
EtO
EtOCH2
EtO
CH2Si
OEt
OEt
OEt
Tetraethoxysilane (TEOS)
Bis(triethoxysilyl)ethane (BTEE)
4 Si
EtO
EtO OEtEtO
1
Anal. Chem. 2003, 75, 6781-6788
• 药典推荐 利妥昔,曲妥珠,贝伐
鉴别-肽图
• 肽药分析方法开发首选 肽药不同pH选择性优化-小分子药物经典选择 相比硅胶基质,峰形更对称
©2017 Waters Corporation 7 COMPANY CONFIDENTIAL
CSH—简化流动相开发,改善肽峰形
• 耐受性与BEH颗粒相同
• 甲酸即可保持峰形对称
LCMS灵敏度更高(肽谱)
方法使用维护简单,运行效率更高
• 峰形更窄
分离度更高(肽谱,肽药) 上样量更大,制备效率更高(肽药) 制备纯化时可直接避免引入TFA (肽药)
Peptide
150
200
250
300
350
400
450
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
Pc,4σ
Percent TFA
150
200
250
300
350
400
450
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10P
c,4σ
Percent TFA
12
10
8
6
4
2
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
Fold
Decre
ase in
MS P
eak A
rea
Percent TFA0.05
0.05
0.00
0.10
0.10
0.00
% TFA
% FA0.05
0.05
0.00
0.10
0.10
0.000.05
0.05
0.00
0.10
0.10
0.00
Low Mass LoadHigh Mass Load
CSH C18
BEH C18
A B CCSH C18
BEH C18
6 µg of mixture 0.6 µg of mixture
CSH130 C18 1.7 µm
BEH130 C18 1.7 µm
0.05 0.05
0.10 0.00
0.00 0.10
% TFA % FA
FA TFA
©2017 Waters Corporation 8 COMPANY CONFIDENTIAL
Peptide BEH C18, 130Å
0.002
0.02
0.038
0.002
0.02
0.038
7 8 9 10 11 12 13 14
0.1%TFA
Peptide HSS T3, 100Å
HSS T3 增强极性肽保留
Peptide BEH C18, 130Å
0.005
0.03
0.055
0.005
0.025
0.045
7 9 11 13 15 17
0.1% FA
Peptide HSS T3, 100Å
• 主要保留机理仍为反相
-100%水流动相时保留最强
• 减少C18覆盖率
-极性分子可以嵌入C18链之间,与烷基链和硅醇
基都发生作用,增加了选择性
-消除疏水塌陷,保证了方法的稳定性
©2017 Waters Corporation 9 COMPANY CONFIDENTIAL
Outline
肽分离常见问题与解决方案
肽分析应用推荐
如何保证分析的稳定
工具与资源
总结
©2017 Waters Corporation 10 COMPANY CONFIDENTIAL
肽图分析条件:填料孔径,柱温要求
• 对于富含脯氨酸的肽段,要得到对称的峰形,必须尝试提高柱温(60℃以上)。
*柱温不会改变其它肽段的峰形。
• 孔径的影响300Å 孔径对肽图中大分子多肽的分离效果更好
*峰B与峰C之间的区域,大分子肽段较多,300Å分离效果明显优于130Å;C峰高也明显超过130Å色谱柱
Peptide BEH C18,130 Å
Peptide BEH C18,300 Å
©2017 Waters Corporation 11 COMPANY CONFIDENTIAL
肽药与质量标准
• 50个及以下AA组成
• 更容易穿透到组织内部
• 免疫原性更小
• 研发成功率是小分子2倍
2017年10月FDA建议合成肽类药物可以按照ANDA途径申报 • 提供理化参数,一,二级结构,聚合物,生物活性数据 • 鉴定超过0.1%的所有相关杂质 • 仿制药中的杂质不得高于原研水平 • 新杂质,不得超过0.5% • 低于0.5%的新杂质,需对其进行表征,并提供完整数据,证明其对原研的活性和免疫原性风险不会产生根本影响
特立帕肽 利拉鲁肽 奈西立肽 替度鲁肽 胰高血糖素
©2017 Waters Corporation 12 COMPANY CONFIDENTIAL
肽药杂质
工艺过程杂质 • FMOC没有脱除,或产生外消旋化 • 多插入一个氨基酸,如Gly,Ala • 氧化,半胱氨酸,蛋氨酸,组氨酸,赖氨酸,色氨酸等 • 二聚,多聚体 • 合成,纯化所用试剂(TFA) • 交叉污染-清洁认证
产物稳定性杂质 • 降解-β消除 • 环缩二氨酸 • 焦谷氨酸化 • 琥珀酰亚胺
*合成杂质—去消旋,脱酰胺杂质更受重视
• HPLC-UV鉴定,tR
• MS检测-单四极杆,QDa
亮丙瑞林
FMOC
2-氯代三苯甲基氯
©2017 Waters Corporation 13 COMPANY CONFIDENTIAL
LC Separation Conditions for RP Column Screening+AutoBlend ——柱筛选(分离选择性比较),TFA体系vsFA体系,流速与柱长
