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WHO/BS/2014.2237
ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION
Geneva, 13 to 17 October 2014
WHO International Collaborative Study of the proposed 1st International
Standard for human Proinsulin
Melanie Moore*, Jackie Ferguson, Peter Rigsby, Jason Hockley and Chris Burns
National Institute for Biological Standards and Control,
Blanche Lane, South Mimms,
Potters Bar, Herts, EN6 3QG, UK
*Corresponding author: Melanie Moore
+44 (0) 1707 641000
[email protected] or [email protected]
NOTE:
This document has been prepared for the purpose of inviting comments and suggestions on the
proposals contained therein, which will then be considered by the Expert Committee on
Biological Standardization (ECBS). Comments MUST be received by 4 October 2014 and
should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:
Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to
the Responsible Officer: Dr Jongwon Kim at email: [email protected].
© World Health Organization 2014
All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20
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The designations employed and the presentation of the material in this publication do not imply the expression of any opinion
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arising from its use. The named authors alone are responsible for the views expressed in this publication.
WHO/BS/2014.2237
Page 2
Summary
The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS)
has recognized (2010) the need for a replacement for the International Reference Reagent (IRR) for
human proinsulin, coded 84/611. We report here the characterization of a candidate standard for
human proinsulin in an international collaborative study carried out by seventeen laboratories in
nine countries. Phase I of the study confirmed by amino acid analysis and UV spectroscopy that the
primary calibrant contained 220 micrograms per vial. The candidate standard, in ampoules coded
09/296, was then calibrated by HPLC analysis in terms of the primary calibrant (phase II) and
further characterized by immunoassay in comparison to 84/611 (phase III). Estimates from the
HPLC calibration indicated the content of 09/296 to be 7.0 micrograms per ampoule. The results
of this study also indicate that the candidate standard shows appropriate immunological activity and
appears sufficiently stable on the basis of a thermally accelerated degradation study, and is thus
suitable to serve as an International Standard for immunoassays of human proinsulin.
Introduction Human proinsulin is a peptide of 86 amino acids, 9390 Da, which is synthesized by pancreatic
beta cells before being enzymatically cleaved to insulin and C-peptide. Elevated serum
concentrations of proinsulin are indicative of insulinomas, diabetes mellitus and other disorders
of glucose metabolism. Measurements of proinsulin in serum and plasma by immunoassays
contribute to the diagnosis of insulinoma and are used to monitor beta cell dysfunction.
The current IRR for human proinsulin, coded 84/611 was established in 1986 and comprises
ampoules of recombinant human proinsulin with a content of 6µg/ampoule assigned through
comparison to local standards in three independent laboratories by ELISA or RIA [1]. Stocks of
84/611 are exhausted and there is a requirement to replace this standard and a proposal for a new
WHO International Standard for human proinsulin was endorsed by the WHO Expert Committee
on Biological Standardisation (ECBS) at its meeting held in October 2010. The proposed
standard was prepared using a batch of human proinsulin which was filled into ampoules, coded
09/296, following procedures recommended by WHO [2]. An international collaborative study
was organised by NIBSC to assign a mass content to the standard and to evaluate the suitability
of 09/296 to serve as an International Standard for the calibration of immunoassays of human
proinsulin.
The international collaborative study was set up in three phases. In phase I, a primary calibrant
prepared from the same bulk material as 09/296 was assigned a mass content by quantitative
amino acid analysis and UV spectroscopy. This value was then used in phase II of the study to
calibrate the candidate standard 09/296 in terms of the primary calibrant by HPLC. In this study,
the effect of accelerated thermal degradation on the candidate standard was also assessed by
HPLC in order to enable a prediction of its long term stability. In phase III, 09/296 was
compared to the current standard 84/611 by immunoassay to assess its suitability to serve as an
International Standard. Participants were also requested to determine the proinsulin
concentration of five human serum samples in order to assess the impact of the introduction of
the candidate preparation on the routine measurement of proinsulin in native samples.
The aims of the study, therefore, were:
Phase I: to assign a mass value to the primary calibrant, PC01, by amino-acid analysis (AAA)
and by UV spectroscopy.
WHO/BS/2014.2237
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Phase II: to calibrate the preparation, 09/296, in terms of the primary calibrant by HPLC and to
assess the effect of accelerated thermal degradation by HPLC.
Phase III: to obtain additional confirmatory data from immunoassays as to its suitability to serve
as a standard, to establish correlation with the current IRR, 84/611.
Participants
Seventeen laboratories in nine countries took part in the study and are listed alphabetically, by
country, in Table 1. Throughout the study, each participating laboratory is referred to by a code
number. These code numbers were randomly assigned and do not reflect the order of listing.
Table 1: List of participants in order of country
AUSTRALIA Dr Kevin Grant
TGA Laboratories, PO Box 100, Woden, ACT 2606.
BELGIUM Alain Bosseloir
ZenTech S.A., Liège Science Park, Avenue du Pré-Aily, 10 4031 Angleur.
BRAZIL Professor Sergio Dalmora
Department of Industrial Pharmacy, Federal University of Santa Maria,
97105-900-Santa Maria – RS.
CANADA Dr Michel Girard and Barry Lorbetskie
Centre for Biologics Research, Banting Building, Tunney's Pasture, Ottawa, Ontario.
CHINA Dr Chenggang Liang
National Institutes for Food and Drug Control, No. 2 Tiantan Xili, Beijing 100050.
NORWAY Rolf Hovik
Pharmaceutical Department, Biological Section, Norwegian Medicines Agency, Sven
Oftedals vei 6, NO-0950 Oslo.
SWEDEN Robert Gunnarsson, Eleni Karamihos and Hanna Ritzen
Mercodia AB, Sylveniusgatan 8A, SE-754 50, Uppsala.
UK Mr Tristan Coleman and Dr John Fox
AltaBioscience, Building Y10, University of Birmingham, Edgebaston, Birmingham, B15
2TT.
UK Dr Adrian Bristow, Dr Carl Dolman, Dr Jackie Ferguson, Dr Melanie Moore, Richard
Tiplady and Dr CT Yuen
National Institute for Biological Standards and Control, Blanche Lane,
South Mimms, Potters Bar, Herts, EN6 3QG.
UK Mr Gareth Dunseath
Diabetes Research Network Wales Laboratory, Diabetes Research Group,
3rd
floor, Institute of Life Sciences, Swansea University, Singleton Park,
Swansea, SA2 8PP.
UK Dr David Halsall and Keith Burlington
Core Biochemical Assay Laboratory, Box 232, Addenbrooke’s Hospital, Cambridge
university Hospital’s NHS Foundation Trust, Hills Road, Cambridge, CB2 0QQ.
UK Mr Peter Sharratt
Protein and Nucleic Acid Facility, Department of Biochemistry,
University of Cambridge, 80, Tennis Court Road, Cambridge, CB2 1GA.
UK Dr Gwen Wark
WHO/BS/2014.2237
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Surrey Pathology Services, SAS Peptide Hormones Section, Clinical Laboratory, Royal
Surrey County Hospital,Guildford, Surrey, GU2 7XX.
UK Dr Stuart Woodhead Invitron Limited,Wyastone Business Park, Wyastone Leys, Monmouth, NP25 3SR.
USA Mr Larry Dangott and Ms Jinny Johnson
Protein Chemistry Laboratory, Texas A&M University, Department of Biochemistry and
Biophysics, Rooms 440-442, 300 Olsen Blvd, TAMU 2128, College Station, TX 77843-
2128.
USA Dr Christine E. Onthank
Diabetes & Obesity, ALPCO Diagnostics, 26-G Keewaydin Drive, Salem, NH 03079.
USA Dr Jack Presley and Dr John Schulze
Molecular Structure Facility, Rm 8 Hutchinson Hall, University of California,
Davis, CA95616.
Bulk Materials and Processing
Human proinsulin, Lot RS30019, prepared by recombinant DNA technology in E. coli was
generously donated to the WHO by Eli Lilly and Co, Indianapolis, USA, through the good
offices of Dr. B. Frank. The material was reported by the manufacturer to be 97% pure by HPLC
and the amino-acid analysis of the material was consistent with the sequence of human
proinsulin.
