Beckman Coulter PA 800 plus User guide

A51969AA
April 2009
Beckman Coulter, Inc.
4300 N. Harbor Blvd.
Fullerton, CA 92835
Application Guide
PA 800 plus
Pharmaceutical Analysis
System
Carbohydrate Labeling and Analysis

Application Guide
PA 800 plus Pharmaceutical Analysis System
Carbohydrate Labeling and Analysis
A51969AA (April 2009)
Copyright © 2009 Beckman Coulter, Inc.
Beckman Coulter, Inc. grants a limited non-exclusive
license to the owner or operator of a PA 800 plus
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of any portion or all of the online help and electronic
documents shipped with the PA 800 plus instrument.
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• Beckman Coulter®
• 32 Karat™
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A51969AA iii
Revision History
Initial Issue, A51969AA, April 2009
32 Karat Software version 9.1
PA 800 plus Software version 1.1
PA 800 plus Firmware version 9.0

A51969AA
iv
Revision History

A51969AA v
Safety Notices
Symbols and Labels
Introduction
The following is a description of symbols and labels used on the Beckman Coulter PA 800 plus
Pharmaceutical Analysis System or shown in this manual.
WARNING
If the equipment is used in a manner not specified by Beckman Coulter, Inc., the
protection provided by the instrument may be impaired.
General Biohazard Symbol
This caution symbol indicates a possible biohazard risk from patient specimen contamination.
Caution, Biohazard Label
This caution symbol indicates a caution to operate only with all covers in position to decrease risk
of personal injury or biohazard.

A51969AA
vi
Safety Notices
Symbols and Labels
Caution, Moving Parts Label
This caution symbol warns the user of moving parts that can pinch or crush.
High Voltage Electric Shock Risk Symbol
This symbol indicates that there is high voltage and there is a risk of electric shock when the user
works in this area.
Class 1 Laser Caution Label
A label reading “THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE
DATE OF MANUFACTURE” is found near the Name Rating tag. The laser light beam is not visible.
Sharp Object Label
A label reading “CAUTION SHARP OBJECTS” is found on the PA 800 plus.
CAUTION
PARTS MOVE
AUTOMATICALLY
A015081L.EPS
144557-AB
A016352L.EPS
A016350L.EPS
CLASS 1 LASER PRODUCT
THIS PRODUCT CONFORMS TO
APPLICABLE REQUIREMENTS OF
21 CFR 1040 AT THE DATE OF
MANUFACTURE.
MANUFACTURED:
726024-C
CAUTION
SHARP OBJECTS
A16558-AA
A016351L.EPS

A51969AA vii
Safety Notices
Symbols and Labels
Recycling Label
This symbol is required in accordance with the Waste Electrical and Electronic Equipment (WEEE)
Directive of the European Union. The presence of this marking on the product indicates:
1. The device was put on the European Market after August 13, 2005.
2. The device is not to be disposed of via the municipal waste collection system of any member
state of the European Union.
It is very important that customers understand and follow all laws regarding the proper
decontamination and safe disposal of electrical equipment. For Beckman Coulter products bearing
this label, please contact your dealer or local Beckman Coulter office for details on the take back
program that facilitates the proper collection, treatment, recovery, recycling, and safe disposal of
this device.
Disposal of Devices Containing Mercury Components
This product contains a mercury-added part. Recycle or dispose of according to local, state, or
federal laws. It is very important that you understand and comply with the safe and proper disposal
of devices containing mercury components (switch, lamp, battery, relay, or electrode). The mercury
component indicator label can vary depending on the type of device.