LC System: - ACQUITY H-Class Bio Columns: - ACQUITY UPLC Peptide BEH C18 Column, 300Å, 1.7 µm 2.1 mm X 150 mm (P/N 186003687) - ACQUITY UPLC Peptide BEH C18 Column, 130Å, 1.7 µm 2.1 mm X 150 mm (P/N 186003556) - ACQUITY UPLC Peptide CSH C18 Column, 130Å, 1.7 µm 2.1 mm X 150 mm (P/N 186006938) - ACQUITY UPLC Peptide HSS T3 Column, 100Å, 1.8 µm 2.1 mm X 150 mm (P/N 186008756) Mobile phase A: - 0.1% formic acid in H2O - 0.1% TFA in H2O Mobile phase B: - 0.1% formic acid in Acetonitrile - 0.1% TFA in Acetonitrile Scouting Flow Rate: - 0.3 mL/min Temperature: - 60C Detection: - 214nm
Peptide CSH C18 Column, 130Å
Peptide HSS T3 Column, 100Å
©2017 Waters Corporation 14 COMPANY CONFIDENTIAL
【Case Study】Secretin-分泌素
Sequence:
– H-His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser- Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH₂ acetate salt
Aspartimide formation
MW: 3055.45 (27 aa)
pI: 9.85 (drug bank)
Purity: 87% (Bachem – requested at this purity)
©2017 Waters Corporation 15 COMPANY CONFIDENTIAL
Secretin MW: 3055.45 (27 aa)
0.002
0.02
0.038
0.002
0.02
0.038
0.002
0.02
0.038
0.002
0.02
0.038
7 9 11 13
0.005
0.025
0.045
0.005
0.03
0.055
0.005
0.035
0.065
0.005
0.025
0.045
7 9 11 13 15 17
0.1% formic acid
0.1%TFA
Peptide BEH C18, 300Å, 1.7 µm
Peptide BEH C18, 130Å, 1.7 µm
Peptide CSH C18, 130Å, 1.7 µm
Peptide HSS T3, 100Å, 1.8 µm
不同改性剂分离效果差异大 UV检测无法确认最终结构
©2017 Waters Corporation 16 COMPANY CONFIDENTIAL
MS Technology is Increasingly Being Incorporated into New Methodologies
©2017 Waters Corporation 17 COMPANY CONFIDENTIAL
Secretin MW:3055.45 (27aa)UV,0.1% FA
0.005
0.025
0.045
0.005
0.03
0.055
0.005
0.035
0.065
1
1
1,11
2
2
2
3
3
3
4
4
4
4 4
5
5
5
6
6
6
12,13
12,13
12,13
14
7
7
7
8
8
8
10,9
9
10
11
11
11,9
10
0.005
0.025
0.045
7 8 9 10 11 12 13 14 15 16 17
1
2
3
4 4 5 6 12,13 7
8
11,9
10
11
Peptide BEH C18, 300Å 1.7 µm
Peptide BEH C18, 130Å 1.7 µm
Peptide CSH C18, 130Å 1.7 µm
Peptide HSS T3, 100Å 1.8 µm
©2017 Waters Corporation 18 COMPANY CONFIDENTIAL
Impurity Retention time (min) for
Peptide BEH C18, 130A
Mass D Mass Putative identity
Peak 1 12.27 2714.601 -339.102 fragment [4-27]
Peak 2 12.05 3124.699 70.997 Ala insertion
Peak 3 12.72 3079.72 26.01 26 Da
Peak 4 12.66, 12.46 3065.70 11.99 12 Da
Peak 5 13.08 2916.58 -137.13 His deletion
Peak 6 12.72 (peak 3), 12.21 3079.66 25.96 26 Da
Peak 7 11.40 3035.70 -18.00 main peak, -18 Da, has +1 Da shift?
Peak 8 11.11 3035.70 -18.00 has +1 Da shift?
