1.

Objective
1.1. This document provides a procedure for the analysis of Asfotase Alfa by Reverse Phase
High Pressure Liquid Chromatography (RP-HPLC).
2. Scope
2.1. This procedure applies RP-HPLC analysis of Asfotase Alfa drug substance, drug product
samples, and the following in-process samples (Capto Adhere Pool, HIC Pool, UF/DF2
Retentate and Protein A Pool) at Alexion Dublin Manufacturing Facility (ADMF).
3. Roles and Responsibilities
Table 1: Roles and responsibilities
Role Responsibility
Area supervisor or designee Ensure the analyst is properly trained on the procedure
Analyst Implement this procedure as described

3.1. Area supervisor or designee:

3.1.1.Ensure the analyst is properly trained on the procedure.
3.2. Analyst:
3.2.1.Implement this procedure as described.
4. Equipment and Materials
4.1. Equipment
4.1.1.Waters HPLC Alliance e2695 and a UV detector
4.1.2.Waters Empower 3 Chromatography Software
4.1.3.Calibrated pipettes
4.2. Materials
Note: Equivalent equipment and materials may be used.
4.2.1. HPLC grade Acetonitrile, Honeywell Cat. # AH015
4.2.2. HPLC grade water, Honeywell, Cat. # AH365
4.2.3. Trifluoroacetic acid (TFA), J.T. Baker, Cat. # 9470
4.2.4. Sodium Chloride
4.2.5. Sodium Phosphate, Monobasic, Monohydrate
4.2.6. Sodium Phosphate, Dibasic, Heptahydrate
4.2.7. 12 x 32 mm Screw Neck Total Recovery Vial, with Cap and Preslit PTFE/Silicone
Septa, 1 mL Volume, Waters, Cat. # 186000385c
4.2.8. Sodium Hydroxide (Fisher, SA48-1)
4.2.9. Hydrochloric acid (Fisher, AC124260010)
4.2.10. 0.2µm PES filter unit (Thermo Cat. # 567-0020)
4.2.11.
4.2.12. 12 x 32 mm Screw Neck Cap and PTFE/silicone Septum, Waters, Cat. #
186000274
Note: Column substitutions may not be used.
 4.2.13. Applied Biosystems POROS R1 Column, 10 μm, 2.1 x 100 mm, 0.3 mL (Applied
Biosystems Cat. # 1-1014-16)

5. TERMS, ACRONYMS AND ABBREVIATIONS

Table 2: Terms, Acronyms and Abbreviations
Term / Acronym / Abbreviation Definition / Explanation
ADMF Alexion Dublin Manufacturing Facility
QC Quality Control
SDS Safety Data Sheet
RP-HPLC Reversed-Phase High-performance liquid chromatography

