admin 管理员组文章数量: 1184232
2024年12月27日发(作者:linux驱动)
HPLC columns
MAbPac SEC-1 columns
Product manual
2
Contents
Introduction 4
Introduction to the MAbPac SEC-1 column 4
MAbPac SEC-1 operating limits and specifications 4
Formats of the MAbPac SEC-1 columns 4
Getting started: Step-by-step procedure 5
Visually inspect the column 5
Mobile phase selection 5
Set-up the LC system 5
Operational guidelines 5
Reproduce the chromatogram in the lot validation report 5
Real sample analysis 5
Column care 9
Column storage 11
Operating pH range: pH 2.5 to 7.5 26
Operating temperature limit: up to 30 ºC 36
Pressure limit 11
Flow rate 26
Injection volume 26
Column washing procedure 36
Example applications 9
Separation of mAbs and their aggregates 11
Separation of mAb fragments 26
Separation of mAbs using ionic strength eluents 36
Separation of mAbs using volatile buffers 11
Ruggedness of MAbPac SEC-1 26
Aggregate analysis 36
Frequently asked questions 47
Ordering information 47
Introduction
Introduction to the
MAbPac SEC-1 column
Thermo Scientific
™
MAbPac
™
SEC-1 is a size exclusion
chromatography (SEC) column specifically designed for
separation and characterization of monoclonal antibodies (mAbs).
The stationary phase is designed to handle different eluent
conditions containing both high and low ionic strength mobile
phases, as well as mass spectrometry friendly volatile eluents.
SEC is a common chromatographic technique for separating
biomolecules based on their sizes. On the SEC column, very
large analytes (>1000 kDa) are excluded by the pores thus eluting
in the void, whereas smaller analytes (<1000 Da) pass through
the pores and elute according to their sizes – larger analytes elute
earlier than smaller analytes.
The MAbPac SEC-1 is based on high-purity, spherical, porous
(300 Å), 5 μm silica particles that are covalently modified with a
proprietary diol hydrophilic layer. This proprietary process brings
an extremely low level of non-desired interaction sites. The 7.8
mm and 2.1 mm small internal diameter (I.D.) columns are packed
into stainless steel housing while the 4.0 mm I.D. format columns
are packed into PEEK housing.
MAbPac SEC-1 operating limits
and specifications
Table 1. Operating conditions
Flow rate range (recommended)
Shipping solution/
Long term storage solution
Typical buffers
Solvents compatibility
Detergent compatibility
Temperature range
Pressure limit
pH range
760 – 1,000 μL/min for the 7.8 mm I.D. columns
200 – 300 μL/min for the 4.0 mm I.D. columns
50 – 75 μL/min for the 2.1 mm I.D. columns
20% acetonitrile in deionized water
Phosphate buffer with NaCl, e.g. 50 mM phosphate buffer (pH 6.8) + 0.3 M NaCl
Good’s buffer with NaCl, e.g. 20 mM MES buffer (pH 6.1) + 0.3 M NaCl
Ammonium formate or ammonium acetate solutions, pH 5 – 7
Compatible with 100% organic solvents
Compatible with 0.1% SDS, though binding will be irreversible and it is recommended that
the column be dedicated to this application
20 – 30 °C
1,000 psi for 300-mm long columns
600 psi for 150-mm long columns
2.5 – 7.5
3
Introduction
Table 2. Physical characteristics
Bonding chemistry
Silica substrate
Particle size
Pore size
Column housing
Separation range for globular proteins
Exclusion limit for globular proteins
(continued)
Diol
Spherical, high-purity porous silica
5 μm
300 Å
PEEK for 4.0 mm I.D. columns
SST for 7.8 mm and 2.1 mm I.D. columns
10,000 - 1,000,000 Dalton
>1,000,000
Calibration curve
The MAbPac SEC-1 column has a wide molecular operating
range, as shown in the molecular weight calibration curve.
MAbPac SEC-1 column, 4 × 300 mm
Cat. no 074696
50 mM sodium phosphate buffer
Mobile phase
(ph 6.8) + 0.3M NaCl
Flow rate0.20 mL/min
Inj. volume5 µL
Temperature25 °C
DetectionUV, 220 nm
1. Thyroglobulin
2. γ-Globulin
3. BSA
4. Ovalbumin
Analytes
5. Trypsin inhibitor
6. Myoglobin
7. Ribonuclease A
8. CytchromeC
10,000,000
1,000,000
M
o
l
e
c
u
l
a
r
w
e
i
g
h
t
100,000
Protein
γ-Globulin aggregate
Thyroglobulin dimer
Thyroglobulin
γ-Globulin dimer
BSA dimer
BSA
Ovalbumin
Trypsin inhibitor
Myoglobin
Ribonuclease A
Cytochrome C
*Tentative, not actual
Retention
time (min)
7.6
8.0
9.0
10.3
11.4
12.6
13.3
14.1
14.4
14.7
14.6
MW (kDa)
7,000*
1,338
669
300
134
67
43
22
18
14
12
10,000
1,000
67891
Retention time (min)
4
Getting started:
Step-by-step procedure
Thermo Fisher Scientific recommends that you perform an
efficiency test on your MAbPac SEC-1 column before use.
