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WHO international collaborative study of the proposed 1st international standard for human C-peptide: Expert Committee on Biological Standardization: Geneva, 12 to 16 October 2015

25 novembre 2015 Organisation mondiale de la santé

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WHO/BS/2015.2256 ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 12 to 16 October 2015

WHO International Collaborative Study of the Proposed 1st International Standard for human C-peptide Melanie Moore, Jackie Ferguson, Tom Dougall, Peter Rigsby and Chris Burns National Institute for Biological Standards and Control Blanche Lane, Potters Bar, Herts, EN6 3QG, UK 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 28 September 2015 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 Hye-Na Kang at email: kangh@who.int. © World Health Organization 2015 All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased 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: bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site: (http://www.who.int/about/licensing/copyright_form/en/index.html). 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.

WHO/BS/2015.2256 Page 2

Summary The World Health Organisation (WHO) Expert Committee on Biological Standardisation (ECBS) has recognised (2010) the need for a replacement for the International Reference Reagent (IRR) for human C-peptide, coded 84/510. We report here the preparation of a candidate for the 1st International Standard for human C-peptide and its characterisation in an international collaborative study carried out by 24 laboratories in 10 countries. Phase I of the study confirmed by amino acid analysis that the primary calibrant contained 209 µg per vial. The candidate standard, in ampoules coded 13/146, was then calibrated by HPLC analysis in terms of the primary calibrant (Phase II) and further characterised by immunoassay in comparison to 84/510 (Phase III). Estimates from the HPLC calibration from all laboratories were in good agreement, and indicated the content of 13/146 to be 8.64 µg per ampoule. The results of this study also indicate that the candidate standard showed 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 C-peptide. In addition, the results of an assessment of the commutability of the candidate standard with a small cohort of patient samples, indicates that the candidate standard 13/146 is likely to be commutable with serum and urine patient samples as measured by current immunoassays. It is therefore proposed that the candidate standard 13/146 be established as the 1st WHO International Standard for human C-peptide, with an assigned potency of 8.64 µg/ampoule with expanded uncertainty of 8.21-9.07 (95% confidence; k=2.45).

Introduction C-peptide is a single chain peptide (MW 3020) of 31 amino acids synthesised in pancreatic beta cells as the connecting peptide for the A and B chains of insulin in the proinsulin molecule. Proinsulin is converted to insulin and C-peptide by a process of enzymatic cleavage, and these are then secreted into the circulation by the beta cells. As insulin and C-peptide are secreted in equimolar amounts, measurements of C-peptide can provide a reliable indication of pancreatic insulin secretion. In addition, C-peptide has a longer half-life (~30min) than insulin (~5min) and so higher concentrations persist in the peripheral circulation and measurement of plasma Cpeptide levels may be a more reliable indication of pancreatic insulin secretion than the level of insulin itself. The measurement of C-peptide has a number of important clinical uses where the monitoring of beta cell function and endogenous insulin levels are required (reviewed in [1]). A key role for C-peptide measurement is in the differentiation between Type I and Type II diabetes and identifying patients with maturity-onset diabetes of the young (MODY). As diabetes patients are at increased risk over time of cardiovascular damage, retinopathy, neuropathy and nephropathy, early diagnosis and careful monitoring of insulin resistance is very important. In particular, C-peptide levels may be useful for the monitoring of beta cell capability over time in Type II diabetes patients, and help determine the establishment of insulin deficiency. In Type I diabetes, residual beta cell function as measured by C-peptide may also be of prognostic value [2], [3], [4] . C-peptide measurements can be used to aid in the diagnosis of hypoglycaemia and insulinoma, and monitoring of treatment [5]. C-peptide also has the potential to serve as a marker