Definitions and Abbreviations

ADMF: Alexion Dublin Manufacturing Facility
5.1. SDS: Safety Data Sheet
6. General RequirementsSafety Information
6.1. Environment, Health & Safety
6.1.1.Wear appropriate personal protective equipment when working in the laboratory.
6.1.2.
6.1.3.Be familiar with the SDS for all chemicals used in this procedure.
7. Procedure
7.1. Preparation of Mobile Phases, Buffers, and Solutions
NOTE: Complete QC reagent preparation forms FORM-0114730 ALXN-FRM-
0006772 General Solution Preparation Form Logbook for ALXN-SOP-0004632 SOP-
0118852ALXN-SOP-0004632 Alexion Ireland Management of Prepared Reagents
for Quality Control and label each solution prepared according to SOP-
0118852ALXN-SOP-0004632 Alexion Ireland Management of Prepared Reagents
for Quality Control.
Solutions may be proportionally scaled up or down as required.
7.1.1.Asfotase Alfa RP-HPLC Mobile Phase A (0.2 % TFA in purified water)
7.1.1.1. Add 1000 mL purified water and 2 mL TFA to a glass container and stir until
homogeneous.
7.1.1.2. Store at room temperature. Expiration date is 2 weeks from the date of
preparation.
7.1.2.Asfotase Alfa RP-HPLC Mobile Phase B (0.2 % TFA in ACN)
 7.1.2.1. Add 1000 mL of acetonitrile and 2 mL TFA to a glass container and stir until
homogeneous.
7.1.2.2. Store at room temperature. Expiration date is 2 weeks from the date of
preparation.
7.1.3.RP-HPLC Column Storage Solution (90 % Acetonitrile in HPLC Water)
7.1.3.1. Measure 900 mL of Acetonitrile using a graduated cylinder and transfer to
a 1 L glass container.
7.1.3.2. Measure 100 mL HPLC grade water using a graduated cylinder and transfer
to the same container with the acetonitrile.
7.1.3.3. Ensure contents are adequately mixed.
7.1.3.4. Store at room temperature. Expiration date is 1 month from date of
preparation.
7.1.4.Asfotase Alfa formulation buffer.
7.1.4.1. For each litre of buffer, add the following to ~800 mL of purified water:
7.1.4.1.1. 5.50 g Sodium Phosphate, Dibasic, Heptahydrate
7.1.4.1.2. 0.62 g Sodium Phosphate, Monobasic, Monohydrate
7.1.4.1.3. 8.76 g Sodium Chloride
7.1.4.2. Ensure the pH is 7.4 ± 0.1 by adjusting with Hydrochloric Acid or Sodium
Hydroxide, if needed.
7.1.4.3. Bring the volume to 1 L with purified water.
7.1.4.4. Verify pH is 7.4 ± 0.1.
7.1.4.5. Filter through a 0.2 μm PES filter
7.1.4.6. Expiry is one month when stored at 2 – 8 ºCºC.

7.2. Preparation of Reference Standard, LOQ Standard and Sample

7.2.1. Record all assay information on FORM-0115324ALXN-FRM-0007367 Data Capture
Form for ALXN-QD-0016287 ADMF: Reverse Phase HPLC for Asfotase Alfa.Data
Capture Form for MET-0019654ALXN-QD-0016287.
7.2.2.
7.2.3. Dilute reference standard and samples to 1 mg/mL with Asfotase Alfa Formulation
Buffer.
NOTE: Use forward pipetting mode without pre-wetting to pre-wetting to dispense
Asfotase Alfa Buffer to all tubes. Use reverse pipetting mode to dispenseaspirate the
reference standard and , and , and test sample. Use forward pipetting mode
without pre-wetting to dispense and reference standard dilution.
7.2.3.1. For example, a 40 mg/mL reference standard, bulk drug substance or drug
product sample is diluted as follows:
20 μL of sample + 780 μL of Formulation Buffer
7.2.3.2. For example, a 100 mg/mL reference standard, bulk drug substance or drug
product sample is diluted as follows:
10 μL of sample + 990 μL of Formulation Buffer
7.2.3.3. Record solution scheme on FORM-0115324.ALXN-FRM-0007367
 NOTE: If the sample concentration is less than 1 mg/mL run neat, adjust
injection volume to achieve a 10µg load. For example: a sample with the
concentration of 0.75 mg/mL:
10 µg Load / 0.75 mg/mL = 13 µL injection
7.2.3.4. From the 1mg/mL reference standard dilution, prepare a 0.01 mg/mL LOQ
Standard by diluting 100-fold using Asfotase Alfa Formulation Buffer.
7.2.3.5. For example: Dilute 1:100 by adding 10 μL of 1mg/mL reference standard and
990 μL of formulation buffer.
7.2.3.6. Record dilutions on FORM-0115324ALXN-FRM-0007367
7.3. HPLC Column and System Start-Up
7.3.1. Operate HPLC separations module per SOP-0118875ALXN-SOP-0004655 Alexion
Ireland Operation and Maintenance of the Waters e2695 Separations Module,
Waters 2489 UV/VIS Detector & Waters 2475 Fluorescence Detector.
7.3.2.Flush or prime pump / line with HPLC grade water.
7.3.3. Place Asfotase Alfa RP-HPLC Mobile Phase A on “Pump A”.
7.3.4. Place Asfotase Alfa RP-HPLC Mobile Phase B on “Pump B”.
7.3.5. Place RP-HPLC Column Storage Solution on “Pump C”.
7.3.6. Place the needle and seal wash lines into Asfotase Alfa RP-HPLC Mobile Phase B
and prime all the lines.
7.3.7. Prime the injector before starting the sample run.
7.3.8. Attach the Applied Biosystems POROS R1 Column to the HPLC system.
7.3.9. Equilibrate the column with 80 % Asfotase Alfa RP-HPLC Mobile Phase A / 20%
Asfotase Alfa RP-HPLC Mobile Phase B at 0.5 mL/min for a minimum of 30 minutes
or until a stable baseline is achieved prior to the first injection.
7.3.10. Purge the injector with initial gradient conditions.
7.4. HPLC / PC System Set-Up
7.4.1. Refer to SOP-0118817ALXN-SOP-0004597 Alexion Ireland Data Acquisition and
Processing of Chromatograms for use of the Waters Empower 3 Chromatography
Data System.
7.4.2.Load the ALXN-QD-0016287 MET-0500048 project.
7.4.3.Asfotase Alfa RP-HPLC Instrument Method:
7.4.3.1. Flow Rate: 0.5 mL/min
7.4.3.2. Degasser Unit: On
7.4.3.3. Run time: 50 minutes
7.4.3.4. Detector: Ultraviolet absorption, 220 nm
7.4.3.5. Column Temperature: 50 ºC ± 5 ºC
7.4.3.6. Sample Tray Temperature: 5 ºC ± 3 ºC

