Beckman Coulter PA 800 plus Application Manual
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Beckman Coulter PA 800 plus Application Manual

Beckman Coulter PA 800 plus Application Manual

Pa 800 plus pharmaceutical analysis system
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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

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  • Page 1 Application Guide PA 800 plus Pharmaceutical Analysis System Carbohydrate Labeling and Analysis A51969AA April 2009 Beckman Coulter, Inc. 4300 N. Harbor Blvd. Fullerton, CA 92835...
  • Page 2 Copyright © 2009 Beckman Coulter, Inc. Beckman Coulter, Inc. grants a limited non-exclusive license to the owner or operator of a PA 800 plus instrument to make a copy, solely for laboratory use, of any portion or all of the online help and electronic documents shipped with the PA 800 plus instrument.

  • Page 3: Revision History

    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...
  • Page 4 Revision History A51969AA...

  • Page 5: Safety Notices

    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.

  • Page 6: Caution, Moving Parts Label

    THIS PRODUCT CONFORMS TO APPLICABLE REQUIREMENTS OF 21 CFR 1040 AT THE DATE OF MANUFACTURE. MANUFACTURED: 726024-C A016350L.EPS Sharp Object Label A label reading “CAUTION SHARP OBJECTS” is found on the PA 800 plus. CAUTION SHARP OBJECTS A16558-AA A016351L.EPS A51969AA...

  • Page 7: Recycling Label

    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.

  • Page 8: Restriction Of Hazardous Substances (Rohs) Labels

    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.

  • Page 9: Alerts For Warning, Caution, Important, And Note

    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).
  • Page 10 Safety Notices Symbols and Labels A51969AA...

  • Page 11: Table Of Contents

    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...
  • Page 12 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...

  • Page 13: Carbohydrate Labeling And Analysis Kit

    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.

  • Page 14: General Precautions

    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).

  • Page 15: Storage Conditions

    Carbohydrate Labeling and Analysis Kit Overview of the Procedure Description Part Number Bench top micro-centrifuge Water bath Centrifugal vacuum evaporator Sodium Cyanoborohydride, 1 M/THF Sigma-Aldrich 296813 Sigma-Aldrich ercaptoethanol M7154 Double Deionized (DDI) water filtered through 0.2 μm filter N-glycosidase F (PNGase F) New England Biolabs P0704S Nonidet®...

  • Page 16: Enzymatic Release Of The N-Linked Oligosaccharides

    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.

  • Page 17: Preparing The Labeling Reagents

    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.

  • Page 18: Preparing The Standards

    Carbohydrate Labeling and Analysis Kit Performing the Labeling Reaction Preparing the Standards 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.
  • Page 19 Carbohydrate Labeling and Analysis Kit Performing the Labeling Reaction 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.

  • Page 20: Preparing The Pa 800 Instrument

    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 •...

  • Page 21: Installing And Pre-Conditioning The N-Cho Capillary

    Installing and Pre-conditioning the N-CHO Capillary Installing and Pre-conditioning the N-CHO Capillary • Prior to using the PA 800 plus system for carbohydrate analysis, the N-CHO capillary must be installed in a capillary cartridge. To properly install the capillary, refer to the Capillary Cartridge sections in the PA 800 plus System Maintenance User’s Guide (A51964AA).

  • Page 22: Clean The Interface Block

    Carbohydrate Labeling and Analysis Kit Preparing the Buffer Trays Clean the Interface Block Clean the electrodes, opening levers, capillary tips, and interface block carefully using a Kimwipe soaked with DDI water. Do this on a weekly basis or when changing chemistries on the PA 800 system.
  • Page 23 Carbohydrate Labeling and Analysis Kit Preparing the Buffer Trays Figure 5 Universal Vials and Caps 1. Universal Vial Cap 2. Maximum Fill Level 3. Universal Vials Cover each vial with a cap as shown in Figure 5. Caps are not intended for re-use and should be disposed of after the buffer used is exhausted.

  • Page 24: Sample Vial Setup

    Carbohydrate Labeling and Analysis Kit Sample Vial Setup Figure 6 Buffer Tray Configuration Sample Vial Setup Before placing the 200 microliter sample vials into the universal vials, ensure that no bubbles are at the bottom of the sample vials. If bubbles exist, centrifuge the sample vials for two minutes at 1,000 g and repeat if necessary.