WHO/BS/2015.2256 Page 3 of residual pancreatic tissue after pancreatectomy, or to verify effectiveness of pancreatic islet cell transplantation [6]. Human C-peptide concentrations are measured by immunoassay from patient serum, plasma or urine. The current IRR for human C-peptide, coded 84/510, for the standardisation of C-peptide immunoassays, was established in 1986 and comprised ampoules of recombinant human Cpeptide with a content of 10 µg per ampoule. This content was assigned through comparison to local standards in 5 independent laboratories by a total of 7 radioimmunoassay systems [7]. Stocks of 84/510 are now exhausted and a new International Standard is required for the calibration of C-peptide immunoassays. A proposal for a new WHO International Standard for human C-peptide was endorsed by the WHO Expert Committee on Standardisation (ECBS) at its meeting held in November 2010. A candidate standard was prepared using a batch of synthetic human C-peptide which was filled into ampoules, coded 13/146, following procedures recommended by WHO [8]. An international collaborative study was organised by NIBSC to assign a mass content to the standard and to evaluate the suitability of 13/146 to serve as an International Standard for the calibration of immunoassays of human C-peptide. The candidate standard, 13/146, was estimated to contain approximately 8 µg of formulated synthetic C-peptide. As this cannot be accurately measured directly by current physicochemical methods and in order to calibrate the preparation 13/146 in mass units in addition to characterizing its immunoreactivity, the collaborative study was devised to comprise three phases: For phase I, a primary calibrant (PC01) was established consisting of a limited number of ampoules containing approximately 250 µg C-peptide. This primary calibrant was then assigned a value using amino acid analysis. In phase II, the candidate standard 13/146, was calibrated in terms of the primary calibrant by HPLC. In this phase of the 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. The purpose of phase III was then to provide confirmatory data of the candidate standard’s immunoreactivity by immunoassay, and its suitability to serve as an international standard by comparison with the current standard 84/510 and existing local standards. As the candidate standard consisted of formulated synthetic Cpeptide, human serum and urine samples were also incorporated into this phase in order to assess the impact of the introduction of the candidate standard on the routine measurement of C-peptide in native samples. The aims of the study, therefore, were: Phase I 1. To assign a mass value to the primary calibrant, PC01, by amino acid analysis (AAA).

WHO/BS/2015.2256 Page 4 Phase II 2. To calibrate the candidate preparation, 13/146, in terms of the primary calibrant by HPLC to assign a value in mass units. 3. To assess the effect of accelerated thermal degradation by HPLC. Phase III 4. To demonstrate the suitability of the preparation 13/146 to serve as the International Standard for human C-peptide by examining its behavior in immunoassays. 5. To assess the relationships among existing local standards, the 1st IRR 84/510 and the proposed IS 13/146 by immunoassay. 6. To evaluate the likely commutability of the proposed IS 13/146 by examining its relationship with human serum and urine samples by immunoassay.

Participants Twenty four laboratories in ten 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 BELGIUM BELGIUM Dr Stefaan Marivoet TOSOH, Transportraat 4, 3980 Tessenderlo, Belgium Mr Michel Hars Diasource Immunoassays, Rue de bosquet, 2-1348 Louvain-La-Neuve, Belgium Dr Chenggang Liang and Dr Yang Zhen NIFDC, No. 2 Tiantan Xili, DongChen District, Beijing, 100050, China Dr Michael Girard and Mr Barry Lorbetskie Regulatory Research Division, Centre for Biologics Evaluation, Biologics and Genetics Therapies Directorate, Health Products and Food Branch, Health Canada, Ottawa, Canada, K1A 0R9 Dr Lars Nygaard CMC Analytical Support, Novo Nordisk A/S, Novo Nordisk Park, Building B6 2.046, DK-2760 Malov, Denmark Dr Verena Hofmann and Mr Erich Schneider Roche Diagnostics, GmbH, Nonnenwald 2, 82377 Penzberg, Germany Dr Renu Jain, Dr Gaurav Pratap Singh, Tara Chand National Institute of Biologicals, A-32, Sector 62, Institutional Area, NOIDA, U.P.-201307, India Yukari Nakagawa and Toshimi Murai Pharmaceutical and Medical Device Regulatory Science of Japan, PMRJ, 21-2 Hiranomachi, Chuo-ku, Osaka 541-0046, Japan