A51969AA
viii
Safety Notices
Symbols and Labels
Restriction of Hazardous Substances (RoHS) Labels
These labels and materials declaration table (the Table of Hazardous Substance's Name and
Concentration) are to meet People's Republic of China Electronic Industry Standard SJ/T11364-2006
"Marking for Control of Pollution Caused by Electronic Information Products" requirements.
RoHS Caution Label
This logo indicates that this electronic information product contains certain toxic or hazardous
elements, and can be used safely during its environmental protection use period. The number in the
middle of the logo indicates the environmental protection use period for the product. The outer
circle indicates that the product can be recycled. The logo also signifies that the product should be
recycled immediately after its environmental protection use period has expired. The date on the
label indicates the date of manufacture.
RoHS Environmental Label
This logo indicates that the product does not contain any toxic or hazardous substances or
elements. The "e" stands for electrical, electronic and environmental electronic information
products. This logo indicates that this electronic information product does not contain any toxic or
hazardous substances or elements, and is green and is environmental. The outer circle indicates
that the product can be recycled. The logo also signifies that the product can be recycled after being
discarded, and should not be casually discarded.

A51969AA ix
Safety Notices
Symbols and Labels
Alerts for Warning, Caution, Important, and Note
WARNING
WARNING indicates a potentially hazardous situation which, if not avoided, could
result in death or serious injury. The warning can be used to indicate the
possibility of erroneous data that could result in an incorrect diagnosis (does not
apply to all products).
CAUTION
CAUTION indicates a potentially hazardous situation, which, if not avoided, may
result in minor or moderate injury. It may also be used to alert against unsafe
practices. The caution can be used to indicate the possibility of erroneous data
that could result in an incorrect diagnosis (does not apply to all products).
IMPORTANT IMPORTANT is used for comments that add value to the step or procedure being performed.
Following the advice in the IMPORTANT notice adds benefit to the performance of a piece of equipment
or to a process.
NOTE NOTE is used to call attention to notable information that should be followed during installation, use,
or servicing of this equipment.

A51969AA
x
Safety Notices
Symbols and Labels

xi
Contents
Revision History, iii
Safety Notices,v
Symbols and Labels,v
Introduction,v
General Biohazard Symbol,v
Caution, Biohazard Label,v
Caution, Moving Parts Label,vi
High Voltage Electric Shock Risk Symbol,vi
Class 1 Laser Caution Label,vi
Sharp Object Label,vi
Recycling Label,vii
Disposal of Devices Containing Mercury Components,vii
Restriction of Hazardous Substances (RoHS) Labels,viii
RoHS Caution Label,viii
RoHS Environmental Label,viii
Alerts for Warning, Caution, Important, and Note,ix
Carbohydrate Labeling and Analysis Kit,1
Overview,1
Safety Information,1
General Precautions,2
Materials and Reagents,2
Contents of this Kit,2
Materials Needed but Not Provided in Kit,2
Storage Conditions,3
Overview of the Procedure,3
Releasing Oligosaccharides From Glycoproteins,3
Enzymatic Release of the N-Linked Oligosaccharides,4
Working with the Supernatant,4
Principle of the Labeling Method,4
Preparing the Labeling Reagents,5
Choosing the Proper Fluorophore,5

xii
Contents
Preparing the Reagents,5
Preparing the Standards,6
Performing the Labeling Reaction,6
Preparing the PA 800 Instrument,8
Installing and Pre-conditioning the N-CHO Capillary,9
Clean the Interface Block,10
Insert the Cartridge,10
Preparing the Buffer Trays,10
Sample Vial Setup,12
Running the Samples,13
Initial Conditions,13
LIF Detector Initial Conditions,14
Time Program,15
Evaluation of Results,17
System Shutdown and Capillary Storage,18
Short Term Storage (<24 hours) of Capillary,18
Long Term Storage (>24 hours) of Capillary,18
Troubleshooting Procedures,19
Appendix,20
LIF Detector Calibration Procedure,20
Setting the Calibration Corrector Factors (CCFs),20
Automatic Calibration,21
CCF Troubleshooting Procedures,24
CCF <1,24
CCF >1,24
No Step Change Detected,24