Peak 9 10.99 3190.77 137.07 main peak, His insertion
Peak 10 10.81 3190.7155 137.013 His insertion
Peak 11 10.69 3106.656 52.9535 main peak, Fe adduct, His insertion
Peak 12 13.70 2829.5496 -224.1529 Fragment [3-27], +42 Da
Peak 13 13.77 3095.67 41.9675 42Da, His deletion, Fragment [3-27]
Secretin MW:3055.45 (27aa)UV,0.1% FA
©2017 Waters Corporation 19 COMPANY CONFIDENTIAL
Sequence:
– pGlu-Pro-Ser-Lys-Asp-Ala-Phe-Ile-Gly-Leu-Met-NH2
MW: 1188.41 (11 aa)
pI: 4.23 (drug bank)
Purity: > 90
Eledoisin-章鱼唾腺精
©2017 Waters Corporation 20 COMPANY CONFIDENTIAL
Eledoisin MW: 1188.41 (11 aa) UV, 0.1% TFA
0
0.025
0.05
0
0.025
0.05
0
0.025
0.05
0
0.025
0.05
8 9 10 11 12 13 14 15 16 17 18
1
1
1
1
2,3
3
3,10,11
2
2,3
4
4
4
4
5
5
5
5
6,7
6,8
6
6 7,8
7
8 9
9
9 8
7 9
10
11
10 11
10 11
Peptide BEH C18, 300Å 1.7 µm
Peptide BEH C18, 130Å 1.7 µm
Peptide CSH C18, 130Å 1.7 µm
Peptide HSS T3, 100Å 1.8 µm
©2017 Waters Corporation 21 COMPANY CONFIDENTIAL
Eledoisin MW: 1188.41 (11 aa) LC/MS Peak identification, 0.1% TFA mobile phases
Impurity Retention time (min) for CSH
C18, 130 A Mass D Mass Putative identity
Peak 1 12.11 1302.634 115.023 main peaks, Asp insertion
1116.552 -71.059 Ala insertion
Peak 2 12.43 1104.55 -83.06 main peaks, unknown 1284.62 97.01 Pro insertion
Peak 3 12.61 1258.651 71.040 Ala insertion 1100.56 -87.05 Ser deletion
Peak 4 12.95 1188.5908 0.9794 NH2->OH Peak 5 11.84 1187.611 0 racemics Peak 6 11.73 1274.6318 87.0204 Ser insertion Peak 7 11.61 1244.6226 57.0112 Gly insertion, or tBu
Peak 8 11.43 874.4775 -313.1339 Fragment [1-3] 896.4481 -291.1633 unknown
Peak 9 11.24 1090.5336 -97.0778 Pro deletion
Peak 10 12.64 1165.5852 -22.0262 unknown 1143.6005 -44.0109 Asp -> Ala, Met -> Ser
Peak 11 12.65 1100.5624 -87.049 Ser deletion
©2017 Waters Corporation 22 COMPANY CONFIDENTIAL
Eledoisin-MW:1188.41(11aa)UV,0.1%TFA:流速与梯度
0
0.025
0.05
9 10 11 12 13 14 15
0
0.02
0.04
13 14 15 16 17 18 19 20 21 22
Peptide CSH C18, 130 A, 1.7 um 2.1 x 150 mm 0.3 ml/min, 18-38B in 20 min 2 ug inj.
Peptide CSH C18, 130 A, 1.7 um 2.1 x 150 mm 0.2 ml/min, 20-28B in 30min 2 ug inj.
Post p/v: 2.76
Pre p/v: 1.22
Pre p/v: 1.05
Post p/v: 3.00
Effect of Flow Rate and Gradient Slope
©2017 Waters Corporation 23 COMPANY CONFIDENTIAL
Eledoisin -MW:1188.41(11aa)UV,0.1%TFA:柱长
0
0.02
0.04
0
0.02
0.04 0
0.02
0.04
2.1 x 50 mm 20-28 B in 10 min 0.67 ug injection
2.1 x 75 mm 20-28 B in 15 min 1 ug injection
2.1 x 100 mm 20-28 B in 20 min 1.34 ug injection
2.1 x 150 mm 20-28 B in 30 min 2 ug injection
0
0.02
0.04
5 10 15 20 25
Peptide CSH C18 Column, 130Å, 1.7 µm
Effect of Column Length
©2017 Waters Corporation 24
UPLC Through HPLC Scalability:方法转换 Eledoisin UV, 0.1% TFA
0
0.025
0.05
25.5 26.5 27.5 28.5 29.5 30.5
0
0.025
0.05
99 104 109 114 119
Peptide CSH C18, 130 A, 1.7 um 2.1 x 150 mm 0.2 ml/min, 16-26 B in 30 min 1 ug injection
Peptide CSH C18, 130A, 3.5 um 2 x 4.6 x 150 mm 0.47 ml/min, 16-26 B in 124 min 9.6 ug injection
0
0.025
0.05
53 56 59 62
