MAbPac SEC-1 柱产品手册说明书

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

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