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WHO/BS/07.2071
ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Geneva - 8 to 12 October 2007
A Collaborative Study to Establish
The 2nd
International Standard Low Molecular Weight Heparin for Molecular Weight
Calibration
Barbara Mulloy1, Alan Heath
1 and Marie-Emmanuelle Behr-Gross
2
1 National Institute for Biological Standards and Control, South Mimms, Herts EN6 3QG, UK.
2 EDQM, Strasbourg, France
© World Health Organization 2007
All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail: [email protected]).
The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication.
WHO/BS/07.2071
Page 2
SUMMARY
An international collaborative study involving fourteen laboratories has taken place, organised by the
European Directorate on Quality of Medicines (EDQM) with NIBSC (in its capacity as a World
Health Organisation Laboratory for Biological Standardisation) to provide supporting data for the
establishment of a replacement for the 1st International Reference Reagent (IRR) Low Molecular
Weight Heparin for Molecular Weight Calibration, and replacement Low Molecular Mass Heparin
for Calibration CRS.
Using a Broad Standard Table derived from results provided by the participants, molecular weight
parameters were determined for seven different low molecular weight heparins using both the current
IRR (90/686) and the candidate replacement calibrant (05/112).
For both 90/686 and 05/112 inter-laboratory CVs were within the range 1.0-6.6% for mean
molecular weights, and within the range 0.3-1.0% for proportion of the sample below a molecular
weight of 8000 g/mole. Most mean molecular weights calculated using 05/112 are lower than those
calculated using 90/686 by less than 1%, with a few measurements between 1 and 2% lower.
Proportions of the samples below 8000 are almost identical using either 90/686 or 05/112 as
calibrant. On the basis of this study a recommendation can be made for the establishment of 05/112
as a replacement for the 1st International Reference Reagent Low Molecular Weight Heparin for
Molecular Weight Calibration. Unlike the 1st IRR (90/686) however, the candidate material 05/112 is
not suitable for use with the method of Neilsen [1].
INTRODUCTION
Low Molecular Weight (LMW) Heparin, a partially depolymerised derivative of unfractionated
heparin, is widely used to prevent and treat thrombosis. The anticoagulant activities of LMW
Heparin depend critically on molecular weight, and the molecular weight distribution is a defining
characteristic of each LMW Heparin product. In the European Pharmacopoeia (Ph. Eur.) a general
monograph (heparins, low-molecular-mass, 0828) and specific monographs for each product include
limits on molecular weight parameters (Dalteparin sodium (1195), Enoxaparin sodium (1097),
Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)).
All the currently practicable methods of assessing the molecular weight distribution of LMW
Heparin are based on size exclusion chromatography (SEC) (also sometimes referred to as gel
permeation chromatography (GPC)). The size exclusion chromatography system must be calibrated,
to generate a relationship between retention time and molecular weight, and the calibrant for any
particular type of macromolecule should be as close as possible in structure to the samples to be
characterized. Though heparin is a linear polysaccharide based on a simple disaccharide repeating
unit, it is heterogeneous in sequence (due to variation in substitution) and polydisperse in size; it has
become conventional to define the molecular weight of a disaccharide repeat unit of heparin as 300
g/mole.
Heparin molecular weight calibrants have been in use for over 12 years, since the establishment of
the 1st International Reference Reagent Low Molecular Weight Heparin for Molecular Weight
Calibration (1st IRR) and the Ph. Eur. calibrant, the 1
st CRS Low Molecular Mass Heparin for
Calibration. In 2004, it was recognised by the EDQM and by NIBSC that stocks of both calibrants
were low, and a decision was taken to replace the calibrants in a joint NIBSC/EDQM project.
WHO/BS/07.2071
Page 3
Recent developments in the production of LMW Heparin, in particular the introduction of “generic”
and/or “biosimilar” candidate products, make it essential to ensure that internationally recognised
calibrants for determination of molecular weights of LMWH products continue to be made available.
The Ph. Eur. CRS calibrants are established for use in methods described in the appropriate
monographs and are not characterised for other methods: the replacement IRR is characterised for
use with a standard method of polymer molecular weight determination, as a “Broad Standard”
calibrant. As both the replacement Ph. Eur. and WHO calibrants are prepared from the same bulk
material and characterised in the same study, results from either method are comparable.
A report on the joint NIBSC/EDQM study for EDQM, PA/PH/BIO (06) 42, was the basis of the
recent establishment of the Heparin Low-Molecular-Mass for Calibration Chemical Reference
Substance batches 2 and 3. This current report for ECBS presents those results of the study which
are relevant to the proposed establishment of a replacement for the 1st IRR LMW Heparin for MW
Calibration.
AIMS OF THE STUDY
The project BSP069 was jointly organised between EDQM and NIBSC and intended to provide data
for the establishment of WHO and Ph. Eur. replacement calibrants to be used for identification and
characterisation by size exclusion chromatography (SEC) of LMM heparins.
A batch of low molecular mass heparin prepared by Leo Pharmaceuticals using a similar process
(derived from porcine mucosal heparin by depolymerization with heparinase I) as for current
calibrant batches was subjected to preliminary studies at NIBSC. The results indicated that the
method of Nielsen [1] is not applicable to this material. This method depends on a specific
characteristic of the calibrant material: namely, that the ultraviolet (UV) absorbance at 234 nm is
proportional to the molar concentration, and the refractive index increment (RI) is proportional to the
concentration by weight, so that the ratio of the responses from UV and RI chromatography detectors
is proportional to the molecular weight.
A preliminary study was therefore undertaken jointly between NIBSC and the EDQM laboratory to
find out whether the Ph. Eur. monograph method (0828, Identification, method C) could be used to
characterise this material, as it relies on the same physical properties of the calibrant as does the
method of Nielsen [1]. The 1st IRR LMW Heparin for MW Calibration (90/686) was established
largely for use with the Nielsen method [2], but since that time a Broad Standard Table for 90/686
has been prepared and published [3] and is now in more common use than the Nielsen method.
The preliminary study was conducted in early 2005, and led to the planning of a two-phase
collaborative study, aimed at characterising the proposed candidate replacement for the 1st IRR
LMW Heparin for MW Calibration (05/112), and the proposed candidate Heparin LMM for
Calibration batches 2 and 3 (cCRS2 and cCRS3) which were all prepared from the same batch.
Phase 1
Three laboratories took part in this stage of the project, the aim of which was to furnish provisional
values for a Broad Standard Table for 05/112 and a value for the number-average relative molecular
mass of the proposed replacement Ph. Eur. calibrants for use in Phase 2 of the study. Secondly, a
Broad Standard Table was prepared from the data for use with 05/112 in Phase 2 of the study.
WHO/BS/07.2071
Page 4
Phase 2
The aim of Phase 2 was to test the performance of the candidate calibrant materials, and to provide a
comparison with the established calibrants, in a larger number of laboratories, and over a panel of
LMM heparins with varying molecular mass distributions. The Ph. Eur. monograph method was
used for the established and candidate CRSs (Mn value from Phase 1), and the Broad Standard
method was used for the 1st IRR 90/686 and the candidate replacement 05/112 (Broad Standard
Table from Phase 1).