Peptide CSH C18, 130 A, 2.5 um 4.6 x150 mm + 4.6 x 75 mm 0.65 ml/min, 16-26 B in 66 min 7.2 ug injection
UPLC
UHPLC
HPLC
©2017 Waters Corporation 25 COMPANY CONFIDENTIAL
Outline
肽分离常见问题与解决方案
肽分析应用推荐
如何保证分析的稳定
工具与资源
总结
©2017 Waters Corporation 26 COMPANY CONFIDENTIAL
To Help Ensure Consistency, Every Batch of Peptide BEH, CSH, and HSS Material is QC Tested with the Tryptic Digest of Cytochrome c
©2017 Waters Corporation 27 COMPANY CONFIDENTIAL
Batch to Batch / Column to Column Consistency CSH130 C18 Batch Reproducibility (Formic Acid)
ACQUITY UPLC® Peptide CSH 130 C18, 1.7 µm XSelect® Peptide CSH 130 C18, 2.5 µm
Date Acquired 12/14/2012 10:32:58 PM EST; Batch_Number 107
Date Acquired 7/12/2013 10:07:36 PM EDT; Batch_Number 121
Date Acquired 7/26/2014 9:54:35 AM EDT; Batch_Number 125
Date Acquired 10/20/2015 6:13:46 PM EDT; Batch_Number 134
Date Acquired 7/29/2016 1:48:35 PM EDT; Batch_Number 140
AU
0.00
0.50
1.00
AU
0.10
0.90
AU
0.00
0.50
1.00
AU
0.00
0.20
0.40
0.60
AU
0.00
0.50
1.00
Minutes
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
2012: Batch #107
2013: Batch #121
2014: Batch #125
2015: Batch #134
Date Acquired 12/19/2012 3:39:30 PM EST; Batch_Number 101
Date Acquired 1/9/2013 4:50:42 PM EST; Batch_Number 102
Date Acquired 10/7/2015 11:18:46 AM EDT; Batch_Number 133
Date Acquired 3/10/2016 11:08:32 AM EST; Batch_Number 137
Date Acquired 2/28/2017 5:57:42 PM EST; Batch_Number 142A
U
0.00
0.50
AU
0.00
0.50
1.00
AU
0.00
0.20
0.40
0.60
AU
0.00
0.20
0.40
0.60
AU
0.00
0.50
1.00
Minutes
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
2016: Batch #137
2012: Batch #101
2013: Batch #102
2015: Batch #133
• Batch-to-Batch Reproducibility
• Cytochrome C Digest, 0.1% Formic Acid
• Each new column will perform comparably to one previously used
• COA and high quality analytical standards are available to customer
©2017 Waters Corporation 28 COMPANY CONFIDENTIAL
3 different batches of bulk media
Also exists for Peptide Columns
Same column dimension (mostly 2.1 x 150 mm, 1.7 µm)
Mimic real life of a method during your Method Validation
Contact us for different dimensions
Method Validation Kits Minimize Risk after Method Validation
AU
-0.010
0.000
0.010
AU
-0.010
0.000
0.010
AU
-0.010
0.000
0.010
Minutes
1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20
Batch 1
Batch 2
Batch 3
©2017 Waters Corporation 29 COMPANY CONFIDENTIAL
为了确保分析方法的持续的可靠性与重现性,有必要测试和评估来自
不同批次填料的色谱柱!!
填料批次!!
©2017 Waters Corporation 30 COMPANY CONFIDENTIAL
Outline
肽分离常见问题与解决方案
肽分析应用推荐
如何保证分析的稳定
工具与资源
总结
©2017 Waters Corporation 31 COMPANY CONFIDENTIAL
资源
©2017 Waters Corporation 32 COMPANY CONFIDENTIAL
沃特世耗材中心:沃!助力讲堂(2018)
©2017 Waters Corporation 33 COMPANY CONFIDENTIAL
总结
肽反相分离的关键是尽可能消除两性基团与填料的相互作用
-流动相中加TFA是常见的优化方式
-杂化颗粒拖尾明显优于硅胶颗粒,杂化表面带电颗粒(CSH)更是大大改善了肽的峰形与载量,尤
其当分析检测需要在甲酸体系或者弱缓冲盐体系时。
BEH色谱柱已成为蛋白药物肽图分析的经典选择
肽药质量标准要求越来越高,分析挑战 -UPLC越来越多用于肽药的质量分析,提高鉴定准确度,更适用于工艺的高通量要求
-制备色谱是肽药的关键手段,用于杂质鉴定,定制肽纯化。严格的质控简化了分析-制备的放大,
沃特世特有的OBD制备柱技术提高了制备的效率
©2017 Waters Corporation 34 COMPANY CONFIDENTIAL
谢谢!