  • Page 25: Running The Samples

    Carbohydrate Labeling and Analysis Kit Running the Samples Figure 7 Micro Vial Inside a Universal Vial 1. Universal Cap 3. Universal Vial 2. Micro Vial 4. Micro Vial inside Universal Vial Running the Samples Initial Conditions Table 2 Test Run: Initial Conditions Capillary Temperature: 20°...

  • Page 26: Lif Detector Initial Conditions

    Carbohydrate Labeling and Analysis Kit Running the Samples Figure 8 Initial Condition Tab LIF Detector Initial Conditions Table 3 Test Run: LIF Detector Initial Conditions Detection Laser Induced Fluorescence Wavelength Excitation - 488 nm, Emission - 520 nm Data Rate 4 Hz Dynamic Range 100 RFU (relative fluorescence units)

  • Page 27: Time Program

    Carbohydrate Labeling and Analysis Kit Running the Samples Figure 9 LIF Detector Initial Conditions Tab NOTE Prior to use, calibrate the LIF detector using the procedure under LIF Detector Calibration Procedure. For comparable results, the detection system should be calibrated at system start-up or whenever a capillary is replaced.
  • Page 28 Carbohydrate Labeling and Analysis Kit Running the Samples Wait 0.2 minutes with vials BI:A4 and BO:A4, see Figure 10. This step dips the capillary in water to protect against sample carry over. Change the rinse water vials if they are contaminated. Separate Step - 20 minutes from vial BI:C1 to vial BO:C1 (buffer), see Figure 10.

  • Page 29: Evaluation Of Results

    Carbohydrate Labeling and Analysis Kit Evaluation of Results Figure 11 Typical Current Profile Field Strength Generated 598 V/cm Typical Current, see Figure 11 under 20 μA NOTE Prior to use, calibrate the LIF detector using the procedure under LIF Detector Calibration Procedure.

  • Page 30: System Shutdown And Capillary Storage

    Carbohydrate Labeling and Analysis Kit System Shutdown and Capillary Storage Figure 12 Electropherogram - Glucose Ladder System Shutdown and Capillary Storage Short Term Storage (<24 hours) of Capillary Perform a three minute, 30 psi rinse with water. The capillary may be stored on the instrument with the capillary ends immersed in water.

  • Page 31: Troubleshooting Procedures

    Carbohydrate Labeling and Analysis Kit Troubleshooting Procedures Whenever the capillary has not been used for three hours or longer, rinse the capillary by performing a 3 minute, 30 psi rinse with water and then a 10 minute rinse with Carbohydrate Separation Buffer before performing a separation.

  • Page 32: Appendix

    Carbohydrate Labeling and Analysis Kit Appendix Appendix LIF Detector Calibration Procedure The PA 800 LIF detector calibration kit procedure is used to calibrate the detection system for carbohydrate analysis. This will ensure consistent results from day to day and from capillary to capillary, as detection is measured in relative fluorescence units (RFU).

  • Page 33: Automatic Calibration

    Carbohydrate Labeling and Analysis Kit Appendix Figure 13 32 Karat Software Configuration dialog Select from the Instrument Configuration window to display the LIF Calibration Wizard Calibration Wizard. See Figure Automatic Calibration Choose Auto as the calibration option, as shown in Figure 14 and click Next...
  • Page 34 Carbohydrate Labeling and Analysis Kit Appendix Figure 14 Calibration Wizard Screen - Auto Select Set up the target RFU value. The target value for N-CHO-coated capillary is 7, as shown in Figure 15 and click Next Figure 15 Calibration Wizard Screen - Step 2 A51969AA...
  • Page 35 Carbohydrate Labeling and Analysis Kit Appendix Fill one buffer vial with 1.5 mL DDI water and another with 200 μL of diluted LIF Performance Test Mix (1:1 with DDI water) in a micro vial. Place them in the buffer vial positions as indicated Figure Place an empty vial in the waste position.

  • Page 36: Ccf Troubleshooting Procedures

    • Verify that the laser provided with the PA 800 was used. • Verify that the correct filters are installed in the LIF detector. • Contact a Beckman Coulter service representative. CCF >1 Table 6 CCF >1...
  • Page 37 Verify that the correct filters are installed in the LIF detector. Verify that the laser provided with the PA 800 system is connected and on. Run the calibration test with a new capillary and chemistries. If there is still no step change detected, contact a Beckman Coulter service representative. A51969AA...
  • Page 38 © 2009 Beckman Coulter, Inc. All Rights Reserved...

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