The present document reports the outcomes of Phase 2 of EDQM project BSP069 for the candidate
replacement calibrant 05/112, and the 1st IRR 90/686.
PARTICIPANTS
A list of participants is given in Appendix 1 at the end of this report. Each laboratory is referred to in
this report by an arbitrarily assigned number, not necessarily representing the order of listing in the
Appendix. Fourteen laboratories agreed to take part in the study, of which 3 participated in phase 1
and 12 provided full sets of results for Phase 2 (one laboratory participated in both phases). The
participants were located in 7 European countries, the United States of America, Australia and
China. Six heparin manufacturers, seven regulatory control laboratories, and one academic
laboratory took part.
SAMPLES
Samples included in the study were:
1st IRR low-molecular-weight heparin for molecular weight calibration (90/686)
Candidate 2nd IRR low-molecular-weight heparin for molecular weight calibration (05/112)
Heparin low-molecular-mass for Calibration CRS batch 1 (CRS1), 25 mg of fractionated heparin
Candidate Heparin LMM for Calibration Batch 2 (cCRS2)
Candidate Heparin LMM for Calibration Batch 3 (cCRS3)
Samples coded A-G inclusive: low molecular weight heparin samples.
BULK MATERIAL AND PROCESSING
The proposed LMW Heparin for MW Calibration (NIBSC 05/112) is a specially prepared sample
derived from porcine mucosal heparin by depolymerization with heparinase I. It was manufactured
and donated by Leo Pharmaceutical Products Ltd. A/S of Ballerup, Denmark (Lot no. 346542).
The proposed calibrant material was filled into sealed, siliconized glass 5 ml DIN ampoules
ampoules at NIBSC on the 3rd
June 2005 using the method of Campbell et al. [5], and was coded
NIBSC 05/112. 8,000 ampoules are available to WHO.
WHO/BS/07.2071
Page 5
The residual moisture content of 05/112 is 0.287%. The mean dry weight for 05/112 is 23.5 mg.
05/112 (8,000 ampoules) is stored at -20 °C; proposed storage and shipment conditions are -20 °C
and ambient temperature respectively.
Product summary for 05/112:
Code number 05/112
Presentation Sealed, siliconized glass 5 ml DIN
ampoules
Number of ampoules available 8000
Date filled 3rd
June 2005
Precision of fill: CV of fill mass (%,
n=136)
0.15
Residual moisture after lyophilization and
secondary desiccation (%, n=6)
0.287%
Mean dry weight (g, n=6) 0.0235
Storage conditions -20 ºC
Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK
Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK
No excipients, bulking agents, buffers or stabilizers have been included.
The calibrant is not used for in vitro diagnostic purposes.
Heterogeneity and Stability
Six ampoules of 05/112 were chosen at random and molecular weight profile determinations made in
duplicate using the method described in the accompanying report. There was no systematic
difference between the ampoules (Table 1).
An accelerated degradation study of the 1st IRR LMW Heparin for MW Calibration, 90/686, was
unable to detect any alteration in mean molecular weights in ampoules held at 56 ºC for the eight
years 1992-2000 (Fig. 1). A similar study has been initiated for 05/112. Ampoules held at elevated
temperatures (37 ºC and 56 ºC) will be monitored at regular intervals throughout the life of the
material. Measurements after 9 months indicate no degradation of samples kept at +37 ºC and +56 ºC
compared with samples kept at -20 ºC.
COLLABORATIVE STUDY
The study was planned and designed as Phase 2 of EDQM project BSP069. The candidate
replacement IRR 05/112, and the 1st IRR 90/686 were compared as calibrants in size exclusion
chromatography of low molecular weight heparins, using a published Broad Standard Table for
90/686 [3] and for 05/112 a Broad Standard Table derived from the results of Phase 1 of the study
(Table 2A).
Methods
WHO/BS/07.2071
Page 6
All the laboratories used both the Ph. Eur. monograph method and the Broad Standard Tables for
identification and characterisation of LMM heparins by SEC. Variations between SEC experimental
methods used by the participants were relatively minor, being limited to different manufacturers of
gel permeation columns and chromatography hardware and software, and different eluant solutions,
as follows:
Column types: No. of Labs (12 in total)
Tosoh-Haas TSK SWXL 2000 7
Tosoh-Haas TSK SWXL 3000/2000 in series 3
BioSep-SEC-S 2000/3000 in series 1
Hema Bio 1000/Bio 40 in series 1
Eluants:
0.2-0.3M sodium sulfate pH 5.0 10
0.1M ammonium acetate 2
Study Design
Participants were requested to carry out 2 independent tests on different days, including in each test
all the samples and references provided and using an experimental protocol based on the Ph. Eur.
Method [4].
RESULTS AND STATISTICAL ANALYSIS
Phase 2 of the study: Results reported by participants
Twelve laboratories each provided two independent sets of data. Chromatograms of all the samples
and calibrants were recorded, and the number–average molecular weight (Mn), weight-average
molecular weight (Mw) and weight percentage with molecular weight below 8000 were reported.
Calculated results and methodological details as well as the listing of raw data files (corresponding to
UV and RI chromatograms) were reported using the electronic reporting sheets provided by the study
organisers. Raw data was also provided by 9 laboratories (laboratories 1, 2, 4, 5, 6, 9, 10, 11 and 12)
though the proprietary nature of the file formats involved precluded the use of raw data from three of
these laboratories.
The molecular weight parameters reported are listed in Tables 3A-G for samples A-G respectively.
Statistical analysis consisted in calculation of mean, standard deviation (SD) and coefficient of
variation (CV), over the full data set. The same data are presented in a graphical format in Figures 2-
8 (left-hand side) for samples A-G respectively.
Mean relative molecular weights (Mn and Mw)
Mn values calculated using 05/112 were 0-5% higher than using 90/686. Mw values calculated using
05/112 were 2-5% higher than using 90/686. CVs over the full data set (two values from each
WHO/BS/07.2071
Page 7
laboratory) for Mn vary between 3.7% and 8.8% for 90/686, and 4.9% and 9.5% for 05/112. CVs for
Mw vary between 3.0% and 9.8% for 90/686, and 4.8% and 10.2% (after the exclusion of apparent
anomalies – see below). There is no apparent dependence of CV upon calibration method or
calibrant.
Weight percentage with a relative molecular weight below 8000.
This value requires the assignment of a single molecular weight point in the chromatogram, and
calculation of the proportions of the integrated area of the chromatogram on either side of that point.
Mean values using 05/112 as calibrant are 2-4% lower than using 90/686, and 0-1% lower than
CRS1. CVs over the full data set vary between 1.2 and 2.8% for 90/686, and between 1.0% and 2.1%
for 05/112 for this parameter (after the exclusion of apparent anomalies – see below), again with no
apparent dependence on calibration method.
Anomalies
Laboratory 1 reports high values for Mm, especially for samples C and G, using 90/686 and 05/112
as standards (see Fig. 4 and Fig. 8).
Laboratory 2 reports high values for %<8000 for most of the samples (this is particularly noticeable
for samples A (Fig. 2), C (Fig. 4), F (Fig. 7) and G (Fig. 8)).
Tables 3A-G include statistics calculated both with and without these two sets of results.