Preparation of the Primary Calibrant, PC01
One vial of human proinsulin, Lot RS30019, nominally containing approximately 11.9 mg
human proinsulin was reconstituted in a 5 ml volume of 10 mM Sodium Phosphate pH 7.0,
0.5 % (w/v) trehalose and diluted to a final volume of 23.8 ml with the same buffer. The human
proinsulin solution was distributed into ampoules on 01 June 2010 as 0.5 g aliquots using a
Hamilton M510B dispenser. The ampoule contents were freeze dried using a Virtis Genesis
25EL, and flame sealed after vacuum was released to dry nitrogen. All processes were performed
within the Standards Processing Division of NIBSC. Ampoules were stored at -20°C at NIBSC.
A final total of 38 ampoules of primary calibrant, PC01, were obtained with a mean fill mass of
0.4991 g (CV 0.01%, n = 3). Each ampoule of human proinsulin primary calibrant, PC01, contained
approximately 250 µg human proinsulin, 0.41 mg di-sodium hydrogen phosphate anhydrous,
0.29 mg sodium di-hydrogen phosphate monohydrate and 2.5 mg trehalose.
Preparation of ampoules of the Candidate Standard, 09/296
Three vials of human proinsulin, Lot RS30019, each nominally containing approximately 12 mg
human proinsulin were reconstituted in a 5 ml volume of 10 mM Sodium Phosphate pH 7.0, 0.5
% (w/v) trehalose and diluted to a final volume of 1800 ml with the same buffer. The human
proinsulin solution was filtered through a 0.45 µm filter and distributed into ampoules on 08
March 2010 as 0.5 g aliquots using a Bausch & Strobel Filling Machine AFV5090. The ampoule
contents were freeze dried and sealed under nitrogen using a Serail CS15 freeze dryer according
to procedures recommended by the WHO [2]. All processes were performed within the
Standards Processing Division of NIBSC. Ampoules were stored at -20 °C at NIBSC.
A total of 2972 ampoules of human proinsulin candidate standard, 09/296, were obtained with a
mean fill mass of 0.5176 g (CV 0.44%, n = 107), a mean dry weight of 0.0032 (CV 6.88 %, n = 4),
WHO/BS/2014.2237
Page 5
mean residual moisture as determined by Karl Fischer titration of 0.9602 % (CV 17.02 %, n = 12)
and a mean head space oxygen of 0.07% (CV% 12.46, n = 12). Although NIBSC does not routinely
perform an automated 100% post-sealing check on container integrity, visual checks on sealing
quality and container integrity were made at the time of sealing. Further checks on container
integrity and sealing quality will be made whilst labeling and again at the time of picking material
for dispatch to customers. After processing, quality control and collaborative study use, a final total
of 2733 ampoules of human proinsulin candidate standard, 09/296, are now available to WHO.
NIBSC will act as custodian of the preparation which is stored under assured temperature controlled
conditions within the Institutes’ Centre for Biological Reference Materials, at Potters Bar,
Hertfordshire, UK.
Although participants were provided with a nominal content of 10 µg human proinsulin for the
candidate standard, our knowledge of the actual proinsulin content of the primary calibrant (220 µg
per ampoule), suggests that each ampoule of human proinsulin candidate standard, 09/296 would be
expected to contain approximately 8.5 µg human proinsulin, 0.41 mg di-sodium hydrogen
phosphate anhydrous, 0.29 mg sodium di-hydrogen phosphate monohydrate and 2.5 mg trehalose.
Collaborative Study for the calibration of 09/296
Materials provided for the collaborative study
The preparations of human proinsulin provided to participants at each phase of the study are
listed in Table 2. In addition to the ampouled preparations, participants in Phase III of the study
were provided with five human serum samples, coded A-E. Participants were requested to assay
the serum samples in the same immunoassays as the ampouled preparations and their house
standards. Serum samples coded A, B and C, from individual human donors, were obtained from
Dr G. Wark (Surrey Pathology Services, Guildford, UK). Each serum sample was tested at
NIBSC and found to be non-reactive for HCV RNA, anti-HIV 1/2 and HBsAg and the inclusion
of the serum samples in this study was approved by local ethics committee. Serum samples D
and E, from individual human donors, were obtained from First Link UK (Wolverhampton, UK).
Samples were certified non-reactive for HIV 1/2, HIV p24, HBsAg, anti-HCV and Syphilis TP
by the supplier.
Table 2: Human proinsulin preparations supplied to participants in the collaborative study.
Human Proinsulin preparation Ampoule unitage and
nominal content Allocation in study
IRR 84/611 6 µg/ampoule Phase III
Primary calibrant, PC01
Assumed 240 µg/ampoule
for phase I (initially
corrected to 214
µg/ampoule) for Phase II
Phases I and II
Candidate standard 09/296 stored at -20°C
Assumed content
approximately 10
µg/ampoule
Phases II and III
Accelerated thermal degradation (ATD)
samples of 09/296 stored at +4C, +20C,
+37C and +45C for 14 months
Contents assumed identical
to 09/296 Phase II
WHO/BS/2014.2237
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Study design and methods
The collaborative study was organised by NIBSC. Participants were provided with samples
based on the assays provided, assay capacity and sample availability. Some thermally
accelerated degradation samples were only available in limited numbers. The study protocols for
each phase of the study, shown in Appendices 2-4, and instructions for use were provided with
the samples.
Phase I: Amino acid analysis and UV spectroscopy for value assignment of PC01 Participants were requested to derive estimates of the human insulin content of the primary
calibrant, PC01, by amino acid analysis using their in-house method. Three vials of the primary
calibrant, which were available in very limited numbers, were provided and participants were
asked to carry out triplicate analytical runs for each vial. Participants providing UV
spectroscopy analyses were provided with 2 ampoules of the primary calibrant and were asked to
use the method normally run in the laboratory and to use the extinction coefficient provided in
their calculations.
Phase II: HPLC for purity evaluation and potency assay of 09/296 Using the assigned value of 220 µg per ampoule for the primary calibrant, PC01, derived from
the data provided in Phase I, participants in Phase II were requested to provide, in triplicate,
estimates of the proinsulin content of the candidate standard 09/296, its accelerated thermal
degradation samples and frozen baselines by comparison with PC01, using the HPLC method
used in house. An outline HPLC protocol was provided for guidance. Participants were also
requested to provide raw data and sample chromatograms of PC01, if possible, in addition to
their own estimates of proinsulin content per ampoule.
Phase III: Immunoassay of candidate standard, 09/296 Participants were requested to carry out the assay(s) normally in use in their laboratory and, where
possible, to perform at least two independent assays, using fresh ampoules, each assay to include all
of the preparations allocated at preferably no less than five dose levels in the linear part of the dose-
response curve. Handling instructions for the materials were included in the study protocol. In
instances where there was not a fresh ampoule for subsequent assays, it was suggested that fresh
dilutions be made from frozen stock solutions. Where dilutions of a stored stock solution were used,
participants were asked to provide details of its storage and identification of the initial preparation.
Participants were also asked to include human serum samples, A-E as below, in each assay and
these were provided in 1ml aliquots.
A Normal non-fasting serum sample 1
B Normal fasting serum sample
C Type 1 diabetic fasting serum sample
D Normal non-fasting serum sample 2
E Normal non-fasting serum sample 3
Participants were asked to ensure that all assays include their local standard where possible and to
provide details of the assay method used, including dilution steps, together with all raw assay data
WHO/BS/2014.2237
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in the form of clearly annotated optical densities, counts, etc. for central computation at NIBSC.
Participants’ own estimates of activity as calculated by the method normally used in their laboratory
were also requested.