The purpose of column performance validation is to ensure
no damage has occurred during shipping. Steps 1 – 5 below
outline the necessary steps to perform this validation test.
Test the column using the conditions described on the Quality
Assurance (QA) report enclosed in the column box. Repeat the
test periodically to track the column performance over time.
Note that slight variations may be found on two different HPLC
systems due to system electronic, hardware, plumbing, operating
environment, reagent quality, column conditioning, and operator
technique.
Visually inspect the column
Report any visible damage upon receiving the column to Thermo
Fisher Scientific immediately. Depending upon the nature of the
damage, we may request that you return the damaged column
back to us for a replacement column.
Mobile phase selection
The MAbPac SEC-1 column can be used with a variety of mobile
phases. The columns are normally used with 20 – 100 mM buffer
(pH 5.0 – 7.5) containing 0.1 – 0.3 M salt. A typical mobile phase
for protein separations is 50 mM phosphate buffer (pH 6.8) + 0.3
M NaCl. Ammonium formate and ammonium acetate buffer can
also be used when coupling the column to a mass spectrometer
and a volatile buffer is needed, in which case 100 mM buffer
concentration is a good starting point. It is highly recommended
that the mobile phase is pre-filtered with a 0.2 μm pore size
membrane filter, or/and an in-line filter containing installed
membrane on HPLC system.
Set up the LC system
Use a standard LC system equipped with a LC pump, a column
oven, a UV detector (210 – 220 nm and/or 280 nm) and an
injector (or an autosampler). It is required that the system be
optimized for low dead volume; usage of I.D. tubing (especially
between the injector and detector) and a proper detector flow
cell is required for best results. See Table 1 for recommend
chromatographic set up. The system should be thoroughly
primed before use. It is recommended the column is run at room
temperature (20 to 30 °C) to achieve better column lifetime.
Table 2. Recommended chromatographic setup
Column ID (mm)2.14.0
7.8
Flow rate
(μL/min)
50 – 75200 – 300760 – 1,000
UV flow cell
Micro
Semi-micro
Analytical
(180 nL)
(2.5 μL)
(11 μL)
Tubing ID (μm)50 – 7575 – 100150 – 250
Sample
injection size (μL)
1 – 25 – 1020 – 50
Operational guidelines
•
Avoid any sudden pressure surge
•
Operate within column specifications (see “Operating
conditions”)
•
Follow the direction of flow that is marked on the column
•
Column conditioning is recommended upon initial column
use: inject 4 successive injections of the sample, or 100 μg
BSA, or other proteins of choice
•
Reverse flow should be avoided except for removal of inlet
blockage (see “Column care”)
•
It is recommended that MAbPac SEC-1 columns be stored in
20% acetonitrile in deionized water. If stored in an aqueous
buffer solution, made sure the pH of storage solution is
between 4 and 6 and contain 0.05% sodium azide.
5
Getting started:
Step-by-step procedure
(continued)
•
Buffers are recommended during use of this column: typically
a combined concentration of 20 mM (to avoid any possible
undesirable interactions with the packing material).
•
The use of a guard column is recommended when injecting
dirty samples to protect the analytical column and to extend
the column lifetime.
•
If necessary, run several blanks between injections to
minimize carry-over.
•
Salt concentration should not exceed 0.5 M.
•
Solvent compatibility: This column is compatible with 100%
organic solvents (i.e., acetonitrile and methanol). However,
take precautions not to precipitate any salts used in the
mobile phase present on the column.
•
Detergent compatibility: Compatible with 0.1% SDS, though
binding will be irreversible and it is recommended that the
column be dedicated to this application.
Reproduce the chromatogram in the lot
validation report
Perform the column QA test using the conditions described in the
QAR, and compare the result with the reported values.
The column should be fully equilibrated before any injection.
At least three injections should be made to assess the
reproducibility. Once you are satisfied with the column
performance report result, proceed to the next step.