Table 2: Gradient:
RP-HPLC RP-HPLC
Time (min.) Flow (mL/min)
Mobile Phase Mobile Phase
 A (%) B (%)
0 0.5 80 20
10.0 0.5 80 20
30.0 0.5 35 65
30.1 0.5 10 90
40.0 0.5 10 90
40.1 0.5 80 20
50.0 0.5 80 20

7.4.4. Set up the instrument method in Empower 3.

7.4.5.Set up the sample set method as suggested below.
7.4.6. Test Injections of each component of the system suitability may be run prior to the
sample sequence.
7.4.7. Insert an injection of RS after every 10 sample injections. Bracketing reference
standard injections may be used to assess the validity of the system throughout
the assay.
7.4.8. Notify the area manager or supervisor if any abnormalities in the pressure trace or
chromatograms are observed during the running of the assay. Refer to Appendix 1
for typical chromatogram and pressure trace. Total pressure may vary between
HPLC Systems. Refer to Appendix 1 for sample pressure trace.

Table 3: Example Sequence:

No. of Injection
Vial Sample Sample Type
Injections Volume
1 Conditioning Injection(RS) Standard 1 10 µL
2 Mobile Phase A Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
1 LOQ Standard Standard 6 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
4 Reference Standard Standard 5 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
5 Test Sample 1 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
6 Test Sample 2 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
7 Test Sample 3 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
8 Test Sample 4 Unknown 1 10 µL
 3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
9 Test Sample 5 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
10 Test Sample 6 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
11 Test Sample 7 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
12 Test Sample 8 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
13 Test Sample 9 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
14 Test Sample 10 Unknown 1 10 µL
3 Asfotase Alfa Formulation Buffer Unknown 1 10 µL
1 Reference Standard Standard 1 10 µL