Assay methods contributed
Summaries of the methods contributed by each laboratory are listed in Tables 3-5
Table 3: Assay methods - Phase I
Table 4: Assay methods - Phase II
Table 5: Assay methods - Phase III
Lab Number
Assay type Comment
1 Amino acid
analysis
In-house method, standard acid hydrolysis, post
separation derivatisation
2 Amino acid
analysis
UV Spectroscopy
In-house method, standard acid hydrolysis
As protocol (Appendix 2)
3
Amino acid
analysis
UV Spectroscopy
In-house method, standard acid hydrolysis
As protocol (Appendix 2)
4 Amino acid
analysis
In-house method: standard acid hydrolysis, pre-column
derivatisation. (Results were excluded from the analysis
as a result of charring during hydrolysis)
5 Amino acid
analysis
In-house method, standard acid hydrolysis
Lab Number
Assay type Comment
3 HPLC As protocol (Appendix 3)
6 HPLC As protocol (Appendix 3)
7 HPLC HPLC: As protocol (Appendix 3)
uHPLC: in-house method
8 HPLC As protocol (Appendix 3)
9 HPLC As protocol (Appendix 3)
10 HPLC As protocol (Appendix 3)
Lab Number
Assay type Comment
3a Immunoassay Mercodia Proinsulin ELISA
WHO/BS/2014.2237
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Statistical analysis
An independent statistical analysis of all immunoassay data was performed at NIBSC. Estimates
of the relative immunoreactivity of 09/296, 84/611 and local standards were obtained by fitting a
parallel-line model comparing log assay response to log concentration using responses in a linear
section of the response range [3]. Assay validity was assessed by analysis of variance with non-
linearity and non-parallelism considered significant at the 1 % level (p < 0.01). The proinsulin
concentrations of the patient serum samples were estimated by reading from the relevant
standard curve. Calculations were performed using the EDQM software CombiStats Version 5.0.
Laboratory means for immunoassays were calculated as unweighted geometric means and
overall means were calculated as the unweighted geometric mean of laboratory means.
Variability between laboratories has been expressed using geometric coefficients of variation
(GCV = {10s-1} × 100% where s is the standard deviation of the log10-transformed estimates).
The relative contents of the accelerated thermal degradation samples determined by HPLC were
used to fit an Arrhenius equation relating degradation rate to absolute temperature assuming
first-order decay [4] and hence predict the degradation rates when stored at -20°C.
Results
Amino acid analysis and UV spectroscopy of primary calibrant PC01
Results obtained from amino acid analysis (4 labs) and UV spectroscopy (3 labs) of primary
calibrant PC01 are shown in Table 6. The mean results from each lab were combined to give a
final estimated content of 220 μg/ampoule (95% confidence limits: 206 – 234).
HPLC of candidate standard 09/296
Results obtained from HPLC of the candidate standard 09/296 (8 labs) are shown in Tables 7 and
A1. These are as reported by participants and assume a content of 214 μg/ampoule for the
primary calibrant PC01 as suggested in the study protocol. In order to maintain a consistent
approach to the analysis of participant's HPLC data, calibration of the candidate standard in
terms of PC01 used the peak area of the main proinsulin peak. A minor contaminant eluting
approximately 2.5 mins later than the main proinsulin peak was reported by three laboratories
and comprised 1.2 - 2.4 % of the total peak area of the candidate standard stored at -20°C. A
leading edge to the main proinsulin peak was observed in the candidate standard only when
analysed by UHPLC. Both were excluded from the calibration. Table 8 shows the final result for
3b Immunoassay TECO Medical Intact Proinsulin immunoassay
11 Immunoassay Mercodia proinsulin ELISA
12 Immunoassay In-house intact proinsulin immunoassay performed on
Perkin Elmer Auto DELFIA
13 Immunoassay Mercodia proinsulin ELISA
14 Immunoassay Invitron Intact Proinsulin immunoassay
15 Immunoassay Invitron Intact Proinsulin immunoassay
16a
16b
Immunoassay
Immunoassay
Alpco Total Proinsulin immunoassay
Alpco Intact Proinsulin immunoassay
17 Immunoassay Zentech Intact Proinsulin immunoassay
WHO/BS/2014.2237
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the candidate standard 09/296, corrected for the content of PC01 determined in Phase I (220
μg/ampoule). Uncertainty in the value assigned to PC01 and the homogeneity of filling weight
(%CV) were combined with the HPLC standard error to give a combined uncertainty estimate.
This gave a final estimate of 7.0 μg/ampoule with an expanded uncertainty (95% confidence;
k=2.36) of 6.4 – 7.7.
Stability of 09/296
The stability of 09/296 ampoules was assessed in the HPLC phase of the study. Participants were
asked to estimate the proinsulin content of accelerated thermal degradation samples of 09/296
that had been stored at elevated temperatures of 4⁰C, 20⁰C, 37⁰C and 45⁰C for a period of 14
months. HPLC estimates of the proinsulin content of these ampoulesare summarized in Table 7.
Mean estimates showed losses of 4.2%, 3.7%, 5.8% and 13.9% at storage temperatures of 4°C,
20°C, 37°C and 45°C respectively. Analysis gave a predicted loss of 0.013% per year when
stored at -20°C.
Immunoassays
Immunoassay results are summarised in Tables 9 and 10 with all individual assay estimates
shown in Appendix Tables A2 and A3. Assay 2 by laboratories 16a and 16b was excluded from
further analysis as they represented anomalously low values for ampoules of the candidate
standard which were not observed in assay 1 and suggested a technical error. Estimates where
the assay response for the serum sample did not lie within the range of the standard curve are
highlighted. Values reported for sample C were at, or were close to, the limit of detection for
both intact and total assays when expressed relative to the kit standards and also relative to the
candidate standard, which is as expected for a serum sample from a type-1 diabetic patient. For
intact assays, normal fasting proinsulin levels are expected to be <10pmol/L which is supported
by the results for sample B (normal fasting serum sample). For one of the total proinsulin assays
used in the study, manufacturer’s data indicate the mean proinsulin level in normal fasting
subjects is expected to be approximately 10 pmol/L and this is also supported by the results for
sample B. As expected, proinsulin levels are elevated in serum samples from normal non-fasting
subjects when assayed by both assay types and there is good agreement between all data whether
they are expressed relative to 09/296 or relative to the kit standards.
Mean estimates of the immunoreactivity of 84/611 relative to 09/296 ranged from 0.476 (lab 16b)
to 0.881 (lab 14) with an overall geometric mean of 0.659 (95% confidence limits 0.571 – 0.762;
GCV 22.4%). This is slightly lower than would be expected given their respective proinsulin
contents of 6 µg/ampoule and 7 µg/ampoule respectively (expected relative immunoreactivity is
0.857). However, the proinsulin content of 84/611 was assigned in a limited study (two
laboratories) in the late 1980s and it is likely that the assigned content of 6 µg/ampoule may be
an overestimation. Data from this study suggest that the actual content of 84/611 may be closer
to 4-5 µg/ampoule. Interestingly, this discrepancy doesn’t appear to have been translated into
manufacturer’s calibration of their assays since the reported values for the serum samples in
terms of kit standards are in good agreement with the values reported in terms of the candidate
standard. In addition, mean potency estimates for 09/296 in pmol/L determined relative to kit
standards ranged from 637,608 (lab 13) to 1,161,767 (lab 12) with an overall geometric mean of
801,384 (95% confidence limits 681,873 – 941,842; GCV 25.3%). Conversion of this value
gives 7.3 µg/ampoule with 95% confidence limits of 6.2 – 8.6.
Conclusions and Recommendations
WHO/BS/2014.2237
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Measurements of serum proinsulin by immunoassays contribute to the diagnosis of insulinoma
and are used to monitor beta cell dysfunction. Stocks of the current IRR for proinsulin, used to
calibrate these immunoassays, are exhausted and this report summarises the international
collaborative study to establish a new WHO International Standard. The study was conducted in
three phases; phase I involved the assignment of a value to a primary calibrant in mass units by
AAA and U-V spectroscopy and phase II applied this value to the calibration of a candidate
standard by RP-HPLC assay. In phase III, the candidate standard was compared to the current
standard by current immunoassays to assess its suitability to serve as an International Standard.
Participants were also requested to also determine the proinsulin concentration of five human
serum samples in order to assess the impact of the introduction of the candidate preparation on
the routine measurement of proinsulin in native samples.
Because of the limited availability of the recombinant proinsulin used in this study, it was only
possible to prepare a small number of ampoules of PC01. As a result, only a small number of
expert laboratories contributed AAA and UV absorbance data for the calibration of PC01. These
data suggested PC01 to contain 220 μg proinsulin per ampoule (relative standard error of 2.7%)
which is a slightly lower content than would have been expected based on the nominal mass
content of the starting materials. Accordingly, in Phase II of the study, all laboratories’ data were
corrected for this value for the primary calibrant.