Note
Due to various reasons, such as difference of LC
systems, mobile phases, etc, you may observe
somewhat different separation from that in the report.
6
Real sample analysis
Once the column performance is satisfactorily confirmed in
“Reproduce the chromatogram in the lot validation report", the
column is ready for real sample analysis.
Note
It is recommended that the column performance test
be performed periodically to monitor the condition of
the column. Please compare it to initial performance
test to note any changes. If the results are comparable
and you are satisfied with the column performance,
continue to use the same column for your applications.
If you are not satisfied please proceed to the column
washing procedure (see “Column washing procedure”).
Column care
Column storage
The column can be stored in the mobile phase for short-
term storage. For long-term storage (more than 5 days), it is
recommended to store the column in a solution containing 20%
acetonitrile in deionized water.
Operating pH range: pH 2.5 to 7.5
The column lifetime is heavily affected by the pH of the buffer
and other chromatographic conditions. To obtain better column
lifetime, it is recommended to use mobile phases with pH
between 5.0 and 7.0.
Operating temperature limit: up to 30
o
C
Based on our experimental data, this column can be used at 30 °C.
The typical operating temperature for most applications is in the
range of 20 – 25 °C.
Pressure limit: 1,000 psi for 300 mm long
column; 600 psi for 150 mm long column
It is extremely important not to impose a sudden column
pressure surge. Although the column pressure is rated up to
1,000 psi for a 300 mm long column, the typical operating
pressure at 300 μL/min should not exceed 800 psi.
Flow rate
Please refer to Table 1 for recommended flow rate. Please note
that the analyte efficiency may be affected at a higher flow rate.
Injection volume
Please refer to Table 1 for recommended injection volume. Large
injection volume may affect resolution.
Column washing procedure
Note
The following procedure is designed for the 4.0 mm
I.D. column. Please adjust the flow rate for different
I.D. columns (760 μL/min for 7.8 mm I.D., or 50 μL/min
for 2.1 mm I.D.).
Particulates in the sample or the mobile phase will
plug the column inlet frit. If solvent flow appears to be
restricted (high column back-pressure), check first to
see that solvent flow is unobstructed up to the column
inlet. If the column has the restriction, there may be
particulate matter on the inlet frit. An attempt should
be made to remove any inlet debris by back-flushing
25 to 30 mL of mobile phase through the column
(at 200 μL/min). If this fails to return the column to near
its original operating pressure, consider replacing the
column.
Make sure that the mobile phases and samples are
free of such particulates by filtering the eluents and
samples with a 0.2 μm filter prior to use. In the event
that column washing/cleaning is needed, the following
procedure can be used as a guideline:
1. Wash the column with 100% D.I. water at 200 μL/min for 30
minutes.
2. Wash the column with 80% methanol at 200 μL/min for 30
minutes.
3. Wash the column with 0.1 M ammonium acetate pH 5.0 buffer
at 200 μL /min for 30 minutes.
4. Wash the column with the mobile phase at 200 μL/min for at
least 30 minutes.
7
Example applications
Separation of mAbs and their
aggregates
Size-exclusion chromatography (SEC) is a well-accepted
technique for the detection and accurate quantification of protein
aggregates in biological drug products. The MAbPac SEC-1
column is specially designed for analysis of mAbs and their
aggregates (Figure 1a, 1b and 1c). When mAbs are produced
from mammalian cell culture, it may contain significant amounts
U
50
A
m
mAbmonomer
40
30
20
Aggregates
10
0
Time (min)
02.5
57.5
1012.5
1518
Figure 1a. Analysis of mAb and aggregates (7.8 × 300 mm)
150
U
A
m
mAbmonomer
125
100
75
50
Aggregates
25
0
Time (min)
02.5
57.5
1012.5
1518
Figure 1b. Analysis of mAb and aggregates (4.0 × 300 mm)
8
of dimers, trimers and other higher order aggregates. The
formation of aggregates may originate from elevated temperature,
shear strain, surface adsorption, high protein concentration
or other unknown reasons. Studies show that the aggregates
present in drug products can cause severe immunogenic and
anaphylactic reactions.