CHINA CANADA

DENMARK

GERMANY INDIA

JAPAN

WHO/BS/2015.2256 Page 5 JAPAN Mr Hirohito Honda Sanyo Chemical Industries Ltd. Medical Care Intracorporation, 11-1 Ikkyo Nomoto-cho, Higashiyama-ku, Kyoto, 605-0995, Japan Mr Yoshiyuki Kitamura Fujirebio INC, Product Lifecycle Management Department, 51 Komiya-cho, Hachioji-shi, Tokyo, 192-0031, Japan Ms Sakae Tazoe Kyowa Medex Co., Ltd, 600-1, Minami-ishiki, Nagaizumi-cho, Sunto-gun, Shizuoka, 411-0932, Japan Mr Koji Maki TOSOH Corporation, 2743-1, Hayakawa, Ayase-shi, Kanagawa, 252-1123, Japan Dr Hanna Ritzen and Ms Eleni Karamihos Mercodia AB, Sylveniusgatan 8A, SE-754 50 Uppsala, Sweden Mr Peter Sharratt PNAC Facility, Dept. of Biochemistry, 80 Tennis Court Road, Cambridge, CB2 1GA, UK Dr Marion Peak and Mr Tristan Coleman AltaBioscience, Building Y10, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK Dr Adrian Bristow and Dr Jackie Ferguson National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QG, UK Dr Gwen Wark and Mr Nick Porter UK NEQAS, Surrey Pathology Service, SAS Peptide Hormones Section, Clinical Laboratory, Royal Surrey County Hospital, Guildford, Surrey, GU2 7XX, UK Dr John Schulze University of California, Davis, Molecular Structure Facility, 1414 GBSF, 451 E. Health Sciences Dr., University of California, Davis, Davis, CA 95616, USA Brian Gregg, Bassem Azizeh, Charles Petersen and Rashmi Dalvi Bachem Americas, Inc., 3132 Kashiwa Street, Torrance, CA 90505, USA Alokesh Ghosal, Mark Hesselberg and Michael Ambrose US Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20852, USA Dr Brittany Lindburg Abbott Laboratories, 100 Abbott Park Road, Bldg AP8 Rm 606, Abbott Park, IL 60064-3500, USA Dr Jyh-Dar Lei Siemens Healthcare Diagnostics Inc., 511 Benedict Avenue, Tarrytown, NY 10591, USA

JAPAN

SWEDEN UK

USA

USA USA USA

USA

WHO/BS/2015.2256 Page 6 USA Nirja Patel ALPCO, 26 Keewaydin Drive, Suite G, Salem, NH-03079, USA

Bulk Material and Processing Human C-peptide bulk material was generated by custom peptide synthesis. A total of 100mg of synthesised and purified human C-peptide was purchased from Almac (Elvingston Science Centre, Scotland, UK). The peptide, with the sequence shown in Table 2, was produced using solid-phase peptide synthesis, cleaved and de-protected, then purified by RP-HPLC prior to lyophilisation to yield the final product. The 100 mg total was lyophilised as a 25mg and 75mg batch for trial and definitive fills respectively, and was reported to be >98% pure by RP-HPLC, with approximately 80% of the expected peptide content.

Table 2 Amino acid sequence of C-peptide obtained from Human Insulin sequence, NCBI reference P01308 C-peptide sequence EAED LQVG QVELG GGPG AGSL QPLA LEGS LQ

Preparation of the Primary Calibrant, PC01 C-peptide from the 75 mg bulk was used to produce the primary calibrant, PC01. 25 mg of Cpeptide was dissolved in a 30ml volume of 10mM sodium phosphate pH 7.0, 0.5% (w/v) trehalose and diluted to a final volume of 50 ml with the same buffer. The human C-peptide solution was distributed to 3ml crimp top vials on 14th June 2013 as 0.5 ml aliquots using a Hamilton Micro lab 500 series. The vial contents were freeze dried using a Virtis Genesis dryer and sealed under nitrogen using a Telstar freeze dryer. Vials were crimped with a hand held crimper manually and ampoules stored at -20⁰C. A total of 100 ampoules of primary calibrant PC01 were obtained, with a mean fill mass of 0.49373 g (CV 0.71%, n=17) and mean moisture content of 2.21% (CV 24.94%, n=3). Each ampoule of human C-peptide primary calibrant contained approximately 0.41 mg di-sodium hydrogen phosphate anhydrous, 0.29 mg sodium di-hydrogen phosphate monohydrate, 2.5 mg trehalose and 250 µg human C-peptide. Further analysis on the bulk material suggested that it contained approximately 80% of the expected peptide content and as a result it was anticipated that the primary calibrant would contain less than the predicted 250 µg C-peptide.

Preparation of ampoules of the candidate standard, 13/146 C-peptide from the 75 mg bulk was also used to produce the candidate standard. Approximately 30 mg C-peptide was dissolved in 100 ml of 10mM sodium phosphate pH 7.0, 0.5% (w/v) trehalose and diluted to a final volume of 1500 ml with the same buffer. The human C-peptide solution was dispensed into 3ml ampoules on 14th June 2013 using a Bausch and Strobel filling

WHO/BS/2015.2256 Page 7 machine (serial AFV5090). The ampoule contents were freeze dried and sealed under nitrogen using freeze dryer CS15 with cycle FD0134vol (SS399) according to procedures recommended by the WHO [8]. Ampoules were stored at -20⁰C.