Comments from participants on the initial report
The comments of participants on the draft Phase 2 report and on recommendations to establish the
candidate materials as IRR and CRS were sought. Although several laboratories approved the
proposal, three laboratories expressed concerns, including the following:
1. The range of results for some of the measurements was very large, and overall inter-laboratory
variation was too great.
2. The difference between results calculated against the 1st (90/686) and the proposed 2
nd (05/112)
IRRs, though within one standard deviation, was systematic.
3. The methodology gave rise to practical problems in that, with the Ph. Eur. recommended eluant (a
sodium salt based buffer), some overlap was observed between calibrant and salt peaks.
4. The methodology also gave rise to problems due to the sparse description of method of calculation
in the Ph. Eur.
In response to these comments, a detailed study was carried out at NIBSC, using the raw data
supplied by six of the laboratories. Items 3 and 4 of these comments apply only to method described
in the Ph. Eur. [4].
Recalculation of results
Raw data from six laboratories was processed in a consistent way at NIBSC, as follows. For the
proposed replacement calibrant, 05/112, a new Broad Standard Table was derived from Phase 2
WHO/BS/07.2071
Page 8
results for 05/112 molecular weight distributions calculated using the 1st IRR, 90/686. A consensus
table was obtained by combining data from all six laboratories, each with 2 runs, giving 12 data sets
in total. For each laboratory and run, a table of molecular weight (MW) and „mass percentage with a
relative molecular mass below MW‟, based on MW values ranging from around 500 to over 30,000
was available. Each table had between 500 and 1000 entries.
The observed „mass percentage with a relative molecular mass below MW‟ was plotted against log
MW for each data set, and good agreement was noted. A consensus Broad Standard Table (Table
2B) was derived in the following manner. A set of 18 MW values between 600 and 18000 were
selected to be the basis of the broad table. At each of these individual MW values, the „mass
percentage with a relative molecular mass below MW‟ was determined as the median observed value
of percent below MW from the 12 data sets, after performing a linear interpolation between
neighbouring MW values for each of the data sets when necessary.
There were no significant anomalies: all the recalculated results are summarized in Tables 4A-G for
samples A-G respectively, and displayed graphically in Figures 2-8 (right-hand side) for samples A-
G respectively.
Mean relative molecular masses (Mn and Mw)
Mn values calculated using 05/112 are about 1% lower than using 90/686. Mm values calculated
using 05/112 are on average 0.3% lower than using 90/686 or CRS1.
Coefficients of variation for Mn vary between 1.06% and 3.54% for 90/686, and 3.34% and 6.60%
for 05/112. CVs for Mm vary between 0.76% and 2.76% for 90/686, and 1.34% and 2.60% for
05/112.
Mass percentage with a relative molecular mass below 8000.
Mean values using 05/112 as calibrant are on average 0.1% lower than using 90/686. Values
calculated using 90/686 are on average 2.3% higher than those using CRS1. CVs vary between 0.4%
and 0.9% for 90/686, and between 0.3% and 1.0% for 05/112.
DISCUSSION
The material from which cCRS2, cCRS3 and 05/112 have been prepared is not suitable for the
calibration method of Nielsen [1]. The ratio of the UV and RI responses at any point in the
chromatogram are not proportional to the molecular weight at that point. The deviation from
proportionality is small, but sufficient to cause serious discrepancies at high molecular weights.
The 1st IRR LMW Heparin for MW Calibration (NIBSC 90/686) was manufactured and donated by
Novo Nordisk. Leo Pharma subsequently took over the manufacture of heparinase-digested LMW
Heparin from Novo (Tinzaparin) and has manufactured the proposed replacement calibrant material
to correspond as closely as possible to the 1st IRR LMW Heparin for MW Calibration. Subtle
differences in the manufacturing method may be responsible for differences between the old and new
batches of calibrant material.
WHO/BS/07.2071
Page 9
Continuity of the measurement system cannot be taken for granted simply by calibration of the
proposed new calibrant against the 1st IRR. The two preparations are similar in molecular weight
distribution; a calibrant should have a wider distribution than the unknown to avoid reliance on
extrapolation. Therefore, 7 different LMW heparin samples have been tested by the study
participants using the Broad Standard Table methods with both current and candidate calibrants.
Results reported by the participants (12 laboratories) for the LMW heparin samples were based on
the Broad Standard Table (Table 1A) determined in phase 1 of the study. They showed discrepancy
with results obtained with current calibrants, and large interlaboratory variations in Mn and Mm
values. A refined Broad Standard Table (Table 1B), based on data from 6 laboratories in Phase 2 of
the study, was therefore used to recalculate data from the same six laboratories.
The centrally recalculated results are not directly comparable with those reported by participants, as
only a subset of the data was used (6 laboratories) and the calculations were all performed in the
same way. However, the results indicate that removal of differences in method of calculation reduce
the inter-laboratory variation. This source of inter-laboratory variation is therefore not a property of
the calibrant material, or of the experimental data. The use of reputable, specialist chromatography
software for the calculation of molecular weight parameters for polydisperse polymers is desirable.
PARTICIPANTS RESPONSE
A full report of the study, containing all the data for both the EDQM and WHO calibrants, was
prepared for the EDQM (document PA/PH/BIO(06)42), and circulated to all participants. All the
participants agreed to the establishment of 05/112 as a replacement calibrant for 90/686, using the
Broad Standard Table shown in Table 2B.
RECOMMENDATION
Recalculation of Phase 2 results from six of the laboratories demonstrates that the revised Broad
Standard Table (Table 2B) for 05/112 generates results for unknowns in close agreement with those
from 90/686. Establishment of 05/112, with this Table, is therefore recommended to the WHO
Expert Committee on Biological Standardisation.
Appendix 2 includes the draft Instruction For Use and Material Safety Data for this preparation.
WHO/BS/07.2071
Page 10
ACKNOWLEDGEMENTS
The organisers would like to thank all the participants in the study for their contribution.
Special thanks should also be given to Leo Pharmaceuticals for donation of material and G. van
Dedem and K. Johansen for scientific advice. The manufacturers of LMM heparin are warmly
thanked for the donation of bulk materials for production of study samples.
The contribution of staff at both NIBSC and EDQM production units is gratefully acknowledged.
The collaborative study was run in the framework of the Biological Standardisation Programme
(internal project number number: BSP069) of the EDQM with the support of the organising institutes
(EDQM, NIBSC) and of the Council of Europe and the European Commission. A. Heath (NIBSC,
UK) was in charge of the statistical evaluation and Dr. M-E. Behr-Gross (EDQM) coordinated the
study.
REFERENCES
[1] Nielsen JI. A convenient method for molecular mass determination of heparin Thromb Haemost
1992; 68: 478-80.
[2] Van Dedem G, J. Nielsen JI. Determination of the molecular mass of low molecular mass (LMM)
heparin, Pharmeuropa 1991(3): 202-18.
[3] Mulloy B, Gee C, Wheeler SF, et al. Molecular Weight measurements of low molecular weight
heparins by gel permeation chromatography. Thromb Haemost 1997; 77: 668-74.
[4] Heparins, low-molecular-mass, monograph 0828 – minor revision adopted in November 2006,
PA/PH/Exp.6/T (06) 47 COM. Strasbourg, France: Council of Europe; to be published in Ph. Eur. 6th
edition, June 2007.