MAbPac SEC-1 column, 5 μm, 7.8 × 300 mm
Cat. no088460
Mobile phase
50 mM sodium phosphate pH 6.8,
in 300 mM NaCl
Flow rate760 μL/min
Inj. volume10 µL
Temperature30 °C
Detection280 nm
Sample:mAb (1 mg/mL)
MAbPac SEC-1 column, 5 μm, 4.0 × 300 mm
Cat. no074696
Mobile phase
50 mM sodium phosphate pH 6.8,
in 300 mM NaCl
Flow rate200 μL/min
Inj. volume5 µL
Temperature30 °C
Detection280 nm
Sample:mAb (1 mg/mL)
Example applications
(continued)
70
U
A
m
mAb monomer
60
50
40
30
20
Aggregates
10
0
Time (min)
02.5
57.5
1012.5
1518
Figure 1c. Analysis of mAb and aggregates (2.1 × 300 mm)
Separation of mAb fragments
Full characterization of mAb includes determination of mass of
the mAb fragments, such as heavy chain (HC) and light chain
(LC) generated by reduction of inter chain disulfide bonds, as well
as Fab and Fc generated by papain digestion. Using denaturing
eluent containing 20% acetonitrile, 0.1% TFA, and 0.05%
formic acid, SEC enables analysis of mAb (Figure 2a), baseline
separation of HC and LC (Figure 2b), as well as partial separation
of Fab and Fc (Figure 2c). It serves as a platform method for mAb
fragment analysis. In addition, this eluent is compatible with direct
mass spectrometry detection.
MAbPac SEC-1 column, 5 μm, 2.1 × 300 mm
Cat. no088789
Mobile phase
50 mM sodium phosphate pH 6.8,
in 300 mM NaCl
Flow rate50 μL/min
Inj. volume1 µL
Temperature30 °C
Detection280 nm
Sample:mAb (1 mg/mL)
9
Example applications
100
mAb
A
50
(continued)
-10
70
HC
m
A
U
LC
B
-10
100
Fab
C
50
Fc
-10
3.07.512.5
Time (min)
Figure 2. mAb and mAb fragments analysis using denaturing eluent
17.5
25.0
MAbPac SEC-1 column, 5 μm, 4.0 x 600 mm
Cat. no074696
Mobile phase 20% acetonitrile, 0.1% FA, 0.05% TFA
Flow rate200 μL/min
Inj. volume5 µL
Temperature30 °C
Detection280 nm
A: mAb
Sample:
B: mAb reduction by DTT
C: mAb digestion by papain
Separation of mAbs using high and low
ionic strength eluents
The MAbPac SEC-1 column utilizes a diol hydrophilic layer
prepared by a proprietary process and results in extremely low
level of non-desired interaction sites. Combined with the use of
the non-metal and bio-compatible PEEK column housing, it is
ideal for separating monoclonal antibodies, including monomer,
200
aggregates and mAb fragments, by providing excellent peak
shapes and efficiency for mAbs under both high and low salt
conditions. As shown in Figure 3, separation of mAb with MAbPac
SEC-1 under both high salt condition (0.3 M NaCl in 50 mM
phosphate buffer, upper C-gram) and low salt condition (0.15 M
NaCl in 10 mM phosphate buffer, lower C-gram), displayed good
peak shape and peak efficiencies.
0.3 M NaCl in