In those laboratories carrying out HPLC assay, there was a relatively large variability between
laboratories, which was somewhat surprising, but is likely to result from method-dependent
differences in the calibration. Correcting the mean of all estimates (6.84 μg; 95% CI 6.35 – 7.34)
by the defined content of the primary calibrant (220 μg), gives a final estimate of the content of
the candidate standard of 7.04 μg/ampoule with an associated expanded uncertainty of 6.36 –
7.72. Estimates of purity of the non-degraded preparation ranged from 97% to 100% which
agrees with the information provided by the manufacturer. No correction for this value has been
applied since estimates are probably method- and laboratory- dependent. Users should conduct
their own evaluation if deemed necessary.
The candidate preparation 09/296 appears to be sufficiently stable to serve as an international
reference reagent since the predicted yearly loss of activity at -20°C is 0.013%. These results
indicate that 09/296 is likely be highly stable under long term storage conditions at -20°C.
The preparation has also been shown to have appropriate immunological activity. There was
good agreement in the mean estimates of the immunoreactivity of 84/611 relative to 09/296 as
would be expected for two preparations derived from very similar starting materials. In addition,
comparisons of the reported proinsulin concentrations for the serum samples supplied in the
study indicate that the use of the candidate standard, with its assigned content, would not lead to
a discontinuity in the calibration of these assay methods. A more complete assessment of the
commutability of the candidate standard with a large number of patient samples was not possible
within the scope of this project. However, given the lack of alternative proinsulin starting
materials and the near identical source and processing steps for the candidate standard and the
depleted IRR, 84/611, the establishment of the candidate standard represents a pragmatic
approach to maintaining the appropriate calibration of proinsulin immunoassays used in clinical
diagnosis.
Proposal
WHO/BS/2014.2237
Page 11
It is recommended that the preparation in ampoules coded 09/296 is established as the First
International Standard for human proinsulin with an assigned content of 7.0 µg per ampoule and
an expanded uncertainty (95% confidence; k=2.36) of 6.4 – 7.7.
Acknowledgements
We gratefully acknowledge the important contributions of all the participants; Eli Lilly & Co
Indianapolis, IN 46285, USA who kindly donated the human proinsulin and the Centre for
Biological Reference Materials, NIBSC for preparation of the ampouled materials.
References
[1] Bristow AF and Gaines Das RE. WHO international reference reagents for human proinsulin
and human insulin C-peptide. J Biol Stand. 1988; 16:179-186.
[2] WHO. Expert Committee on Biological Standardization. Fortieth report. Geneva, World
Health Organization, 1990 (WHO Technical Report Series, No.800, 181-214).
[3] Finney DJ. Statistical Method in Biological Assay 3rd Edition. London: Charles Griffin;
1978.
[4] Kirkwood TBL. Predicting the stability of biological standards and products. Biometrics, 33:
1977: 736-742.
WHO/BS/2014.2237
Page 12
Table 6: AAA and UV results for primary calibrant PC01
Method Lab Ampoule Proinsulin content (μg/ampoule) Mean (ampoule) Mean (lab)
AAA
1
A 230.00 230.00 . 230.00 230.83
B 231.00 232.00 . 231.50
C 231.00 231.00 . 231.00
2
A 218.57 217.79 . 218.18 216.36
B 219.41 213.78 . 216.60
C 216.83 211.77 . 214.30
5
A 196.58 204.02 187.18 195.93 193.77
B 183.03 193.03 200.58 192.21
C 203.63 190.52 185.38 193.18
3 . 207.00
UV
2 . 222.00
3a . 231.00
3b . 239.00
Final Estimate (μg/ampoule) 219.99
Standard error 5.92
Standard error % 2.7%
95% confidence limits 205.50 – 234.49
WHO/BS/2014.2237
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Table 7: HPLC results for 09/296 and ATD samples of 09/296
Lab Mean estimates (μg/ampoule)*
A (+20°C) B (+37°C) C (-20°C) D (+4°C) E (+45°C)
8 7.58 7.46 7.78 7.62 7.12
7a 7.10 6.15 6.95 6.35 5.40
7b 6.10 6.30 6.70 6.00 5.50
3 6.29 6.30 6.72 6.83 5.91
9 6.30 6.62 7.15 6.93 6.68
6 7.10 6.51 7.28 6.96 6.13
10a . 5.94 6.28 5.94 5.14
10b 5.67 6.29 5.90 5.82 5.28
Final estimate (μg/ampoule) 6.59 6.45 6.84 6.56 5.89
Standard error 0.26 0.16 0.21 0.22 0.25
Standard error % 3.9% 2.5% 3.0% 3.4% 4.3%
95% confidence limits 5.96 – 7.22 6.06 – 6.83 6.35 – 7.34 6.03 – 7.08 5.30 – 6.49
*PC01 assumed to contain 214 μg/ampoule
Table 8: Assignment of value to candidate standard 09/296 (ampoule code C), correcting
for content of primary calibrant PC01 determined in Phase I
Final Estimate (μg/ampoule) 7.04
HPLC standard error % 3.0%
PC01 standard error % 2.7%
Homogeneity of filling weight CV % 0.4%
Combined standard uncertainty % 4.1%
Standard uncertainty 0.29
Expanded uncertainty (95% confidence; k=2.36) 6.36 – 7.72
WHO/BS/2014.2237
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Table 9: Summary of immunoassay estimates calculated relative to 09/296 (pmol/L for
serum samples A-E and relative potency for 84/611)
Method Lab Serum A Serum B Serum C Serum D Serum E 84/611
Intact
12 5.97 1.52 0.62 2.87 5.03 0.614
16b 14.61 3.71 0.24 8.00 10.84 0.476
14 13.53 3.24 0.42 8.03 10.87 0.881
15 12.68 3.06 0.39 6.97 11.00 0.874
17 6.23 3.06 0.37 3.13 4.45 0.656
3b 5.68 3.17 0.52 3.12 3.98 0.682
Total
16a 43.28 10.50 0.16 25.89 61.39 0.490
11 36.69 8.29 1.79 23.01 60.79 0.680
13 36.93 9.87 2.70 25.32 62.63 0.696
3a 43.32 8.31 1.54 26.36 71.06 0.665
Table 10: Summary of immunoassay estimates (pmol/L) calculated relative to local
standards
Method Lab Serum A Serum B Serum C Serum D Serum E 09/296
Intact
12 9.31 2.20 0.86 4.42 7.78 1,161,767
16b 15.59 3.60 0.20 8.19 11.34 795,016
14 11.73 2.68 0.32 6.84 9.36 654,300
15 11.16 2.42 0.27 5.86 9.58 665,734
17 7.00 3.07 0.36 2.88 3.39 914,026
3b 8.54 4.24 0.48 4.15 5.57 1,156,140
Total
16a 50.82 11.41 0.14 29.57 73.46 822,461
11 33.25 6.89 1.36 20.29 56.75 732,290
13 29.17 6.71 1.59 19.17 52.53 637,608
3a 32.31 6.76 1.31 19.81 51.53 669,137
WHO/BS/2014.2237
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Appendix 1
Table A1: HPLC results for 09/296 and ATD samples of 09/296
Lab Ampoule Injection Mean (μg) Overall sample mean (μg)
8
A-1 7.41 7.58
A-2 7.75
B-1 7.47 7.46
B-2 7.45
C-1 7.84 7.78
C-2 7.73
D-1 7.55 7.62
D-2 7.70
E-1 6.92 7.12
E-2 7.31
7a
A-1 7.3 7.10
A-2 6.9
B-1 5.8 6.15
B-2 6.5
C-1 6.3 6.95
C-2 7.6
D-1 5.7 6.35
D-2 7
E-1 5.2 5.40
E-2 5.6
7b
A-1 6.1
B-1 6.3
C-1 6.7
D-1 6.0
E-1 5.5
3
A-1 6.291
B-1 6.296
C-1 6.724
D-1 6.827
E-1 5.905
9
A 6.297
B 6.619
C 7.146
D 6.932
E 6.681
6
A-1 7.23 7.10
A-2 6.97
B-1 6.86 6.51
WHO/BS/2014.2237
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B-2 6.16
C-1 7.33 7.28
C-2 7.23
D-1 6.79 6.96
D-2 7.13
E-1 5.91 6.13
E-2 6.36
10a
A-1 4.21 4.88 (excluded)
A-2 5.54
B-1 5.89 5.94
B-2 5.98
C-1 6.16 6.28
C-2 6.39
D-1 5.96 5.94
D-2 5.91
E-1 5.34 5.14
E-2 4.94
10b
A-3 5.27 5.67
A-4 6.07
B-3 6.36 6.29
B-4 6.22
C-3 5.98 5.90
C-4 5.82
D-3 5.82 5.82
D-4 ND
E-3 5.36 5.28
E-4 5.20
WHO/BS/2014.2237
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Table A2: Individual immunoassay estimates calculated relative to 09/296 (pmol/L for
serum samples A-E and relative potency for 84/611)
Method Lab Assay Serum
A
Serum
B
Serum
C
Serum
D
Serum
E 84/611
Intact
12
1 5.22 1.20 0.60 2.38 4.16 0.500
2 5.45 1.50 0.64 2.88 5.11 0.634
3 6.58 1.73 0.58 . 5.40 0.661
4 6.77 1.70 0.64 3.46 5.59 0.679
16b 1 14.61 3.71 0.24 8.00 10.84 0.476
14 1 13.53 3.24 0.42 8.03 10.87 0.881
15 1 12.97 3.12 0.46 7.34 11.23 0.886
2 12.40 2.99 0.34 6.62 10.77 0.863
17
1 6.30 3.00 0.37 3.13 4.45 0.661
2 . . . . . .