[5] Campbell PJ. International biological standards and reference preparations. 1. Preparation and
presentation of materials to serve as standards and reference preparations. J Biol Standardisation
1974; 2; 249-67.
WHO/BS/07.2071
Page 11
Table 1: Heterogeneity: Number average molecular weight (Mn) and weight average molecular
weight (Mw) for six ampoules of 05/112 chosen at random in August 2005. Individual duplicate
measurements are listed for each ampoule.
Sample Mn Mw
1 4065 6615
3921 6611
2 4026 6547
3898 6599
3 3997 6500
3906 6601
4 4020 6600
3876 6584
5 3995 6566
3896 6657
6 3958 6562
3860 6589
Mean 3960 6586
SD 68 39
CV 1.7 0.6
WHO/BS/07.2071
Page 12
Table 2A: Phase 1 results: provisional Broad Standard Table for 05/112.
Point Log10(MM) MM % >M %<M
1 2.805 638 99.21 0.79
2 3.085 1216 96.82 3.18
3 3.252 1785 92.72 7.28
4 3.377 2384 87.27 12.73
5 3.474 2979 81.3 18.70
6 3.553 3569 74.91 25.09
7 3.624 4203 67.94 32.06
8 3.682 4807 61.58 38.42
9 3.734 5426 55.29 44.71
10 3.779 6018 49.62 50.38
11 3.819 6594 44.49 55.51
12 3.858 7208 39.46 60.54
13 3.920 8321 31.55 68.45
14 3.987 9713 23.50 76.5
15 4.095 12441 12.42 87.58
16 4.161 14490 7.21 92.79
WHO/BS/07.2071
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Table 2B: Broad Standard Table for 05/112 derived from Phase 2 results vs 90/686
Point Log10(MM) MM % >M % <M
1 2.78 600 0.40 99.60
2 3.08 1200 3.87 96.13
3 3.26 1800 8.94 91.06
4 3.38 2400 14.49 85.51
5 3.48 3000 20.68 79.32
6 3.56 3600 27.20 72.80
7 3.62 4200 33.89 66.11
8 3.68 4800 40.49 59.51
9 3.73 5400 46.83 53.17
10 3.78 6000 52.92 47.08
11 3.82 6600 58.59 41.41
12 3.86 7200 63.89 36.11
13 3.92 8400 72.96 27.04
14 3.98 9600 80.09 19.91
15 4.08 12000 89.21 10.79
16 4.13 13600 92.96 7.04
17 4.19 15600 95.95 4.05
18 4.26 18000 97.77 2.23
WHO/BS/07.2071
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Table 3A Sample A
Values reported by participants
Mn
Mm
%<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
5129 5156
7303 7054
75.7 75.6
2
5145 5127
7646 7047
75.5 76.1
2 1
4514 4443
4727 4338
99.8 96.1
2
4386 4353
4820 4801
97.2 97.1
3 1
4779 5052
5858 6153
81.3 79.3
2
4829 5123
5938 6272
80.3 77.9
4 1
4817 5080
5956 6337
82.3 79.3
2
5022 5239
5969 6292
82.3 79.5
5 1
4958 5250
6021 6410
80.5 76.7
2
4925 5200
5976 6367
80.8 76.9
6 1
4970 5258
6053 6441
79.4 75.5
2
4956 5214
6059 6415
79.3 75.8
7 1
4900 5083
6017 6342
79.5 77.8
2
4805 5084
5945 6322
80.4 76.8
8 1
4810 4962
6000 6172
82.0 81.0
2
4726 5245
5914 6524
82.0 76.0
9 1
4896 4923
5967 6179
80.8 79.2
2
4918 4925
5977 6190
80.8 79.2
10 1
4856 5121
5907 6278
82.7 79.5
2
4850 5101
5932 6321
82.6 79.2
11 1
4733 4772
6051 6300
81.2 78.9
2
4501 4765
6010 6270
80.3 78.6
12 1
4864 5172
6015 6446
81.0 77.5
2
4829 4985
6034 6451
80.9 79.3
Mean
4838 5026
6004 6238
82.0 79.5
SD
178.5 237.5
570.4 565.2
5.4 5.5
CV (%)
3.69 4.73
9.50 9.06
6.58 6.88
Excluding lab 1
Excluding lab 2
Mean
5870 6165
80.5 78.0
SD
359.0 530.4
1.9 1.6
CV (%)
6.12 8.60
2.33 2.04
WHO/BS/07.2071
Page 15
Table 3B Sample
B
Values reported by participants
Mn
Mm
%<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3414 3419
4493 4483
92.0 92.3
2
3442 3435
4632 4528
91.6 91.9
2 1
2666 3257
4001 3879
100.0 99.1
2
3392 3345
3853 3823
99.5 99.5
3 1
3233 3434
4131 4364
94.0 93.2
2
3238 3466
4185 4441
93.6 92.5
4 1
3170 3321
4135 4375
95.2 94.0
2
3171 3242
4222 4425
94.6 93.4
5 1
3355 3535
4242 4492
94.1 92.3
2
3347 3504
4230 4464
94.1 92.4
6 1
3366 3544
4251 4500
93.6 91.8
2
3331 3482
4233 4456
93.6 92.0
7 1
3387 3485
4602 4828
90.1 89.2
2
3260 3434
4243 4499
93.5 91.9
8 1
3087 3167
4120 4232
95.0 94.0
2
3017 3510
3996 4456
96.0 93.0
9 1
3318 3187
4196 4230
94.4 93.8
2
3307 3173
4195 4230
94.3 93.6
10 1
3173 3311
4165 4395
94.8 93.0
2
3221 3343
4246 4486
94.4 93.0
11 1
3099 3023
4152 4223
94.4 93.4
2
2886 3082
3976 4193
94.8 94.1
12 1
3272 3486
4271 4570
94.0 92.5
2
3277 3259
4339 4643
93.7 93.1
Mean
3226 3352
4213 4384
94.4 93.3
SD
178.9 149.8
178.9 219.0
2.1 2.1
CV (%)
5.54 4.47
4.25 5.00
2.18 2.27
Excluding lab 1
Excluding lab 2
Mean
4181 4373
93.9 92.7
SD
148.0 225.7
1.3 1.1
CV (%)
3.54 5.16
1.36 1.16
WHO/BS/07.2071
Page 16
Table 3C Sample
C
Values reported by participants
Mn
Mm
%<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
4930 4923
11273 9904
64.8 65.0
2
4921 4871
11994 9699
64.9 65.5
2 1
4333 4138
4820 5227
98.9 90.0
2
4231 4186
5065 5051
92.5 92.3
3 1
4475 4726
6542 6914
70.9 69.0
2
4479 4771
6595 7018
70.2 67.9
4 1
4418 4629
7114 7864
70.1 67.9
2
4567 4709
7144 7829
70.6 67.8
5 1
4670 4936
6805 7336
69.9 65.9
2
4630 4871
6768 7292
69.8 66.1
6 1
4585 4840
6625 7091
69.9 66.4
2
4542 4761
6655 7086
69.9 66.6
7 1
4613 4778
6839 7343
68.6 66.9
2
4543 4796
6806 7281
69.3 65.9
8 1
4387 4517
7093 7480
70.0 69.0
2
4218 4824
6580 7205
73.0 68.0
9 1
4562 4452
6582 6953
