who/bs/2014.2237 english only expert …...who/bs/2014.2237 page 5 mean residual moisture as...

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

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 alone are responsible for the views expressed in this publication.

mailto:[email protected]


WHO/BS/2014.2237

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

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

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

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

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

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


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


Lab Number

Assay type Comment

3 HPLC As protocol (Appendix 3)


7 HPLC HPLC: As protocol (Appendix 3)

uHPLC: in-house method




Lab Number

Assay type Comment

3a Immunoassay Mercodia Proinsulin ELISA

WHO/BS/2014.2237

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Appendix 3

Phase II Study Protocol


Proinsulin (84/611)

Phase II

INTRODUCTION













assignment of the proposed International Standard, and to assess the immunoreactivity and

commutability of the candidate preparation by proinsulin immunoassay.

AIMS OF THE STUDY




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







calibrant by reverse-phase HPLC.




The aims of the study are therefore:

WHO/BS/2014.2237










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.


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


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

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




Dr Melanie Moore




UK

Tel: 44 (0) 1707 641247

Fax: 44 (0) 1707 641057

WHO/BS/2014.2237

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

Appendix 4

Phase III Study Protocol


Proinsulin (84/611)

Phase III

INTRODUCTION













assignment of the proposed International Standard, and to assess the immunoreactivity and

commutability of the candidate preparation by proinsulin immunoassay.

AIMS OF THE STUDY




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







calibrant by reverse-phase HPLC.




The aims of the whole study are therefore:

WHO/BS/2014.2237










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.


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

Normal serum sample 1 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


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

REFERENCES




Dr Melanie Moore




UK

Tel: 44 (0) 1707 641247

Fax: 44 (0) 1707 641057

WHO/BS/2014.2237

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


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…..



etc….

Serum sample A

Serum sample B

Serum sample C

Serum sample D

Serum sample E

WHO/BS/2014.2237

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

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

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

http://www.bipm.org/en/committees/jc/jctlm/

WHO/BS/2014.2237

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.

who/bs/2014.2237 english only expert …...who/bs/2014.2237 page 5 mean residual moisture as...

Documents