3 6.16 3.12 . . . 0.650
3b 1 5.68 3.17 0.52 3.12 3.98 0.682
Total
16a 1 43.28 10.50 0.16 25.89 61.39 0.490
11 1 36.91 8.23 1.87 22.76 59.89 0.670
2 36.47 8.35 1.71 23.25 61.71 0.690
13 1 39.13 11.46 3.50 27.84 64.10 0.673
2 34.86 8.49 2.08 23.04 61.20 0.720
3a 1 49.21 7.10 1.38 31.93 89.09 0.660
2 38.14 9.72 1.72 21.77 56.67 0.669
Shaded cells indicate that assay response for serum sample was not in range of standard curve
WHO/BS/2014.2237
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Table A3: Individual immunoassay estimates (pmol/L) calculated relative to local
standards
Method Lab Assay Serum
A
Serum
B
Serum
C
Serum
D
Serum
E 09/296
Intact
12
1 9.74 2.01 0.95 4.19 7.65 1,490,700*
2 8.29 2.17 0.90 4.27 7.75 1,194,450
3 9.58 2.37 0.76 . 7.79 1,151,300
4 9.70 2.29 0.83 4.81 7.94 1,140,250
16b 1 15.59 3.60 0.20 8.19 11.34 795,016
14 1 11.73 2.68 0.32 6.84 9.36 654,300
15 1 11.23 2.43 0.31 6.09 9.63 647,781
2 11.08 2.40 0.23 5.64 9.52 684,185
17
1 7.19 3.55 0.49 3.69 5.17 946,424
2 6.93 2.35 0.27 2.25 2.23 .
3 6.90 3.46 . . . 882,738
3b 1 8.54 4.24 0.48 4.15 5.57 1,156,140
Total
16a 1 50.82 11.41 0.14 29.57 73.46 822,461
11 1 34.06 6.93 1.44 20.39 56.95 744,324
2 32.47 6.85 1.29 20.20 56.56 720,450
13 1 29.83 7.00 1.72 19.96 53.44 612,006
2 28.51 6.43 1.46 18.42 51.64 664,281
3a 1 30.24 5.93 1.49 21.01 49.83 608,585
2 34.52 7.72 1.16 18.67 53.29 735,713
Shaded cells indicate that assay response for serum sample was not in range of standard curve
*excluded from further calculation
WHO/BS/2014.2237
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Appendix 2
Phase I Study Protocol
Replacement of the WHO International Reference Reagent for human
Proinsulin (84/611)
Phase I
INTRODUCTION
Proinsulin, the precursor of insulin, is a 9390 MW peptide of 86 amino acids synthesised in
pancreatic beta cells. Proinsulin is converted to insulin and C-peptide by a process of enzymatic
cleavage, and these are then secreted from the beta cell. In healthy individuals proinsulin is
normally found in low concentrations in the plasma. However, elevated proinsulin levels are
found in patients with insulinomas, diabetes mellitus and other disorders of glucose metabolism.
Serum proinsulin measurements are therefore important in the diagnosis of insulinoma, and to
monitor beta cell dysfunction.
Stocks of the current International Reference Reagent (IRR) for human proinsulin, 84/611, are
running low and a new International Standard is required for the calibration of proinsulin
immunoassays. A new preparation of biosynthetic human proinsulin, has been filled into
ampoules, coded 09/296, following procedures recommended by WHO (1). It is intended that an
international collaborative study is organised with expert laboratories to aid in the value
assignment of the proposed International Standard, and to assess the activity and commutability
of the candidate preparation by proinsulin immunoassay.
AIMS OF THE STUDY
Serum proinsulin measurements are reported in molar terms, and therefore there is a requirement
to value assign the International Standard in SI units. The value assigned to the current IRR
84/611, 6µg/amp, is a consensus estimate derived from immunoassay (2). The candidate
standard, 09/296, contains approximately 10 µg of formulated biosynthetic proinsulin. As this
cannot be accurately measured directly by current physicochemical methods, a three phase
international collaborative study is planned in order to calibrate the preparation 09/296 in mass
units and characterise its immunoreactivity.
Phase I involves the establishment of a primary calibrant (PC01), consisting of a limited number
of ampoules containing approximately 250 µg proinsulin. A defined value will be assigned to the
primary calibrant by amino acid analysis and UV spectroscopy.
Phase II will involve calibration of the candidate standard 09/296, in terms of the primary
calibrant by HPLC.
Phase III will provide confirmatory data of the candidate standard’s immunoreactivity by
immunoassay. In addition, patient serum samples will be incorporated into this phase to assess
the commutability of the candidate proinsulin standard in immunoassay systems.
The aims of the study are therefore:
WHO/BS/2014.2237
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1. To calibrate the new preparation, 09/296, by physiochemical methods in mass units,
relative to the primary calibrant, PC01.
2. To demonstrate the suitability of the preparation 09/296 to serve as the International
Standard for human proinsulin by examining its behaviour in immunoassays.
3. To assess the relationships among existing local standards and the proposed IS.
4. To assess the commutability of the proposed IS 09/296 by examining its relationship
with patient samples by immunoassay.
5. To determine the stability of the preparation 09/296 by comparison with ampoules
stored at elevated temperatures.
MATERIALS
Preparations supplied to participants in collaborative study, Phase I.
Recombinant human proinsulin produced in E. coli was weighed and dissolved in a buffer of
10mM sodium phosphate, pH 7.0 and 0.5% trehalose. This solution was dispensed into glass
ampoules (at 0.5 ml per ampoule), lyophilised and sealed. The primary calibrant was prepared
identically to the candidate standard.
The materials for Phase I of this study are listed in Table 1.
Table 1.
Proinsulin preparation Ampoule content
Primary calibrant PC01 Nominally 250 µg per ampoule
TESTS REQUESTED
Handling of material
On receipt, ampoules should be stored at -20°C until use. It is recommended that the contents of
each ampoule are reconstituted in double distilled water for physicochemical analyses.
Phase I
Participants are requested to derive estimates of the proinsulin content of the primary calibrant
PC01 by amino acid analysis following their in-house method. Three vials of PC01 will be
provided and participants are asked to carry out a minimum of two analytical runs for each vial.
Participants are also requested to determine the proinsulin content of the ampoule by U-V
spectroscopy using the method normally run in the participant’s laboratory. The Extinction
Coefficient of proinsulin is 0.65 (1 mg/ml solution of human proinsulin at 278 nm). Appendix 1
gives a suggested laboratory protocol although the method normally used in the participants’
laboratory is acceptable.
WHO/BS/2014.2237
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Proinsulin is a polypeptide of 9390 Da, 86 amino acids. The proinsulin amino acid sequence is
detailed below: FVNQHL CGSHLVEALY LVCGERGFFY TPKTRREAED
LQVGQVELGG GPGAGSLQPL ALEGSLQKRG IVEQCCTSIC SLYQLENYCN
REPORT
A preliminary report, after completion of phase 3 of the project, will be prepared and circulated
to all participants for comment before submission to the Expert Committee on Biological
Standardization of WHO. In the report, participating laboratories will be identified by a
laboratory number only and any requests to treat information in confidence will be respected.