70.9 69.3
2
4573 4465
6605 6986
70.8 69.1
10 1
4419 4622
6786 7323
71.3 68.1
2
4394 4578
6634 7185
72.0 68.7
11 1
4526 4502
6941 7332
70.5 68.2
2
4317 4575
7022 7348
69.9 68.3
12 1
4565 4864
6916 7573
70.1 66.7
2
4561 4574
7056 8145
69.7 68.2
Mean
4519 4663
7053 7353
72.0 69.4
SD
174.0 213.1
1520.3 1017.5
7.6 6.8
CV (%)
3.85 4.57
21.56 13.84
10.51 9.80
Excluding lab 1
Excluding lab 2
Mean
6636 7130
69.9 67.5
SD
582.1 714.0
1.9 1.3
CV (%)
8.77 10.01
2.67 1.90
WHO/BS/07.2071
Page 17
Table 3D Sample
D
Values reported by participants
Mn
Mm
%<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3694 3703
4911 4889
89.2 89.0
2
3724 3718
5027 4903
88.9 89.2
2 1
3401 2917
3767 3563
100.0 99.7
2
2933 2876
3523 3490
98.8 98.7
3 1
3414 3621
4372 4605
92.2 91.2
2
3460 3697
4460 4719
91.5 90.1
4 1
3460 3628
4520 4773
92.4 90.7
2
3460 3550
4451 4631
92.9 91.3
5 1
3643 3844
4590 4866
91.7 89.5
2
3624 3806
4572 4845
91.7 89.5
6 1
3634 3829
4596 4870
91.1 88.8
2
3620 3788
4599 4847
91.0 88.9
7 1
3618 3729
4568 4759
91.2 90.2
2
3550 3744
4499 4766
91.6 89.6
8 1
3379 3471
4339 4479
94.0 93.0
2
3318 3824
4376 4859
93.0 90.0
9 1
3302 3156
4244 4280
93.5 92.7
2
3308 3165
4239 4275
93.6 92.8
10 1
3463 3620
4516 4774
92.6 90.8
2
3476 3616
4520 4774
92.6 90.8
11 1
2969 2883
4045 4130
94.4 93.4
2
2745 2932
3918 4108
94.3 93.6
12 1
3516 3744
4507 4794
92.0 90.1
2
3533 3548
4529 4712
92.0 91.1
Mean
3427 3517
4404 4571
92.8 91.4
SD
243.8 331.5
329.4 400.5
2.5 2.8
CV (%)
7.11 9.43
7.48 8.76
2.66 3.05
Excluding lab 1
Excluding lab 2
Mean
4352 4542
92.1 90.7
SD
292.1 405.8
1.4 1.5
CV (%)
6.71 8.94
1.54 1.66
WHO/BS/07.2071
Page 18
Table 3E Sample
E
Values reported by participants
Mn
Mm
%<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3140 3136
4797 4721
88.6 88.4
2
3171 3157
4868 4693
89.5 88.7
2 1
3363 2815
3925 3871
99.5 97.0
2
3093 3036
3786 3755
98.7 98.6
3 1
2972 3140
4151 4371
91.7 90.7
2
2996 3207
4238 4488
91.0 89.6
4 1
2886 2999
4247 4482
91.9 90.4
2
2821 2850
4181 4354
92.3 90.8
5 1
3074 3229
4292 4551
91.4 89.4
2
3050 3174
4256 4498
91.6 89.7
6 1
3096 3251
4292 4547
91.1 89.0
2
3085 3212
4349 4583
90.4 88.5
7 1
3055 3136
4284 4467
90.8 89.9
2
3005 3158
4253 4507
91.0 89.1
8 1
3696 2737
4063 4162
93.0 93.0
2
2660 3167
3982 4430
94.0 91.0
9 1
3032 2821
4266 4312
91.5 90.6
2
3026 2814
4258 4303
91.5 90.7
10 1
2796 2886
4208 4447
92.2 90.5
2
2814 2883
4215 4451
92.2 90.4
11 1
2783 2666
4209 4293
91.9 90.7
2
2576 2747
4066 4273
92.0 91.1
12 1
2917 3105
4190 4462
91.8 90.1
2
2894 2756
4279 4538
91.4 90.6
Mean
3000 3003
4236 4398
92.1 90.8
SD
227.0 190.4
226.2 222.2
2.4 2.4
CV (%)
7.57 6.34
5.34 5.05
2.60 2.64
Excluding lab 1
Excluding lab 2
Mean
4181 4370
91.5 90.1
SD
137.4 210.1
1.1 1.1
CV (%)
3.29 4.81
1.21 1.17
WHO/BS/07.2071
Page 19
Table 3F Sample
F
Values reported by participants
Mn
Mm
%<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3795 3794
6653 6328
79.5 79.3
2
3825 3804
6703 6176
79.4 79.9
2 1
3861 3461
4309 4376
99.9 96.6
2
3533 3479
4292 4268
97.1 96.9
3 1
3554 3752
5134 5399
83.5 82.0
2
3581 3823
5225 5530
82.6 80.6
4 1
3639 3808
5382 5608
84.1 81.7
2
3443 3505
5224 5509
84.3 82.2
5 1
3689 3885
5279 5628
83.2 80.3
2
3674 3845
5262 5606
83.3 80.3
6 1
3660 3850
5283 5621
82.3 79.5
2
3651 3812
5279 5585
82.3 79.7
7 1
3706 3819
5292 5570
82.6 79.8
2
3603 3775
5196 5517
83.0 80.3
8 1
3337 3407
5140 5312
85.0 84.0
2
3250 3817
4944 5501
87.0 82.0
9 1
3594 3401
5188 5339
83.7 82.5
2
3622 3438
5227 5393
83.5 82.3
10 1
3408 3537
5162 5490
84.9 82.4
2
3423 3532
5149 5484
85.0 82.4
11 1
3503 3413
5356 5554
83.4 81.6
2
3466 3690
5361 5608
82.9 81.5
12 1
3515 3741
5157 5514
84.1 81.5
2
3527 3441
5246 5650
83.8 82.6
Mean
3577 3660
5268 5482
84.6 82.6
SD
148.7 177.9
513.9 422.4
4.6 4.5
CV (%)
4.16 4.86
9.75 7.71
5.42 5.48
Excluding lab 1
Excluding lab 2
Mean
5140 5412
83.3 81.3
SD
287.7 365.0
1.7 1.3
CV (%)
5.60 6.75
1.99 1.55
WHO/BS/07.2071
Page 20
Table 3G Sample
G
Values reported by participants
Mn
Mm
%<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3800 3794
8215 7449
75.5 75.3
2
3819 3791
8877 7420
75.5 76.0
2 1
3940 3522
4468 4663
99.0 93.9
2
3548 3491
4471 4449
95.6 95.4
3 1
3470 3647
5428 5722
80.2 78.6
2
3390 3617
5433 5766
79.9 77.9
4 1
3343 3467
5625 6062
80.9 78.5
2
3406 3454
5582 5949
81.2 78.9
5 1
3609 3794
5527 5911
80.2 77.1
2
3599 3757
5499 5871
80.4 77.4
6 1
3555 3733
5527 5893
79.4 76.3
2
3541 3688
5534 5868
79.3 76.5
7 1
3578 3681
5584 5923
78.9 77.5
2
3353 3708
5546 5907
79.5 76.7
8 1
3173 3260
5197 5461
83.0 82.0
2
3190 3774
5318 5788
83.0 79.0
9 1
3604 3376
5636 5950
79.7 78.4
2
3596 3367
5644 5969
79.7 78.4
10 1
3305 3413
5535 5925
81.2 78.6
2
3321 3408
5496 5900
81.4 78.7
11 1
3382 3268
5567 5805
81.0 79.1
2
3268 3344
5581 5836
80.4 79.0
12 1
3447 3666
5509 5937
80.5 77.8
2
3481 3340
5656 6200