A final total of 2718 ampoules of human C-peptide candidate standard, coded 13/146, were obtained with a mean fill mass of 0.5129 g (CV 0.23%, n = 88), a mean dry weight of 0.0026 g (CV 2.59%, n=6), a mean residual moisture (as determined by Karl Fischer titration) of 2.25% (CV 6.65%, n=12) and a mean head space oxygen of 0.55% (CV 33.3%, n=12). Mean residual moisture levels of 2.25% for 13/146 and 2.21% for PC01 were slightly higher than the target level of <1%. However, these were deemed acceptable, as peptides have been known to have slightly higher residual moisture levels than other formulations. In addition, the ampoules have a very low dry weight of 2.6 mg, which makes residual moisture analysis by Karl Fischer titration more prone to error. To address this second point, Karl Fischer analysis was repeated using three pooled ampoules for analysis, which resulted in a mean residual moisture of 0.63% (CV 28.8%, n=4), providing further evidence that ampoules of 13/146 contain an acceptable residual moisture level. Each filled ampoule contained approximately 0.41 mg di-sodium hydrogen phosphate anhydrous, 0.29 mg sodium di-hydrogen phosphate monohydrate, 2.5 mg trehalose and 10 µg human C-peptide. Taking into account that the bulk product was reported to contain approximately 80% of the expected peptide content, it was expected that the candidate standard would contain less than 10 µg C-peptide. Preliminary analysis by RP-HPLC in-house confirmed this and suggested that the candidate standard contained approximately 8.5 µg per ampoule.

Collaborative Study for the calibration of 13/146 Materials provided for the collaborative study The ampouled preparations of human C-peptide provided to participants at each phase of the study are listed in Table 3. It was also the aim of this study to assess the impact of introducing the candidate standard on the routine measurement of C-peptide in native samples. Although Cpeptide may be measured by some immunoassays from serum, plasma or urine, serum is the common sample matrix used by the majority of commercially available immunoassay kits. Urine samples were included to provide an additional native sample type. Fasting (overnight) and nonfasting serum and urine samples were obtained from volunteers where possible in order to provide a range of C-peptide values. Therefore, in addition to the ampouled preparations, participants in Phase III of the study were provided, where available, with 12 human serum samples and 12 human urine samples plus 4 C-peptide negative serum samples and 4 C-peptide negative urine samples spiked with candidate standard 13/146 to provide (or provide after dilution for urine samples) 5ng/ml, 2.5ng/ml, 1.25ng/ml and 0.625ng/ml C-peptide for inclusion in assays. All serum and urine and spiked samples were coded alphabetically, A-AF.

WHO/BS/2015.2256 Page 8 Serum samples were kindly collected by Dr G. Wark (UK NEQAS, Surrey Pathology Services, Guildford, UK), purchased from FirstLink (UK) Ltd or obtained from NIBSC in-house volunteer blood donors. Serum from UK NEQAS and FirstLink (UK) Ltd was frozen and sent to NIBSC on dry ice, where it was thawed, dispensed into 0.5 ml or 1 ml aliquots and stored at -80⁰C. Blood from in-house NIBSC blood donors was allowed to clot at room temperature for 1 hour, before transferring to 4⁰C overnight. The samples were then centrifuged at 2000xg for 15 mins at 4⁰C, the serum removed and then dispensed into 0.5 ml or 1 ml aliquots and stored at -80⁰C. All serum samples were tested 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 a local ethics committee. Urine samples were purchased from Sera Laboratories International Ltd and obtained from NIBSC in-house volunteer donors. Urine samples from Sera Laboratories were frozen and sent to NIBSC on dry ice, where it was thawed and dispensed into 50 µl aliquots and stored at -80⁰C. Urine from in-house NIBSC donors was dispensed into 50 µl aliquots on the day of donation and stored at -80⁰C. Urine samples purchased from Seralab were tested and found non-reactive for HCV RNA, anti-HIV 1/2, and HbsAg. Urine samples from NIBSC in-house volunteer donors were not tested for viral markers. Inclusion of urine samples in this study was approved by a local ethics committee. In order to maintain sample stability, both serum and urine samples were shipped to participants on dry ice. Participants were requested to store samples at -80’C prior to use in their assay.