REFERENCES
1. WHO Tech Rep Ser No 800, 1990 181-214
2. Bristow A.F. and Gains-Das R.E. (1988) J Biol Standardization 16:179-186
For further information, please contact:
Dr Melanie Moore
National Institute for Biological Standards and Control
Blanche Lane, South Mimms
Potters Bar, Herts. EN6 3QG
UK
Tel: 44 (0) 1707 641247
Fax: 44 (0) 1707 641057
WHO/BS/2014.2237
Page 22
Study Protocol (Phase I) Appendix 1
Laboratory Protocol
Spectrophotometric determination of human proinsulin content of Primary
Calibrant, PC01
For a more accurate determination of proinsulin content, the concentration of the solution
measured should be > 0.2 x the Extinction Coefficient. As the Extinction Coefficient of
proinsulin is 0.65 (1 mg/ml solution of human proinsulin at 278 nm), it is recommended that 2
ampoules of PC01 (0.25 mg/ampoule) are opened and 0.5 ml ddH20 (double distilled water)
added directly to each pre-weighed vial of PC01 to give a final concentration of approximately
0.5 mg/ml. Dissolve the contents by rotating slowly for 5 minutes. Determine the total volume
added by re-weighing the vial and combine each ampoule to give a final volume of approx 1 ml.
Warm up a recently calibrated scanning densitometer and set it to scan between 360 and 240 nm.
Fill a 1 cm path length, 1ml volume, quartz cuvette with ddH20, and scan in a baseline.
Dry the cuvette, add the human proinsulin solution and scan over the same wavelength range
(the blank baseline should be automatically deducted by the machine). Print out the resulting
scan trace.
Turbidity correction
With a straight edge, extrapolate the straight line between 360 nm and 320 nm back towards 240
nm to produce a corrected baseline. Using the x and y axes scales, determine the value of this
baseline at the peak maximum, (which should be on or close to 278 nm). Subtract this baseline
value from the maximum value to give the final corrected values.
Protein content calculation
Multiply the corrected value, obtained above, by the total volume of the sample. Using the
Extinction Coefficient of 0.65 (1 mg/ml solution of human proinsulin at 278 nm), calculate the
total vial content as in the example below:
If A278 corrected = 0.31 and total sample volume = 1.001mls
Total A278 units = 0.31 x 1.001 = 0.31031
Thus total content of two vials = 0.31031 / 0.65 = 0.4774 mg, giving a mean vial content of
0.239 mg
WHO/BS/2014.2237
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Appendix 3
Phase II Study Protocol
Replacement of the WHO International Reference Reagent for human
Proinsulin (84/611)
Phase II
INTRODUCTION
Proinsulin, the precursor of insulin, is a 9390 MW peptide of 86 amino acids synthesised in
pancreatic beta cells. Proinsulin is converted to insulin and C-peptide by a process of enzymatic
cleavage, and these are then secreted from the beta cell. In healthy individuals proinsulin is
normally found in low concentrations in the plasma. However, elevated proinsulin levels are
found in patients with insulinomas, diabetes mellitus and other disorders of glucose metabolism.
Serum proinsulin measurements are therefore important in the diagnosis of insulinoma, and to
monitor beta cell dysfunction.
Stocks of the current International Reference Reagent (IRR) for human proinsulin, 84/611, are
running low and a new International Standard is required for the calibration of proinsulin
immunoassays. A new preparation of biosynthetic human proinsulin, has been filled into
ampoules, coded 09/296, following procedures recommended by WHO (1). It is intended that an
international collaborative study is organised with expert laboratories to aid in the value
assignment of the proposed International Standard, and to assess the immunoreactivity and
commutability of the candidate preparation by proinsulin immunoassay.
AIMS OF THE STUDY
Serum proinsulin measurements are reported in molar terms, and therefore there is a requirement
to value assign the International Standard in SI units. The value assigned to the current IRR
84/611, 6µg/amp, is a consensus estimate derived from immunoassay (2). The candidate
standard, 09/296, contains approximately 10 µg of formulated biosynthetic insulin. As this
cannot be accurately measured directly by current physiochemical methods, a three phase
international collaborative study is planned in order to calibrate the preparation 09/296 in mass
units and characterise its immunoreactivity.
Phase I involves the establishment of a primary calibrant (PC01), consisting of a limited number
of ampoules containing approximately 250 µg proinsulin. A defined value will be assigned to the
primary calibrant by amino acid analysis and UV spectroscopy.
Phase II will involve calibration of the candidate standard 09/296, in terms of the primary
calibrant by reverse-phase HPLC.
Phase III will provide confirmatory data of the candidate standard’s immunoreactivity by
immunoassay. In addition, patient serum samples will be incorporated into this phase to assess
the commutability of the candidate proinsulin standard in immunoassay systems.
The aims of the study are therefore:
WHO/BS/2014.2237
Page 24
1. To calibrate the new preparation, 09/296, by physiochemical methods in mass units,
relative to the primary calibrant, PC01.
2. To demonstrate the suitability of the preparation 09/296 to serve as the International
Standard for human proinsulin by examining its behaviour in immunoassays.
3. To assess the relationships among existing local standards and the proposed IS.
4. To assess the commutability of the proposed IS 09/296 by examining its relationship
with patient samples by immunoassay.
5. To determine the stability of the preparation 09/296 by comparison with ampoules
stored at elevated temperatures.
MATERIALS
Preparations supplied to participants in collaborative study, Phase II.
Recombinant human proinsulin produced in E. coli was weighed and dissolved in a buffer of
10mM sodium phosphate (pH 7.0) and 0.5% (w/v) trehalose. This solution was dispensed into
glass ampoules (0.5 ml per ampoule), lyophilised and sealed. The primary calibrant was prepared
identically to the candidate standard.
The materials for Phase II of this study are listed in Table 1. The candidate standard and its’
accelerated degradation samples will be coded by letter.
Table 1.
Proinsulin preparation Ampoule content
Primary calibrant PC01 214 µg per ampoule as defined in Phase I
Candidate standard 09/296 stored at -20°C Nominally 10 µg proinsulin per ampoule
Accelerated thermal degradation (ATD)
samples of 09/296 stored at +4°C, +20°C,
+37°C and +45°C
Content assumed identical to 09/296
TESTS REQUESTED
Handling of material
On receipt, ampoules should be stored at -20°C until use. It is recommended that the contents of
each ampoule are reconstituted in double distilled water for physiochemical analyses.
Phase II
The primary calibrant, PC01, was assigned the consensus value of 214 µg per ampoule in Phase I
of the study. Using this value, participants are asked to provide, in triplicate, estimates of
proinsulin content of the candidate standard 09/296, and its accelerated thermal degradation
samples, by comparison with PC01, using the HPLC method used in house. An outline HPLC
WHO/BS/2014.2237
Page 25
protocol is provided in Appendix 1 for guidance. Participants are requested to provide raw data
and sample chromatograms of PC01, if possible, in addition to their own estimates of proinsulin
content per ampoule.
REPORT
A preliminary report will be prepared and circulated to all participants for comment before
submission to the Expert Committee on Biological Standardization of WHO. In the report,
participating laboratories will be identified by a laboratory number only and any requests to treat
information in confidence will be respected.
REFERENCES
1. WHO Tech Rep Ser No 800, 1990 181-214
2. Bristow A.F. and Gains-Das R.E. (1988) J Biol Standardization 16:179-186
For further information, please contact:
Dr Melanie Moore
National Institute for Biological Standards and Control
Blanche Lane, South Mimms
Potters Bar, Herts. EN6 3QG
UK
Tel: 44 (0) 1707 641247
Fax: 44 (0) 1707 641057
WHO/BS/2014.2237
Page 26
Study Protocol (Phase II) Appendix 1
Laboratory Protocol
Outline method for HPLC assay of proinsulin content
Column: C18, pore size 250Å, particle size 5µm, dimensions 250 x 4.6mm. Note pore
size is critical, and should be >200Å for good recovery and reduction of peak
trailing.
If available, a chilled autosampler should be used.
Flow rate 0.8 ml/min throughout, at ambient temperature.