80.0 78.8
Mean
3488 3557
5686 5901
81.5 79.4
SD
192.1 182.3
939.9 616.6
5.2 4.9
CV (%)
5.51 5.13
16.53 10.45
6.38 6.16
Excluding lab 1
Excluding lab 2
Mean
5426 5762
80.0 78.0
SD
327.1 414.8
1.8 1.4
CV (%)
6.03 7.20
2.25 1.81
WHO/BS/07.2071
Page 21
Table 4A
Recalculated results Sample A
Mn Mm %<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
5003 5049
6055 6081
80.3 80.0
2
4957 5062
6020 6138
80.6 79.5
5 1
4910 4946
6025 5989
80.5 80.7
2
4947 4893
6009 6017
80.3 80.5
6 1
4970 4992
6053 6060
79.4 79.5
2
4956 4935
6059 6053
79.3 79.5
9 1
4896 4850
5967 5963
80.8 80.6
2
4918 4912
5977 6016
80.8 80.4
11 1
4756 4561
5998 5897
81.4 81.1
2
4732 4498
6011 5861
80.6 81.1
12 1
4852 4862
5919 5936
81.4 80.9
2
4864 4857
5936 5929
81.3 81.2
Mean
4897 4868
6002 5995
80.6 80.4 SD
83.7 173.4
45.5 81.4
0.7 0.6
CV
1.71 3.56
0.76 1.36
0.8 0.8
WHO/BS/07.2071
Page 22
Table 4B
Recalculated results Sample B
Mn Mm %<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3381 3410
4227 4263
94.4 94.3
2
3368 3434
4226 4314
94.4 93.9
5 1
3347 3377
4225 4294
94.1 93.9
2
3368 3332
4298 4212
93.8 94.1
6 1
3366 3361
4251 4223
93.6 93.9
2
3331 3306
4233 4173
93.6 94.1
9 1
3318 3202
4196 4102
94.4 94.4
2
3307 3315
4195 4193
94.3 94.2
11 1
3155 2918
4160 4026
94.5 94.4
2
3200 2932
4148 3991
94.8 95.0
12 1
3268 3280
4159 4169
94.4 94.4
2
3293 3282
4188 4182
94.4 94.4
Mean
3309 3262
4209 4179
94.2 94.3 SD
70.7 169.4
43.2 98.4
0.4 0.3
CV
2.10 5.20
1.03 2.36
0.4 0.3
WHO/BS/07.2071
Page 23
Table 4C
Recalculated results Sample C
Mn Mm %<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
4695 4726
6861 6846
69.5 69.2
2
4674 4763
6755 6885
70.0 69.0
5 1
4605 4615
6656 6655
70.3 70.3
2
4610 4589
6683 6654
70.0 70.3
6 1
4585 4640
6625 6694
69.9 69.8
2
4542 4577
6655 6682
69.9 70.1
9 1
4562 4431
6582 6511
70.9 71.5
2
4573 4606
6605 6635
70.8 70.4
11 1
4549 4298
6873 6739
70.6 70.3
2
4585 4284
6892 6622
70.3 70.9
12 1
4625 4643
6918 6924
70.0 69.6
2
4652 4640
6992 6962
69.8 69.7
Mean
4605 4568
6758 6734
70.2 70.1 SD
48.9 152.4
141.7 139.0
0.4 0.7
CV
1.06 3.34
2.10 2.06
0.6 1.0
WHO/BS/07.2071
Page 24
Table 4D
Recalculated results Sample D
Mn Mm %<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3656 3689
4549 4585
92.2 92.1
2
3666 3740
4481 4574
93.2 92.7
5 1
3628 3650
4577 4574
91.6 91.8
2
3642 3613
4580 4564
91.6 91.6
6 1
3634 3623
4596 4587
91.1 91.2
2
3620 3605
4599 4584
91.0 91.1
9 1
3302 3486
4244 4406
93.5 92.5
2
3308 3601
4239 4516
93.6 92.1
11 1
3507 3278
4491 4367
92.7 92.4
2
3582 3329
4537 4386
92.4 92.6
12 1
3552 3563
4468 4479
92.6 92.4
2
3580 3570
4488 4482
92.6 92.6
Mean
3556 3562
4487 4509
92.3 92.1 SD
126.0 136.9
123.8 83.4
0.9 0.6
CV
3.54 3.84
2.76 1.85
0.9 0.6
WHO/BS/07.2071
Page 25
Table 4E
Recalculated results Sample E
Mn Mm %<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3103 3113
4272 4301
92.7 91.5
2
3091 3156
4279 4363
92.7 91.1
5 1
3055 3086
4264 4303
91.5 91.3
2
3074 3042
4308 4252
91.1 91.5
6 1
3096 3107
4292 4282
91.1 91.2
2
3085 3076
4349 4326
90.4 90.7
9 1
3032 2934
4266 4160
91.5 91.8
2
3026 3039
4258 4260
91.5 91.4
11 1
2810 2557
4150 4019
92.4 92.2
2
2890 2593
4197 4036
92.0 92.3
12 1
2958 2975
4169 4182
92.2 92.0
2
2969 2958
4183 4177
92.1 92.1
Mean
3016 2970
4249 4222
91.8 91.6 SD
92.2 196.0
60.8 109.8
0.7 0.5
CV
3.06 6.60
1.43 2.60
0.8 0.5
WHO/BS/07.2071
Page 26
Table 4F
Recalculated results Sample F
Mn Mm %<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3941 3735
5278 5298
83.4 83.2
2
3653 3775
5269 5371
83.5 82.6
5 1
3672 3691
5255 5257
83.3 83.3
2
3681 3657
5271 5246
83.1 83.3
6 1
3660 3668
5283 5289
82.3 82.5
2
3651 3620
5279 5262
82.3 82.6
9 1
3594 3494
5188 5137
83.7 83.9
2
3622 3643
5227 5240
83.5 83.3
11 1
3534 3270
5314 5194
83.6 83.3
2
3635 3334
5316 5150
83.4 83.8
12 1
3569 3586
5153 5169
84.2 83.9
2
3569 3557
5177 5168
84.1 84.0
Mean
3648 3586
5251 5232
83.4 83.3 SD
103.1 153.1
53.2 70.0
0.6 0.5
CV
2.8 4.3
1.01 1.34
0.7 0.6
WHO/BS/07.2071
Page 27
Table 4G
Recalculated results Sample G
Mn Mm %<8000
Lab Run
90/686 05/112
90/686 05/112
90/686 05/112
1 1
3673 3697
5602 5611
80.0 79.7
2
3653 3715
5496 5602
80.6 79.7
5 1
3631 3660
5525 5600
80.1 79.7
2
3649 3616
5617 5519
79.5 80.2
6 1
3555 3548
5527 5563
79.4 79.3
2
3541 3557
5534 5555
79.3 79.4
9 1
3604 3505
5636 5525
79.7 80.3
2
3596 3609
5644 5652
79.7 79.5
11 1
3446 3169
5552 5426
81.0 80.7
2
3513 3186
5488 5314
80.9 81.4
12 1
3544 3565
5528 5541
80.5 80.2
2
3592 3580
5564 5550
80.4 80.4
Mean
3583 3534
5559 5538
80.1 80.0 SD
66.1 177.5
53.3 90.8
0.6 0.6
CV
1.85 5.02
0.96 1.64
0.7 0.8
WHO/BS/07.2071
Page 28
Figures
Figure 1: Stability of ampoules of the 1st IRR LMWH for MW calibration 90/686 at 56 ºC over the
years 1992-2000. Mp = peak molecular weight, Mn = number average molecular weight, Mw =
weight average molecular weight.