7.4.9. Save the sample set using a unique name as per SOP-0118817ALXN-SOP-0004597.
7.4.10. Proceed with the sample set run.
7.4.11. Sequence may be aborted prior to any samples being injected. Refer to SOP-
0118817ALXN-SOP-0004597.
7.5 HPLC Column and System Shut-Down.
7.5.1 Equilibrate the column and system in Column Storage Buffer i.e. for 60 minutes at
0.5 mL/min.
7.5.2 Remove the column, cap ends and store at ambient temperature.
7.6 Process all data chromatograms using the Asfotase Alfa processing method for MET-
0019654 ALXN-QD-0016287 ADMF: Reverse Phase HPLC for Asfotase Alfa. Integration
parameter guidelines consist of:
 Calculate suitability and Noise
 Void volume 1.0 min
 Baseline noise 14 to 16 min
 Peak Width: 30 Sec
 Threshold: 110.0 µV/sec
7.7 LOQ Parameters:
 Minimum Peak Height: 0 µV
 Minimum Peak Area: 0 µVsec
 Inhibit Integration: 0 to 16 minutes, 22 to 50 minutes
7.7.1 Integrate all peaks present in the sample chromatogram that are not detected in
the initial Formulation Buffer chromatogram.
7.8 Data Analysis
7.8.1 The reference standard main peak in all injections must elute between 20 and 22
minutes. Consult area supervisor or designee if the main peak falls outside of this
range.
7.8.2 Calculate the percent purity for all sample injections. See Section 8.1
7.9 System Suitability Acceptance Criteria
NOTE: Dimer refers to Main peak
 7.9.1 The Reference Standard profiles must be comparable to the chromatogram
examples found in Appendix 1, or if not baseline resolved Appendix 2, as
determined by the approximate number of peaks, peak retention times and peak
intensity in either case.
7.9.2 The peak area % RSD for the main peak of all injections of the Reference Standard
must be ≤ 3%.
7.9.3 The retention time % RSD of the main peak for the initial 5 injections of the
Reference Standard must be ≤ 1 %.
7.9.4 The first injection of the formulation buffer must not possess any peaks greater
than 10:1 signal to noise which would interfere with the analyte or unresolved
impurities. Report as whole number on FORM-0115324ALXN-FRM-0007367.
7.9.5 LOQ Reference Standard injection Dimer measurements must be quantifiable.
7.9.6 The % RSD for the Dimer % area from all Reference Standards injections must be ≤
2.0 %.
7.9.7 The % RSD for the Dimer % area from all LOQ standard injections must be ≤ 1 %.
7.9.8 Theoretical plates must be >20,000.
7.9.9 Symmetry factor must be lower than 2.0.
7.9.10 In the event that the acceptance criteria are not met, inform the area supervisor or
designee and refer to SOP-0119338ALXN-SOP-0005125 Alexion Global Operations
Laboratory Investigation Management.
7.10 Sample Acceptance Criteria
7.10.1 The percent peak response for each BDS and/or DP sample must be 100 ± 15 %.
See section 8.3.
7.10.2 The percent peak response for each in-process sample (Capto Adhere Pool, HIC
Pool, UF/DF2 Retentate and Protein A Pool) must be 100 ± 20 %.
7.10.3 In the event that the acceptance criteria are not met, inform the area supervisor or
designee and refer to SOP-0119338ALXN-SOP-0005125.

8.0 Calculations
8.1 Percent Purity
Total Peak Area−( ∑ of Impurity Peak Areas )
Percent Purity = ∗100
TotalPeakArea
8.2 Calculate the percent peak response for all 10 ug sample injections (only if the system
suitability criteria are met)
8.3 % Peak Response = ( Asample/ BMean) * 100

Where:
A Sample = Total Peak Area from sample injections
Bmean = mean of the total peak area from the reference standard injections