A51969AA 1
Carbohydrate Labeling and Analysis Kit
Overview
Beckman Coulter’s Carbohydrate Labeling & Analysis Kit uses capillary electrophoresis to separate
and quantify oligosaccharides released from glycoproteins. This kit has been developed to work
with the PA 800 plus Pharmaceutical Analysis System, using the 488 nm laser and the 520 nm
emission filter.
This kit contains the reagents, buffer and coated capillaries required to label, separate and quantify
oligosaccharides. This kit also provides a glucose size marker for relative size determination and a
maltose standard for quantitation and mobility characterization of the released oligosaccharides.
For further information, please refer to the following publication:
Quantitative Analysis of Sugar Constituents of Glycoproteins by Capillary Electrophoresis, Fu Tai, A.
Chen, Thomas S. Dobashi and Ramon A. Evangelista, “Glycobiology”, Vol. 8, No. 11, pp. 1045-1052,
1998.
Safety Information
NOTE This application guide has been validated for use in the PA 800 Enhanced Protein Characterization and
the PA 800 plus Pharmaceutical Analysis Systems.
IMPORTANT The Carbohydrate Labeling and Analysis Kit is for laboratory use only.
IMPORTANT In this procedure use 1 M sodium cyanoborohydride in THF, which is a very reactive compound.
Please consult the MSDS before using or disposing of this reagent.
CAUTION
Refer to the Material Safety Data Sheets (MSDS) information, available at
www.BeckmanCoulter.com, regarding the proper handling of materials and
reagents. Always follow standard laboratory safety guidelines.
CAUTION
When installing or removing capillaries from the cartridge, use safety goggles.

A51969AA
2
Carbohydrate Labeling and Analysis Kit
Materials and Reagents
General Precautions
• Prior to using the instrument, turn on the laser and let it stabilize for 15 minutes. Turn it off
when not in use for longer periods of time.
• Do not interchange the capillary (i.e. if a capillary is being used for carbohydrate analysis, do
not use the same capillary in another application).
• When the capillary is not in use or after finishing with all experiments, it is recommended to
run a shutdown method to rinse the capillary and leave the ends immersed in water, to avoid
capillary blockage and to keep the capillary from drying out.
• Prior to using the capillary for the first time, it is recommended to rinse the capillary for 10
minutes at 30 psi pressure with DDI water and then rinse for 10 minutes at 30 psi with the
Carbohydrate Separation Gel Buffer.
Materials and Reagents
Contents of this Kit
Materials Needed but Not Provided in Kit
Table 1 Kit Contents Reorder Number (477600)
Component Quantity Reorder # Code
Carbohydrate Separation Buffer 56 mL 477623
N-CHO Coated Capillary 2 477601
Labeling Dye (APTS) 4x5 mg 501309 L6
Labeling Dye Solvent 1 mL L3
Glucose Ladder Standard 50 mg G20
Quantitation/Mobility Marker (Maltose) 0.18 mg G22
APTS-M (monosaccharide grade) 20 mg 725898
Description Part Number
LIF Performance Test Mix 726022
Universal Plastic Vials (pack 100) A62251
Universal Rubber Vial Caps - blue (pack 100) A62250
200 μL Micro Vials (pack 50) 144709
Assorted pipettors and pipette tips
Spatula
Analytical scale
Vortexer

A51969AA 3
Carbohydrate Labeling and Analysis Kit
Overview of the Procedure
Storage Conditions
The kit components are stable for one year when stored under the following conditions:
• Upon receipt, the kit should be stored at 2 to 8°C.
• Reconstituted Quantitation Control (G22) and labeled glucose ladder standard (G20) should be
stored at -35 to -15°C.
• Reconstituted labeling reagent (APTS) shall be stored at -35 to -15°C. It is stable for 2 weeks after
reconstitution.
Overview of the Procedure
The carbohydrate analysis involves the following three tasks:
• Enzymatic release of the N-linked oligosaccharides from the glycoprotein.
• Labeling of the mixture of released oligosaccharides.
• Analysis of the flouorophore-labeled oligosaccharides by capillary electrophoresis.
Releasing Oligosaccharides From Glycoproteins
IMPORTANT This labeling kit does not contain releasing enzymes.
There are multiple enzymatic and chemical procedures for releasing oligosaccharides from
proteins. In order to successfully label the released oligosaccharide, it is essential that the reducing
termini of the oligosaccharide is not destroyed by the deglycosylation method.
The following is a suggested protocol for N-deglycosylation, using N-Glycosidase F (PNGase F):
Bench top micro-centrifuge
Water bath
Centrifugal vacuum evaporator
Sodium Cyanoborohydride, 1 M/THF Sigma-Aldrich
296813
2-mercaptoethanol Sigma-Aldrich
M7154
Double Deionized (DDI) water filtered through 0.2 μm filter
N-glycosidase F (PNGase F) New England
Biolabs P0704S
Nonidet® NP-40 non ionic detergent
Description Part Number