AD 56 deg. 90/686
2000
3000
4000
5000
6000
7000
1990 1992 1994 1996 1998 2000 2002
year
MW Mp
Mn
Mw
WHO/BS/07.2071
Page 29
Histograms of Mn, Mw, and %>8000 for sample A-G. Results calculated with 90/686 as calibrant
are shown in grey boxes, and with 05/112 in white boxes. Laboratory numbers are indicated in the
boxes.
Fig. 2: Histograms for sample A
Sample A - Mn
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000
2 2 10
11
11
12
3
4
8
9
3
4
5
6
7
7
8
9
1
1
10
12
5
6
Sample A - Mn recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000
11 11
12
12
9
9
1
1
5
5
6
6
Sample A - Mw
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000
2
2
3 10
11
12
4
5
6
7
8
9
10
3
8
9
11
12
4
5
6
7
1 1
Sample A - Mw, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000
11 1
1
11
12
12
5
5
6
6
9
9
Sample A - percent less than 8000
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1
1
6
5 12
7
11
3
4
6
9
10
7
11
12
3
5
8
9
4
8
10 2 2
Sample A - percent less than 8000, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
6 1
1
6
11
11
12
12
5
5
9
9
WHO/BS/07.2071
Page 30
Fig. 3: Histograms for sample B
Sample B - Mn
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
2 11
11
8
10
12
2
3
4
8
9
1
1
10
12
3
4
5
6
7
7
9
5
6
Sample B - Mn recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11 11
12
12
9
1
1
5
5
6
6
9
Sample B - Mw
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
2 2 10
11
11
12
3
4
5
6
8
8
9
9
1
1
10
3
4
5
12
6
7
7
Sample B - Mw, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
11 1
1
11
12
12
5
5
6
6
9
9
Sample B - percent less than 8000
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
7 7 1
1
5
6
10
11
12
3
11
12
3
4
5
6
8
9
9
10
4
8
2 2
Sample B - percent less than 8000, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1
1
11
12
12
5
5
6
6
9
9
11
WHO/BS/07.2071
Page 31
Fig. 4: Histograms for sample C
Sample C - Mn
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
2 10
2
3
4
8
9
10
11
11
12
4
5
6
7
8
9
12
3
6
7
1
1
5
Sample C - Mn recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
11 9 1
11
12
12
5
5
6
6
9
1
Sample C - Mw
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
4000
4400
4800
5200
5600
6000
6400
6800
7200
7600
8000
8400
8800
9200
9600
10000
10400
10800
11200
11600
12000
2 2 3
6
9
10
5
7
11
12
3
6
8
9
4 10
11
5
7
8
12 4 1 1
Sample C - Mw, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000
5
5
6
6
9
9
1
1
11
11
12
12
Sample C - percent less than 8000
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1
1
5
6
12
7
10
11
4
3
7
8
9
12
4
5
6
8
10
11
3
9
2 2
Sample C - percent less than 8000, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1 1
11
12
12
5
5
6
6
11
9
9
WHO/BS/07.2071
Page 32
Fig. 5: Histograms for sample D
Sample D - Mn
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11 11
2
9 10
2
3
4
8
8
9
1
10
12
3
4
5
6
7
1
12
5
6
7
Sample D - Mn recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11 9
9
1
1
11
12
12
5
5
6
6
Sample D - Mw
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
2 2 11 11
9
9
3
8
8
10
12
3
4
5
6
7
1
10
12
4
5
6
7
1
Sample D - Mw, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
9 11
11
12
12
9
1
1
5
5
6
6
Sample D - percent less than 8000
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1
1
6
12
5
7
10
3
4
6
7
12
3
4
5
10
11
8
9
11
8
9
2
2
Sample D - percent less than 8000, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
6
6
1
1
11
12
5
5
11
12
9
9
WHO/BS/07.2071
Page 33
Fig. 6: Histograms for sample E
Sample E - Mn
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11 10
10
11
2
4
8
9
12
3
4
5
6
7
9
1
1
12
3
5
6
7
2 8
Sample E - Mn recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11 11 12
12
5
5
6
9
9
1
1
6
Sample E - Mw
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
2 2
8
10
11
11
12
3
4
5
6
7
8
9
10
12
3
4
7
9
5
6
1
1
Sample E - Mw, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000
11 1
11
12
12
5
6
6
9
9
1
5
Sample E - percent less than 8000
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1 1
5
6
12
4
7
10
11
3
5
6
7
9
10
11
12
3
4
9
8
8
2 2
Sample E - percent less than 8000, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
5
6
6
1
11
11
12
12
5
9
9
1
WHO/BS/07.2071
Page 34
Fig 7; Histograms for sample F
Sample F - Mn
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
10
11
2
8
8
9
10
11
12
3
4
5
6
9
1
1
12
2
3
4
5
6
7
7
Sample F - Mn recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11 9 11
12
12
5
5
6
6
9
1 1
Sample F - Mw
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000
2
2
10
12
3
5
6
7
8
9
10
11
3
4
8
9
11
12
4
5
6
7
1 1
Sample F - Mw, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000
1
1
11
12
12
5
5
6
6
9
9
11
Sample F - percent less than 8000
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1 1
5
6
7
10
11
12
3
4
6
11
5
7
9
12
3
4
8
9
10
8
2 2
Sample F - percent less than 8000, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
6 1
1
11
5
5
6
11
12
12
9
9
WHO/BS/07.2071
Page 35
Figure 8: Histograms for sample G
Sample G - Mn
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11
8
8
10
10
11
12
3
4
4
9
12
2
3
5
6
7
7
9
1
1
5
6
2
Sample G - Mn recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800 5000
11 11 1
1
12
12
5
5
6
6
9
9
Sample G - Mw
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000
2 2 8 3
8
10
11
12
4
5
6
7
9
11
3
6
10
12
4
5
7
9
1
Sample G - Mw, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
M
4000 4200 4400 4600 4800 5000 5200 5400 5600 5800 6000 6200 6400 6600 6800 7000 7200 7400 7600 7800 8000
11 1
1
11
12
12
5
5
6
6
9
9
Sample G - percent less than 8000
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
1 1
6
5 12
7
9
10
11
3
4
6
7
3
5
9
10
11
12
4
8 8 2 2
Sample G - percent less than 8000, recalculated
0
1
2
3
4
5
6
7
8
9
10
11
12
%
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
6
6
1
1
12
5
5
9
9
11
11
12
WHO/BS/07.2071
Page 36
Appendix 1:
LIST OF THE PARTICIPANTS PROVIDING DATA FOR PHASES 1 AND 2 OF THE
STUDY
Dr. T. Zimmermann and Dr. M. Loch
Zentrales Institutt des Sanitätsdienstes der Bundeswehr München
Germany
Dr. A da Silva
INFARMED Biology and Biotechnology Laboratory
Portugal
Dr J. Fareed,
Loyola University Medical Center#Pathology Department
USA
Dr. C Bauer
Momenta Pharmaceuticals, Inc.