Table 3: Human C-peptide ampouled preparations supplied to participants in the collaborative study. Human C-peptide Preparation 1st IRR 84/510 Primary calibrant, PC01 Candidate standard 13/146 stored at -20⁰C Accelerated thermal degradation (ATD) samples of 13/146 stored at +4⁰C, +20⁰C, +37⁰C and +45⁰C for 7 months Accelerated thermal degradation (ATD) samples of 13/146 stored at +4⁰C, +20⁰C, +37⁰C and +45⁰C for 18 months Ampoule unitage and nominal content 10 µg/ampoule Nominal 175 µg/ampoule assumed for Phase I Nominal content approximately 8.5 µg/ampoule assumed for Phase II Contents assumed identical to 13/146 at -20⁰C Allocation in study Phase III Phases I and II Phases II and III

Phase II

Contents assumed identical to 13/146 at -20⁰C

Phase III

WHO/BS/2015.2256 Page 9

Table 4: Human serum and urine samples supplied to participants in Phase III of the collaborative study. Code A B C D E F G H I J K L M N O P Q R S T U V W X Y Z AA AB AC AD AE AF Description Serum from non-fasting normal donor Serum from fasting donor Serum from Type I diabetic donor Serum from non-fasting normal donor Serum from fasting normal donor Serum from fasting normal donor Serum from fasting normal donor Serum from non-fasting normal donor Serum from non-fasting normal donor Serum from non-fasting normal donor Serum from Type I diabetic donor Serum from non-fasting normal donor 13/146 spiked C-peptide negative serum, 5ng/ml 13/146 spiked C-peptide negative serum, 2.5ng/ml 13/146 spiked C-peptide negative serum, 1.25ng/ml 13/146 spiked C-peptide negative serum, 0.625 ng/ml Urine from fasting normal donor Urine from fasting normal donor Urine from non-fasting normal donor Urine from Type I diabetic donor Urine from Type I diabetic donor Urine from non-fasting normal donor Urine from non-fasting normal donor Urine from non-fasting normal donor Urine from non-fasting normal donor Urine from non-fasting normal donor Urine from non-fasting normal donor Urine from non-fasting normal donor 13/146 spiked C-peptide negative urine, 100, 50 or 25ng/ml 13/146 spiked C-peptide negative urine, 50, 25 or 12.5ng/ml 13/146 spiked C-peptide negative urine, 25, 12.5 or 6.25ng/ml 13/146 spiked C-peptide negative urine, 12.5, 6.25 or 3.12ng/ml

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, human serum and human urine samples were only available in limited numbers. The study protocols for each phase of the study, included in Appendices 2-4, and

WHO/BS/2015.2256 Page 10 instructions for use were provided to participants along with samples.

Phase I: Amino acid analysis for value assignment of PC01 Participants were requested to derive estimates of the human C-peptide content of the primary calibrant, PC01, by amino acid analysis using their in-house method. Three vials of the primary calibrant were provided and participants were requested to carry out a minimum of 2 analytical runs for each vial.

Phase II: HPLC for value assignment of 13/146 Using the assigned value of 209 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 C-peptide content of the candidate standard 13/146 and its accelerated thermal degradation samples by comparison with PC01, using the HPLC method used in-house. An outline HPLC protocol was provided for guidance, and an additional ampoule of PC01 was provided in order to optimise HPLC conditions, reconstitution and injection volumes in line with the requirements of their system. Participants were requested to provide raw data and sample chromatograms, if possible, in addition to their own estimates of C-peptide content per ampoule.

Phase III: Immunoassay of candidate standard, 13/146 Due to the limited number of ATD and serum/urine samples that were available to send to participants, phase III of the study was split into two parts. In Phase IIIa, participants were sent ampoules of the candidate standard, 13/146, plus coded ampoules of ATD samples of 13/146 (dependent on availability) as described in Table 3 above. In Phase IIIb, participants were provided with candidate standard 13/146, a frozen baseline of the candidate standard, the 1st IRR 84/510, and 16 serum or 16 urine samples, depending on assay capability. All 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. Participants were requested to include all of the preparations allocated at no less than 5 dose levels in the linear part of the dose-response curve. Specific handling instructions for the materials were included in the study protocol (see Appendix 3) describing a core range of dose levels that were to be included in the assay by all laboratories. 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 provided, participants were asked to include human serum samples and spiked serum samples, coded A-P, and human urine samples and spiked urine samples, coded Q-AF, as described in Table 4 above. Spiked serum and urine samples were provided in order that the final concentration read in respective assays would be 5, 2.5, 1.25 or 0.625 ng/ml. Urine spiked samples were therefore provided at 5x-10x concentration depending on the urine dilution factor

