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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 2 nd International Standard Low Molecular Weight Heparin for Molecular Weight Calibration Barbara Mulloy 1 , 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.

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Page 1: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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.

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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.

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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.

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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.

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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

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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

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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

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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.

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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.

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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.

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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

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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

Page 13: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

WHO/BS/07.2071

Page 13

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

Page 14: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

WHO/BS/07.2071

Page 14

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

Page 15: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 16: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 17: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 18: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 19: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 20: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 21: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 22: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 23: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 24: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 25: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 26: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 27: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 28: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 29: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 30: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 31: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 32: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 33: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

Page 34: EXPERT COMMITTEE ON BIOLOGICAL STANDARDISATION LMWH.pdf · Nadroparin calcium (1134), Parnaparin sodium (1252), Tinzaparin sodium (1271)). All the currently practicable methods of

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

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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

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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

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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

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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.

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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.

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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

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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