50 mM phosphate
bu
版权声明:本文标题:MAbPac SEC-1 柱产品手册说明书 内容由网友自发贡献,该文观点仅代表作者本人, 转载请联系作者并注明出处:http://www.roclinux.cn/p/1735341516a1650168.html, 本站仅提供信息存储空间服务,不拥有所有权,不承担相关法律责任。如发现本站有涉嫌抄袭侵权/违法违规的内容,一经查实,本站将立刻删除。
更多相关文章
精进:如何成为一个很厉害的人--作者:采铜
精进:如何成为一个很厉害的人 作者:采铜 文章目录 精进:如何成为一个很厉害的人序 用更勇敢的方式去生活01 时间之尺 我们应该怎样对待时间活在“全部的现在” 从当下出发&
Transformer作者:指令型智能体的构建之法
来源 | The Robot Brains Podcast OneFlow编译 翻译|徐佳渝、贾川、杨婷 2017年,Google发布的《Attention Is All You Need》论文提
【转载】bat批处理教程 作者:hipi 日期:2006-11-05
编者注:这是我十多年前学习批处理时找到的一篇入门教程。原作者以聊天的语气讲解批处理,算得上是一篇好文章。时过境迁,我已找不到原博文地址了,但文字版的博文
Flutter瘦身减脂运动锻炼APP源码|ChatGPT集成|多语言支持|作者亲测有效
FitBot应用介绍 概述:FitBot是一款集成了Laravel管理后端的Flutter瘦身减脂运动辅助APP源码,支持多语言和ChatGPT集成,兼容Android 14和Flutter 3.19.x。无论您是健身爱好者、初学者,还是
品牌机重装系统:一段充满挑战与成就的技术旅程
.code-box {position: relative;background-color: #2d2d2d;border: 1px solid #444;border-radius: 6px;padding: 20px;margin:
没有NVIDIA控制面板的日子:一段电脑爱好者的曲折探索
图标消失的那个下午 我记得很清楚,那是个周日的下午,阳光透过窗帘洒在桌面上。我刚刚升级了最新的游戏,兴冲冲地点开设置,想要把图形特效拉到极致。然而,当我习惯性地右键点击桌面时,心里咯噔一下——本该出现的“NVIDIA控制面板”选项,不
告别复杂操作,轻松实现DBF文件的快速读取
有个需求,在c#前端读取dbf中的数据。网上搜索到的大部分都是配置ODBC方式的连接去读取的,还得安装驱动。因为最终客户端不能要求每个客户都去安装foxpro驱动,故此此处实现直接用代码去读取dbf数据。 using System;
让硬件精灵帮你:一键识别和匹配驱动程序
简介:”硬件精灵Unknown Device Identifier”是一款专用于识别和解决电脑中未知设备问题的实用工具,广泛应用于系统维护与硬件升级场景。当操作系统因缺少或不兼容驱动而无法识别硬件时,该软件通过扫描硬件ID和供应商ID
电脑突然哑巴了?这四招让您的声音再次洪亮
相信不少朋友都遇到过这种情况:正想看电影、打游戏或者开视频会议,突然发现电脑没声音了。今天我就把我总结的最常见原因和4个超实用的解决方法都分享给大家,保证你看完就能自己搞定! 一、为什么电脑会突然没声音? 让你的电脑没
DIY玩家注意啦!B650M声卡驱动解决音响无声音的大法已出炉
我的这个问题是驱动问题 如果你是个倒霉蛋是硬件问题这个文章无法解决 前提 Win11系统 主板类型 华硕 PRIME B650M-K 问题 音响外放没有声音但是 其他状况正常 本人问题存
UEFI技术宝典:新手到高手的完美跳板
作者简介罗冰:系统安全(特别是物理隔离领域)专家,主导开发网络隔离卡、双网隔离机、国产隔离系统、单向光传输等各类安全产品,拥有十几项发明和实用新型专利。致力于UEFI技术的研究、实践和推广,在CSDN和知乎上设有“UEF
一步到位:从MSDN获取并熟练安装纯净Windows系统的全面教程!
纯净的windows系统在哪下载?其实下载纯净系统无非就两种方式,一种是微软官方下载,另一个就是msdn网站上下载。MSDN并不是微软的官方网站,它是个人性质的原版软件信息收录站点,但该网站上提供的都是原版的系统文件,可放心下载使用。
ACHIEVE MAXIMUM PERFORMANCE ON YOUR PC USING THE OFFICIAL MSDN PURE WINDOWS INSTALLATION PROCESS
纯净的windows系统在哪下载?其实下载纯净系统无非就两种方式,一种是微软官方下载,另一个就是msdn网站上下载。MSDN并不是微软的官方网站,它是个人性质的原版软件信息收录站点,但该网站上提供的都是原版的系统文件,可放心下载使用。
一不小心就挂了?QQ音乐提示没声卡,联想小新用户必看指南!
(着急的读者可以直接拉到最后,看看我使用的解决方法,中途是一些其他人成功的一些解决方案) 今天打开电脑,突然发现扩音器没声音了。放音乐提示我检查声卡是否安装。 在网上搜索,发现,有两种可能,一个是驱动问题,一个是声卡松
游戏体验大提升?揭秘破解卡顿、闪退的秘密,让你的显卡焕然新生!
显卡错误代码43,玩游戏卡顿,电脑花屏,进入不了游戏等,其问题的核心多半是显卡驱动问题。 出了驱动外,显卡过热也会导致这些问题,显卡过热,显卡风扇不转,显卡供电不足等出现的情况也不是没有。 通用解决办法 这个方
Win1803下想彻底关机?先来一次Intel MEI的清零操作!
电脑型号:戴尔 灵越 15 7000系列 Inspiron 7537 2018年5月8日更新,Windows 10 更新到1803后会自动更新Intel MEI到最新版本,可能会再次出现无法彻底关机或睡眠无法唤醒的情况。
实战攻略:让你不再被Realtek HD Audio Driver的安装困扰
解决安装Realtek HD Audio Driver失败的方法今天是郁闷惨了,先是电脑由于ntoskrnl.exe文件丢失或损坏迫使我重装系统,重装系统后又出现声卡驱动无法安装,安装若干遍声音驱动都提示“安装Realtek H
发表评论