WHO/BS/2015.2256 Page 11 used in the assay. Serum and urine samples were provided as 0.5 ml and 0.05 ml aliquots respectively. Participants were asked to ensure that all assays included their local standard, and to provide details of the assay method used, including dilution steps, together with all raw assay data 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 5-7 below. In total, immunoassays from 23 different manufacturers were performed by 15 laboratories in Phase IIIa and b. These included the following C-peptide immunoassay or radioimmunoassays; Abbott ARCHITECT, Alpco, Beijing Bio-Ekon Biotechnology Co.Ltd., Beijing North Institute of Biological Technology, DiaSorin, Diasource, Fujirebio, Guangzhou Darui Anitbody Engineering and Technology Co. Ltd., Kyowa Medex, Mercodia, EMD Millipore, Roche Elecsys, Sanyo SphereLight, Shenzhen Mindray Bio-Medical Electronics Co. Ltd., Sichuan Maccura Biotechnology Co. Ltd, Siemens Immulite 1000, Immulite 2000, ADVIA Centaur XP and ADVIA Centaur CP, and Tosoh AIA PACK C-peptide and C-peptide II assays.

Table 5: Assay methods – Phase I Lab Number 1 2 3 4 5 6 Assay Type Amino acid analysis Amino acid analysis Amino acid analysis Amino acid analysis Amino acid analysis Amino acid analysis Comments In-house method, standard acid hydrolysis In house method, standard acid hydrolysis In-house method, standard acid hydrolysis In-house method, standard acid hydrolysis In-house method, phenol hydrolysis In-house method, standard acid hydrolysis

Table 6: Assay methods – Phase II Lab Number 5 7 8 9 10 Assay Type HPLC HPLC HPLC HPLC HPLC Comments As protocol (Appendix 2) As protocol, with injection of 0.2-2.0 µg PC01 and 1 µg 13/146 and coded ampoules As protocol (Appendix 2), with shorter 150mm column and reduced flow rate As protocol (Appendix 2), with Phenomenex C18 column (5um, 300A) As protocol (Appendix 2)

WHO/BS/2015.2256 Page 12 11 12 HPLC HPLC As protocol (Appendix 2) As protocol (Appendix 2)

Table 7: Assay methods – Phase III Lab Number 10 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 21 22a 22b 23 24a 24b 24c 24d 24e 24f 24g 24h 25 Assay Type Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Radioimmunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Immunoassay Radioimmunoassay Immunoassay Immunoassay Comments

Two immunoassays performed on two different analysers.

One immunoassay performed on two different analysers

Statistical analysis The relative contents of the accelerated thermal degradation samples determined by immunoassay in Phase IIIa were analysed with a parallel line model using EDQM CombiStats Software Version 5.0 [9]. These results and relative contents by HPLC from Phase II were used to fit an Arrhenius equation relating degradation rate to absolute temperature, assuming first-order decay and hence predict the degradation rates when stored at -20°C [10].

WHO/BS/2015.2256 Page 13 Estimated concentrations (ng/ml) for all samples were log10 transformed for the assessment of commutability in Phase IIIb. For each laboratory pair, the mean and standard deviation of the bias was determined for serum and urine samples separately. Pooled standard deviations taken across all laboratory pairs were used to set commutability criteria as ±2SDS or ±2SDU for the acceptable difference in bias between reference materials and serum or urine samples respectively. Between-laboratory variability for individual samples has been assessed using geometric coefficients of variation (GCV = {10s-1}×100% where s is the standard deviation of the log10 transformed estimates).

Results Phase I: Amino acid analysis of primary calibrant PC01 Results obtained from amino acid analysis (6 laboratories) of the primary calibrant PC01 are shown in Table 8. The results from each laboratory are in good agreement, and the mean results from each laboratory were combined to give a final estimated content of 209 µg/ampoule (95% confidence limits: 199-219 µg/ampoule).