Solvents A and B = 0.05 - 0.1% TFA with 80% and 20% acetonitrile respectively.
Preparation of primary calibrant, PC01, standard curve: Reconstitute ampoule
contents in 428µl distilled water to give final concentration 500 µg/ml. Sub aliquots
should be prepared and stored at -20°C for subsequent use. Duplicate injections of 10µl
(5µg), 5µl (2.5µg) and 2µl (1µg), plus blanks, should be sufficient to provide a standard
curve for PC01.
Preparation of coded ampoules: Reconstitute ampoule contents in 100µl double
distilled water. Injections of 25µl (2.5µg) should provide sufficient material for assay.
These should be carried out in triplicate if possible.
Sample gradient for solvents A (80% acetonitrile) and B (20% acetonitrile), time, t, in
minutes: Start t0, 100% B; t2, 100%B; t20, 50% B; t23, 0% B; t25, 0% B; t28, 100%B;
t38, 100% B Stop. Main peak is between 12 and 16 minutes (may vary, depending on
type of column and flow rate used).
Absorbance monitored at 215 nm.
Ampoule content is determined from the ratio of appropriate peak areas (sample area:
PC01 area) or by linear regression analysis.
Example trace for PC01 and 09/296
WHO/BS/2014.2237
Page 27
Appendix 4
Phase III Study Protocol
Replacement of the WHO International Reference Reagent for human
Proinsulin (84/611)
Phase III
INTRODUCTION
Proinsulin, the precursor of insulin, is a 9390 MW peptide of 86 amino acids synthesised in
pancreatic beta cells. Proinsulin is converted to insulin and C-peptide by a process of enzymatic
cleavage, and these are then secreted from the beta cell. In healthy individuals proinsulin is
normally found in low concentrations in the plasma. However, elevated proinsulin levels are
found in patients with insulinomas, diabetes mellitus and other disorders of glucose metabolism.
Serum proinsulin measurements are therefore important in the diagnosis of insulinoma, and to
monitor beta cell dysfunction.
Stocks of the current International Reference Reagent (IRR) for human proinsulin, 84/611, are
running low and a new International Standard is required for the calibration of proinsulin
immunoassays. A new preparation of biosynthetic human proinsulin, has been filled into
ampoules, coded 09/296, following procedures recommended by WHO (1). It is intended that an
international collaborative study is organised with expert laboratories to aid in the value
assignment of the proposed International Standard, and to assess the immunoreactivity and
commutability of the candidate preparation by proinsulin immunoassay.
AIMS OF THE STUDY
Serum proinsulin measurements are reported in molar terms, and therefore there is a requirement
to value assign the International Standard in SI units. The value assigned to the current IRR
84/611, 6µg/amp, is a consensus estimate derived from immunoassay (2). The candidate
standard, 09/296, contains approximately 7 µg of formulated biosynthetic proinsulin. As this
cannot be accurately measured directly by current physiochemical methods, a three phase
international collaborative study is planned in order to calibrate the preparation 09/296 in mass
units and characterise its immunoreactivity.
Phase I involves the establishment of a primary calibrant (PC01), consisting of a limited number
of ampoules containing approximately 250 µg proinsulin. A defined value will be assigned to the
primary calibrant by amino acid analysis and UV spectroscopy.
Phase II will involve calibration of the candidate standard 09/296, in terms of the primary
calibrant by reverse-phase HPLC.
Phase III will provide confirmatory data of the candidate standard’s immunoreactivity by
immunoassay. In addition, patient serum samples will be incorporated into this phase to assess
the commutability of the candidate proinsulin standard in immunoassay systems.
The aims of the whole study are therefore:
WHO/BS/2014.2237
Page 28
1. To calibrate the new preparation, 09/296, by physiochemical methods in mass units,
relative to the primary calibrant, PC01.
2. To demonstrate the suitability of the preparation 09/296 to serve as the International
Standard for human proinsulin by examining its behaviour in immunoassays.
3. To assess the relationships among existing local standards and the proposed IS.
4. To assess the commutability of the proposed IS 09/296 by examining its relationship
with patient samples by immunoassay.
5. To determine the stability of the preparation 09/296 by comparison with ampoules
stored at elevated temperatures.
MATERIALS
Preparations supplied to participants in collaborative study, Phase III.
The 1st IRR for human proinsulin, 84/611, was prepared as previously described (2) and consists
of recombinant human proinsulin produced in E. coli, weighed and dissolved in a buffer
containing serum albumin and lactose. The solution was dispensed into glass ampoules
lyophilised and sealed. The 1st IRR was assigned a proinsulin content of 6 µg per ampoule by
immunoassay.
The candidate standard was prepared from recombinant human proinsulin produced in E. coli,
weighed and dissolved in a buffer of 10mM sodium phosphate (pH 7.0) and 0.5% (w/v) trehalose.
This solution was dispensed into glass ampoules (0.5 ml per ampoule), lyophilised and sealed.
Samples of non-lyophilised solution were stored in liquid nitrogen after dispensing to provide a
frozen baseline.
Patient serum samples were kindly collected by Gwen Wark (UK NEQAS). Serum was frozen
and sent to NIBSC on dry ice, where it was thawed, dispensed into 1 ml aliquots and stored at -
20’C.
The materials for Phase III of this study are listed in Table 1. Patient serum samples will be
coded by letter.
Table 1.
Proinsulin preparation Ampoule content
1st International Reference Reagent for
human Proinsulin, 84/611
6 µg per ampoule
Candidate standard 09/296 Nominally 7 µg proinsulin per ampoule.
Non-lyophilized frozen baseline of candidate
standard, 09/296
Assume same content as lyophilised
candidate 09/296.
Type I diabetic serum sample 1 ml human serum
Type II diabetic serum sample 1 ml human serum
WHO/BS/2014.2237
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Normal serum sample 1 1 ml human serum
Normal serum sample 2 1 ml human serum
Normal serum sample 3 1 ml human serum
As with all material of biological origin, these preparations should be regarded as potentially
hazardous to health. It should be used and discarded according to your own laboratory's safety
procedures.
TESTS REQUESTED
Handling of material
On receipt, ampoules should be stored at -20°C until use. Before opening, ampoules should be
brought to room temperature to minimise moisture uptake. It is recommended that the contents
of each lyophilised ampoule are reconstituted in 1ml PBS plus 0.1% BSA, or the appropriate
assay buffer, to provide a stock solution. Further dilutions should be performed in the
appropriate assay buffer or PBS plus 0.1% BSA, ensuring buffers contain protein cover to
prevent adsorption to ampoules.
Serum samples and frozen baseline ampoules should be thawed at room temperature and mixed
well prior to assay.
Phase III
Participants are requested to carry out the assay(s) normally used in their laboratory and, where
possible, to perform at least two independent assays with each sample provided in duplicate or
triplicate, using fresh ampoules where provided. Each assay should include the current IRR for
proinsulin, 84/611, the candidate standard 09/296 and the frozen baseline of 09/296, at
preferably no less than five dose levels in the linear part of the dose-response curve. Patient
serum samples should be tested in duplicate or triplicate according to the in house method.
Estimates of the proinsulin content of the current IRR for proinsulin, 84/611, the candidate
standard 09/296, the frozen baseline of 09/296, and patient serum samples should be calculated
in comparison with their assay kit standard.
Participants are requested to provide details of the assay method used, including dilution steps,
together with all raw data for each sample in electronic format if possible. Participants’ own
calculated estimates of proinsulin concentration are also requested. A sample table for data
reporting is provided in Appendix 1.
REPORT
A preliminary report will be prepared and circulated to all participants for comment before
submission to the Expert Committee on Biological Standardization of WHO. In the report,
participating laboratories will be identified by a laboratory number only and any requests to treat
information in confidence will be respected.
WHO/BS/2014.2237
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REFERENCES
1. WHO Tech Rep Ser No 800, 1990 181-214
2. Bristow A.F. and Gains-Das R.E. (1988) J Biol Standardization 16:179-186
For further information, please contact:
Dr Melanie Moore
National Institute for Biological Standards and Control
Blanche Lane, South Mimms
Potters Bar, Herts. EN6 3QG
UK
Tel: 44 (0) 1707 641247
Fax: 44 (0) 1707 641057
WHO/BS/2014.2237
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Study Protocol (Phase III) Appendix 1
Example data reporting table
Run 1
Sample Read 1 Read 2 Read 3 Avg
read
Proinsulin
concentration
Baselines
Kit standard, dilution 1
Kit standard, dilution 2 etc….