A51969AA
4
Carbohydrate Labeling and Analysis Kit
Releasing Oligosaccharides From Glycoproteins
Enzymatic Release of the N-Linked Oligosaccharides
1. Aliquot the sample volume in order to have between 25-300 μg of glycoprotein.
2. Dry the glycoprotein solution completely in a centrifugal vacuum evaporator.
3. Add 45 μL of 1X PBS buffer.
4. Add 1.0 μL of 5% SDS (or a volume that gives final concentration of 0.1% SDS).
5. Add 1.5 μL of 1:10 dilution of 2-mercaptoethanol in DDI water (or a volume that gives final
concentration of 50 mM 2-mercaptoethanol).
6. Heat the sample for 5 minutes at 100°C.
IMPORTANT If the denatured protein precipitates, discard the sample and restart this process, repeating
steps 1 through 5, and for step 6, denature the protein by incubating it at 37°C for 10 minutes.
7. Cool the sample to room temperature.
8. Add 5 μL of Nonidet® NP40 non-ionic detergent (or a sufficient volume that gives a final
concentration of 0.75%).
9. Add PNGase F enzyme, according to its activity.
10. React for 15 hours at 37°C.
11. Add approximately 150 μL of cold ethanol (or 3 times the actual reaction mixture volume).
12. Vortex the mixture and place it on ice for complete protein precipitation.
13. Centrifuge the samples at 15,000 rpm for 5 minutes.
14. Withdraw and save the supernatant.
IMPORTANT This solution contains the RELEASED N-LINKED OLIGOSACCHARIDES.
15. Discard the solid. This is the deglycosylated protein.
Working with the Supernatant
There are two options for working with the supernatant:
For quantitative analysis, add an internal standard to the supernatant. See Preparing the Standards
beginning with Step 1a.
For qualitative analysis, bring the supernatant to dryness in the centrifugal vacuum evaporator.
These oligosaccharides are ready to be labeled with APTS. See Performing the Labeling Reaction in
this chapter.
Principle of the Labeling Method
After release (enzymatic or chemical), the oligosaccharides can be labeled with a fluorophore called
8-aminopyrene-1,3,6=, 6-trisulfonate. The stoichiometry of labeling reaction is one APTS molecule
per molecule of oligosaccharide. Figure 1 illustrates the labeling reaction of an N-linked
oligosaccharide with APTS.

A51969AA 5
Carbohydrate Labeling and Analysis Kit
Preparing the Labeling Reagents
Figure 1 Labeling Reaction of an Oligosaccharide with APTS
The efficiency of the labeling reaction is dependent on temperature and the amount of
oligosaccharides. This protocol has been optimized for labeling 5 nmoles or less of total
oligosaccharides.
Samples with amounts higher than 5 nmoles may give a lower reaction yield. Use maltose (G22) as
an internal labeling control and/or as an internal mobility marker.
Preparing the Labeling Reagents
Choosing the Proper Fluorophore
APTS-M is a high-purity fluorophore intended for labeling monosaccharides. APTS-M contains
citric acid as a catalyst. APTS (L6) can be used for oligosaccharides.
Preparing the Reagents
1 Prepare the APTS labeling reagent.
To a vial of APTS labeling dye (L6)
a. Add 48 μL of Labeling dye solvent (15% acetic acid – L3).
b. Vortex for 5 seconds until complete dissolution.
c. Store at -35 to -15°C for up to 2 weeks when not in use.
2 Prepare the APTS-M (Monosaccharide grade) Labeling Dye
a. Add 400 μL of DDI water to the APTS-M vial.
b. Vortex the mixture for 5 seconds until all of the solid is dissolved.
c. Store the reconstituted APTS-M at -35 to -15°C for up to two weeks.