9.0 Reporting Data

 9.1 Record the Percent Purity result calculated in Section 8.1 as the “Raw Result”.
9.2 Any impurities where the % Peak Area is < 1.0% (LOQ) will be excluded from the “Reported
Purity”.
9.3 Where the “Raw Result” is > 99.0%, the “Reported Purity” result for the sample will be
reported as “> 99.0%”.
9.4 Where the Raw Result is < 99.0%, impurities < LOQ are excluded from the Percent Purity
calculation (Section 8.1) and the “Reported Purity” result for the sample will be reported
as Percent Purity adjusted for impurity peaks blow the LOQ.
9.5 There should be no new reference peaks > LOQ (1.0%) present. New reference peaks <
LOQ should be reported as “No New Reference Peaks”.
9.6 The Raw Result, before adjustment for peaks < LOQ, will be used for trending purposes
only.
9.7 Evaluate the test results against the associated specification, if applicable. In the event a
result is unexpected and suspected to not meet the applicable specification, notify the
supervisor. Complete data review and refer to SOP-0119338ALXN-SOP-0005125 for
further investigation.
9.8 Submit completed data package to area supervisor or designee for review and approval.

10.0 Appendices
10.1 Appendix 1, Example Reference Standard Chromatogram and Pressure Trace
10.2 Appendix 2, Example Pro A Sample Chromatogram with unresolved baseline peaks

11.0 References:
Table 4: References

Document Number Document Title

General Solution Preparation Logbook for ALXN-SOP-0004632:
FORM-0114730 Alexion Ireland Management of Prepared Reagents for Quality
Control
Data Capture Form for ALXN-QD-0016287 ADMF: Reverse Phase
FORM-0115324
HPLC for Asfotase Alfa
 Alexion Ireland Operation and Maintenance of the Waters e2695
SOP-0118875 Separations Module, Waters 2489 UV/VIS Detector & Waters
2475 Fluorescence Detector
SOP-0118852 Alexion Ireland Management of Prepared Reagents for Quality
Control
SOP-0119338 Alexion Global Operations Laboratory Investigation Management
SOP-0118817 Alexion Ireland Data Acquisition and Processing of
Chromatograms

FORM-0114730ALXN-FRM-0006772: General Solution Preparation Logbook for SOP-0118852ALXN-

SOP-0004632: Alexion Ireland Management of Prepared Reagents for Quality Control.
11.1
11.2 FORM-0115324ALXN-FRM-0007367: Data Capture Form for MET-0019654.ALXN-QD-0016287
11.3 SOP-0118875ALXN-SOP-0004655: Alexion Ireland Operation and Maintenance of the Waters e2695
Separations Module, Waters 2489 UV/VIS Detector & Waters 2475 Fluorescence Detector.
11.4 SOP-0118852ALXN-SOP-0004632: Alexion Ireland Management of Prepared Reagents for Quality
Control.
11.5 SOP-0119338ALXN-SOP-0005125: Alexion Global Operations Laboratory Investigation Management.
11.6 SOP-0118817ALXN-SOP-0004597: Alexion Ireland Data Acquisition and Processing of
Chromatograms.

12.0 Revision History

Change Type
Version
(New, Revise, Revision Summary Justification
No.
or Admin)

Section 7.9.1 updated to instruct on

integration of non baseline resolved
peaks Update as per Change
4.0 Revise Appendix 2.0 added as an example of Control 80109 and Task
acceptable integration for non baseline 81939
resolved peaks
11.8 updated for correct reference.

Updated firstDoc references throughout

5.0 Admin document to align with Vault Quality Periodic review
Correct typo in section 7.4.2

6.0 Revise Document formatting corrected To align with

throughout as per WI-0029400
TMP-0011828
Table 1 added in section 3 to include
Roles and Responsibilities in table format

Table 2 added in section 4 to include

abbreviations terms and definitions in
 table format

Updated Vault references throughout To align with Vault

document Quality
Addition of “note” to section 7.2.2 for
standardisation of analytical test method
to provide clarification on specific
pipetting techniques i.e., forward or
Refer to PR 150142
‍ reverse pipetting

 7.5.2
7.6.4
Appendix 1, Example Reference Standard Chromatogram and Pressure Trace
(Page 1 of 1)

Note: Dimer refers to Main Peak

Appendix 2, Example Pro A Sample Chromatogram with unresolved baseline peaks
(Page 1 of 1)