84/611 dilution 1
84/611 dilution 2
etc….
09/296 dilution 1
09/296 dilution 2 etc…..
09/296 frozen baseline dilution 1
09/296 frozen baseline dilution 2
etc….
Serum sample A
Serum sample B
Serum sample C
Serum sample D
Serum sample E
Run 2
Sample Read 1 Read 2 Read 3 Avg
read
Proinsulin
concentration
Baselines
Kit standard, dilution 1
Kit standard, dilution 2 etc….
84/611 dilution 1
84/611 dilution 2
etc….
09/296 dilution 1
09/296 dilution 2 etc…..
09/296 frozen baseline dilution 1
09/296 frozen baseline dilution 2
etc….
Serum sample A
Serum sample B
Serum sample C
Serum sample D
Serum sample E
WHO/BS/2014.2237
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Appendix 5: Draft Instructions for use
WHO International Standard
1st International Standard for human Proinsulin
NIBSC Code: 09/292
Instructions for use
(Version 1.0, Dated 01/11/2014)
1. INTENDED USE
The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS)
has recognized (2010) the need for a replacement for the International Reference Reagent (IRR) for
human proinsulin, coded 84/611. A preparation of proinsulin, coded 09/292, was ampouled and
evaluated for its suitability to serve as a WHO International Standard by international collaborative
study. It was established as the 1st International Standard for human Proinsulin by the Expert
Committee on Biological Standardization of the WHO in October 2014. This replaces the
International Reference Reagent, coded 84/611, as the primary reference material for the calibration
of immunoassays of Proinsulin.
2. CAUTION
This preparation is not for administration to humans.
3. UNITAGE 7.0 µg per ampoule with an expanded uncertainty (95% confidence; k=2.36) of 6.4 – 7.7.
4. CONTENTS
Country of origin of biological material: USA
Each ampoule contains the residue, after freeze-drying, of 0.5ml of a solution which contained:
Proinsulin 7 µg
di-Sodium hydrogen phosphate anhydrous 0.41 mg
Sodium di-hydrogen phosphate monohydrate 0.29 mg
Trehalose 2.5 mg
5. STORAGE
Unopened ampoules should be stored at -20°C.
Please note: because of the inherent stability of lyophilized material, NIBSC may ship these
materials at ambient temperature.
6. DIRECTIONS FOR OPENING
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 (labeled)
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.
WHO/BS/2014.2237
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7. USE OF MATERIAL
No attempt should be made to weigh out any portion of the freeze-dried material prior to
reconstitution.
For practical purposes each ampoule contains the same quantity of Proinsulin. Depending on the
intended use, dissolve the total contents of the ampoule in a known amount of a suitable diluent.
If extensive dilutions are prepared, a carrier protein (0.05 - 0.1% w/v BSA or HSA) should be
added. The ampoules do not contain bacteriostat and a solution of the reagent should not be
assumed to be sterile.
8. PREPARATION OF AMPOULES
Ampoules coded 09/292 containing a nominal 8.5µg lyophilized proinsulin, sodium phosphate
and trehalose were prepared according to the methods recommended for international biological
standards. A weighed portion of the proinsulin was dissolved in a sterile solution containing
10 mM sodium phosphate pH 7.0, 0.5 % (w/v) trehalose and diluted to a final volume of 1800 ml
with the same buffer. This solution was passed through a filter (mean pore diameter 0.45μm) and
distributed in 0.5 ml aliquots into ampoules. Filled solutions were lyophilized, and after
secondary desiccation, were sealed under nitrogen by heat fusion of the glass and stored at –20oC
in the dark.
An international collaborative study was conducted in three phases to assign a mass content to
09/292. Phase I involved the assignment of a value to a primary calibrant in mass units by AAA
and UV spectroscopy and phase II applied this value to the calibration of a candidate standard by
reverse phase-HPLC assay. In phase III, the candidate standard was compared to the current
standard by current immunoassays to assess its suitability to serve as an International Standard.
9. STABILITY
Stability based on HPLC analysis of thermally accelerated degradation samples showed a
predicted yearly loss of activity when stored at -20°C of 0.013% and a predicted yearly loss of
proinsulin content of 3.7% at 20°C. These results indicate that 09/292 is likely to be highly stable
under long term storage conditions at -20°C and that the material will also be stable during
normal shipping at ambient temperatures.
NIBSC follows the policy of WHO with respect to its reference materials.
It is the policy of WHO not to assign an expiry date to their international reference materials.
They remain valid with the assigned potency and status until withdrawn or amended. Reference
materials are held at NIBSC within assured, temperature-controlled storage facilities. Unopened
ampoules should be stored on receipt as indicated on the label. In addition, once reconstituted,
diluted or aliquoted, users should determine the stability of the material according to their own
method of preparation, storage and use.
10. REFERENCES
11. ACKNOWLEDGEMENTS Grateful acknowledgements are due to Dr B. Frank and Eli Lilly & Co., Indianapolis, USA for
providing the bulk material and the participants in the collaborative study.
12. FURTHER INFORMATION
Further information can be obtained as follows;
This material: [email protected]
WHO/BS/2014.2237
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WHO Biological Standards: http://www.who.int/biologicals/en/
JCTLM Higher order reference materials:
http://www.bipm.org/en/committees/jc/jctlm/
Derivation of International Units:
http://www.nibsc.org/products/biological_reference_materials/frequently_asked_questions/how_
are_international_units.aspx
Ordering standards from NIBSC:
http://www.nibsc.org/products/ordering_information/frequently_asked_ questions.aspx NIBSC
Terms & Conditions:
http://www.nibsc.org/terms_and_conditions.aspx
13. CUSTOMER FEEDBACK
Customers are encouraged to provide feedback on the suitability or use of the material provided
or other aspects of our service. Please send any comments to [email protected]
14. CITATION In all publications, including data sheets, in which this material is referenced, it is important that
the preparation's title, its status, the NIBSC code number, and the name and address of NIBSC
are cited and cited correctly.
15. MATERIAL SAFETY SHEET
Physical and Chemical properties (at room temperature)
Physical appearance : Freeze dried powder Corrosive: No
Stable: Yes Oxidising: No
Hygroscopic: Yes Irritant: No
Flammable: No Handling: See caution, Section 2
Other (specify)
Toxicological properties
Effects of inhalation: Not established, avoid inhalation
Effects of ingestion: Not established, avoid ingestion
Effects of skin absorption: Not established, avoid contact with skin
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 an appropriate
disinfectant. Rinse area with an appropriate disinfectant followed by water.
Absorbent materials used to treat spillage should be treated as biologically hazardous waste.
16. LIABILITY AND LOSS
In the event that this document is translated into another language, the English language version
shall prevail in the event of any inconsistencies between the documents.
Unless expressly stated otherwise by NIBSC, NIBSC’s Standard Terms and Conditions for the
Supply of Materials (available at http://www.nibsc.org/About_Us/Terms_and_Conditions.aspx
or upon request by the Recipient) (“Conditions”) apply to the exclusion of all other terms and are
WHO/BS/2014.2237
Page 35
hereby incorporated into this document by reference. The Recipient's attention is drawn in
particular to the provisions of clause 11of the Conditions.
17. INFORMATION FOR CUSTOMS USE ONLY
Country of origin for customs purposes*: United Kingdom
* Defined as the country where the goods have been produced and/or sufficiently processed
to be classed as originating from the country of supply, for example a change of state such
as freeze-drying.
Net weight: 3 mg
Toxicity Statement: Non-toxic
Veterinary certificate or other statement if applicable.
Attached: No
18. CERTIFICATE OF ANALYSIS
NIBSC does not provide a Certificate of Analysis for WHO Biological Reference Materials
because they are internationally recognised primary reference materials fully described in the
instructions for use. The reference materials are established according to the WHO
Recommendations for the preparation, characterization and establishment of international and
other biological reference standards
http://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biol
efstandardsrev2004.pdf (revised 2004). They are officially endorsed by the WHO Expert
Committee on Biological Standardization (ECBS) based on the report of the international
collaborative study which established their suitability for the intended use.