A51969AA
6
Carbohydrate Labeling and Analysis Kit
Performing the Labeling Reaction
Preparing the Standards
1 Prepare the Maltose Quantitative Control/Mobility Marker (G22).
a. Add 500 μL of DDI water to the Maltose quantitative control labeled G22. This makes a
solution containing 500 nmoles of maltose, or concentration of 1 nmol/μL.
b. For quantitation purposes, add 5 μL of G22 reconstituted to your released oligosaccharides.
c. Dry down the sample in a centrifugal evaporator and proceed to labeling protocol, as
described in the next section.
d. Store at -35 to -15°C when not in use.
NOTE When using the maltose solution for quantitation purposes, make sure that the solution is
prepared fresh and used right away. Bacterial contamination can digest the sugars, leading to a
miscalculation of your quantitation.
2 Prepare the Glucose Ladder Standard (G20).
a. Weigh and dissolve 5 mg of Glucose Ladder Standard (G20) in 80 μL DDI water in 1.5 mL
microcentrifuge tube. Sonicate if necessary.
b. Aliquote at least ten 2 μL portions of the Glucose ladder standard solution to 0.5 mL
microcentrifuge vials and dry them in a centrifugal vacuum evaporator. The dried glucose
ladder can be stored at room temperature or used immediately.
Performing the Labeling Reaction
1 Label the Sample and/or Standard (G20).
a. Add 2 μL of 1 M sodium cyanoborohydride/THF to the dried oligosaccharide sample.
b. Add 2 μL of APTS Labeling Reagent to the sample.
IMPORTANT Due to the reaction of sodium cyanoborohydride with water, avoid moisture and store
this product under dry conditions. Use a dry needle to dispense this chemical. Pass dry argon
into the chemical bottle through the septum, while dispensing 2 μL from the bottle.
c. Incubate the reaction mixture at 37°C for 4 hours, or at 60°C for 90 minutes, or at room
temperature overnight (mildest reaction).
NOTE A mild labeling condition is necessary to avoid losing the sialic acid residues from your
oligosaccharide chain. You can leave the reaction mixture at room temperature overnight and in
the dark to do this. For Glucose Ladder incubation, select any of the temperature/ time options.
Figure 2 illustrates a summary of the overall labeling protocol.

A51969AA 7
Carbohydrate Labeling and Analysis Kit
Performing the Labeling Reaction
2 Prepare the Sample and/or Standard for capillary electrophoresis.
a. To the 4 μL reaction mixture prepared above add:
• For Glucose Ladder Standard: add 96 μL of DDI water
• For all other samples: add 46 μL DDI water
This solution can be stored at 2 to 8°C.
b. Take an aliquot of 5 μL of the solution above and add 195 μL of DDI water.
c. Mix the contents welland transfer the solution into a micro vial for analysis in the PA 800
system see Sample Vial Setup.
Figure 3 illustrates a summary of the sample preparation for injection.
Figure 2 Labeling Reaction Scheme

A51969AA
8
Carbohydrate Labeling and Analysis Kit
Preparing the PA 800 Instrument
Figure 3 Sample Preparation for Injection
Preparing the PA 800 Instrument
Before proceeding, review the following procedures in the PA 800 plus System Maintenance User’s
Guide (A51964AA).
• Install the LIF Detector
• Calibrate the LIF Detector
• Capillary Cartridge Procedures
• Cleaning the Interface Block and the Opening Levers