USA
Dr G. Rautmann
EDQM
France
Dr S. Sennert
Nordmark Arzneimittel GmbH & Co. KG
Germany
Dr L. Husager
Danish Medicines Authority
Denmark
Dr K. Johansen
Leo Pharma
Denmark
Dr B. Mulloy and Ms. A.-M. Hackett
National Institute for Biological Standards and Control
UK
Dr. Huihong Fan
National Institute for the Control of Pharmaceutical and Biological Products
China
Dr. L. Liverani
WHO/BS/07.2071
Page 37
Opocrin. S.p.A
Italy
Dr T. Freudemann, Dr. R. Domanig
Sandoz GmbH
Austria
Dr V. Dupont, Dr. C. Houiste
Sanofi Aventis
France
Dr C. Loh
TGA
Australia
WHO/BS/07.2071
Page 38
Appendix 2: Proposed Instruction For Use and Material Safety Data
PROPOSED 2ND INTERNATIONAL REFERENCE REAGENT LOW MOLECULAR WEIGHT
HEPARIN FOR MOLECULAR WEIGHT CALIBRATION
05/112
DRAFT VERSION 1, DATED August 6, 2007
This material is not for in vitro diagnostic use
1. CAUTION
THIS PREPARATION IS NOT FOR ADMINISTRATION TO HUMANS.
The preparation does not contain material of human origin.
As with all materials of biological origin, this preparation should be regarded as potentially
hazardous to health. It should be used and discarded according to your own laboratory's safety
procedures. Such safety procedures probably will include the wearing of protective gloves and
avoiding the generation of aerosols. Care should be exercised in opening ampoules or vials, to
avoid cuts.
2. DIRECTIONS FOR OPENING THE DIN AMPOULE
DIN ampoules have an „easy-open‟ coloured stress point, where the narrow ampoule stem joins
the wider ampoule body.
Tap the ampoule gently to collect the material at the bottom (labelled) end. Ensure that the
disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and
against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the
disposable ampoule breaker covering the ampoule stem between the thumb and first finger of the
other hand. Apply a bending force to open the ampoule at the coloured stress point, primarily
using the hand holding the plastic collar.
Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for
example, by the use of suitable gloves and an eye shield. Take care that no material is lost from
the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas at slightly
less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN
ampoule.
3. DIRECTIONS FOR USE
The calibrant is intended for use in the determination of the molecular weight distribution of low
molecular weight heparins by high-performance gel permeation chromatography. It may be used
to calibrate a chromatography system by broad standard calibration; the table appended as
Appendix 1 has been found suitable.
WHO/BS/07.2071
Page 39
4. CITATION
In all publications (or data sheets for immunoassay kits) in which this preparation is used as an
assay calibrant, it is important that the title of the preparation, ampoule code and the name and
address of NIBSC are cited and cited correctly.
3. PRODUCT LIABILITY
Information emanating from NIBSC is given after the exercise of all reasonable care and skill in
its compilation, preparation and issue, but is provided without liability in its application and use.
This product is intended for use as a standard or reference material in laboratory work in relation
to biological research, manufacturing or quality control testing of biological products or in the
field of in vitro diagnostics. It is the responsibility of the user to ensure that he/she has the
necessary technical skills to determine the appropriateness of this product for the proposed
application. Results obtained from this product are likely to be dependent on conditions of use
and the variability of materials beyond the control of NIBSC.
NIBSC accepts no liability whatsoever for any loss or damage arising from the use of this
product, whether loss of profits, or indirect or consequential loss or otherwise, including, but not
limited to, personal injury other than as caused by the negligence of NIBSC. In particular,
NIBSC accepts no liability whatsoever for:-
(i) results obtained from this product; and/or
(ii) non-delivery of goods or for damages in transit.
In the event of any replacement of goods following loss or damage a customer accepts as a
condition of receipt of a replacement product, acceptance of the fact that the replacement is not to
be construed as an admission of liability on NIBSC's behalf.
WHO/BS/07.2071
Page 40
LOW MOLECULAR WEIGHT HEPARIN
NIBSC code: 05/112
MATERIAL SAFETY SHEET
Physical properties (at room temperature)
Physical appearance Solid
Fire hazard None
Chemical properties
Stable Yes Corrosive: No
Hygroscopic Yes Oxidising: No
Flammable No Irritant: Yes
Other (specify) Contains material of porcine origin
Toxicological properties
Effects of inhalation: No adverse effects reported for this material
Effects of ingestion: No adverse effects reported for this material
Effects of skin absorption: No adverse effects reported for this material
Suggested First Aid Inhalation Seek medical advice
Ingestion Seek medical advice
Contact with eyes Wash with copious amounts of water. Seek medical advice
Contact with skin Wash thoroughly with water
Action on Spillage and Method of Disposal
Spillage of ampoule contents should be taken up with absorbent material wetted with a
virucidal agent. Rinse area with a virucidal agent followed by water.
Absorbent materials used to treat spillage should be treated as biologically hazardous
waste.
Compiled by: Barbara Mulloy Date: 23/06/2006
WHO/BS/07.2071
Page 41
APPENDIX 1: BROAD STANDARD TABLE FOR 05/112
(LMW Heparin for Molecular Weight Calibration Proposed 2nd International Reference Reagent)
Point Log10(M) M % >M % <M
1 2.78 600 0.40 99.60
2 3.08 1200 3.87 96.13
3 3.26 1800 8.94 91.06
4 3.38 2400 14.49 85.51
5 3.48 3000 20.68 79.32
6 3.56 3600 27.20 72.80
7 3.62 4200 33.89 66.11
8 3.68 4800 40.49 59.51
9 3.73 5400 46.83 53.17
10 3.78 6000 52.92 47.08
11 3.82 6600 58.59 41.41
12 3.86 7200 63.89 36.11
13 3.92 8400 72.96 27.04
14 3.98 9600 80.09 19.91
15 4.08 12000 89.21 10.79
16 4.13 13600 92.96 7.04
17 4.19 15600 95.95 4.05
18 4.26 18000 97.77 2.23