Table 8: AAA results for PC01 Method Lab Ampoule 1 1 2 3 1 AAA 2 2 3 1 3 2 3 C-peptide content (mg/ampoule) 0.209 0.208 0.207 0.210 0.209 0.208 0.199 0.191 0.193 0.208 0.206 0.207 0.209 0.211 0.207 0.196 0.195 0.191 / / / / / / 0.196 0.193 0.192 Mean (ampoule) 0.209 0.207 0.207 0.210 0.210 0.208 0.197 0.193 0.192 0.194 0.209 0.208 Mean (lab)

WHO/BS/2015.2256 Page 14 1 4 2 3 5 1 1 6 2 3 Final Estimate (mg/ampoule) Standard error Standard error % 95% confidence limits 0.242 0.230 0.220 0.205 0.215 0.216 0.215 0.209 0.004 1.88% 0.199 – 0.219 0.226 0.207 0.209 0.205 0.213 0.214 0.214 / / / 0.205 / / / 0.234 0.219 0.215 0.205 0.214 0.215 0.215 0.215 0.205 0.222

Phase II: HPLC of candidate standard 13/146 Results from HPLC of the candidate standard 13/146 (7 laboratories) are shown in Table 9 and raw data in Table A1. Data is as reported by participants using the peak area of the main Cpeptide peak to calibrate the candidate standard in terms of PC01. Some laboratories (lab 8, 9 and 10) noted an additional minor peak eluting after the main C-peptide peak in ATD samples, and that this peak was increased significantly in sample P (+45⁰C ATD sample). A possible explanation was offered by one participant that the 4 glutamine residues in C-peptide have the potential to form pyroglutamates upon thermal degradation or long term storage, which may lead to a peak with increased retention time. These impurity peaks were excluded from content calculations. Some participants (lab 8, 10 and 11) also noted a “shoulder” peak to the main Cpeptide peak. This was included in calculations by laboratory 8, but excluded from content calculations by laboratory 10. Laboratory 11 provided data both excluding and including the shoulder peak, and showed no difference in final ampoule content estimates. Results for the candidate standard, and its ATD samples, were in good agreement between laboratories, providing a final estimate of 8.64 µg/ampoule for the candidate standard, 13/146. Table 10 shows the final value assignment to the candidate standard, 13/146. Uncertainty in the value assigned to PC01 (% standard error) 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 for 13/146 of 8.64 µg/ampoule with expanded uncertainty of 8.21-9.07 (95% confidence; k=2.45).

WHO/BS/2015.2256 Page 15 Table 9: HPLC results for 13/146 Mean estimates (μg/ampoule)* Lab (-20°C) 5 7 8 9 10 11 12 Final estimate (μg/ampoule) Standard error Standard error % 95% confidence limits 8.59 8.77 8.51 8.61 8.45 8.64 8.89 8.64 0.06 0.66% 8.50, 8.78 N (+4°C) 8.71 8.77 8.58 8.57 8.15 / 9.05 8.64 0.12 1.4% 8.33, 8.95 L (+20°C) 9.10 8.54 8.38 8.42 7.75 / 8.80 8.50 0.19 2.19% 8.02, 8.98 M (+37°C) 8.16 7.91 8.06 7.80 8.15 / 8.10 8.03 0.06 0.73% 7.88, 8.18 P (+45°C) 7.78 7.72 7.65 7.37 7.25 / 7.76 7.59 0.09 1.20% 7.35, 7.82

Table 10: Assignment of value to candidate standard 13/146 Final Estimate (μg/ampoule) HPLC standard error % PC01 standard error % Homogeneity of filling weight CV % Combined standard uncertainty % Standard uncertainty Expanded uncertainty (95% confidence; k=2.45) 8.64 0.66% 1.88% 0.23% 2.01% 0.174 8.21, 9.07

WHO/BS/2015.2256 Page 16

Stability of 13/146 assessed by HPLC Participants were requested to estimate the C-peptide content of accelerated thermal degradation samples of 13/146 that had been stored at elevated temperatures of +4⁰C, +20⁰C, +37⁰C and +45⁰C for a period of 7 months. HPLC estimates of the C-peptide content of these ampoules are summarised in Table 9. Laboratories were in good agreement, giving estimates of 8.64, 8.50, 8.03 and 7.59 µg/ampoule which indicate losses of 0%, 1.6%, 7.1% and 12.2% at storage temperatures of +4⁰C, +20⁰C, +37⁰C and +45⁰C respectively for this time period. Analysis of degradation, as described in the statistical analysis section above, resulted in an acceptable predicted yearly loss of 0.07% per year (equivalent to 0.006 µg/year) when stored at -20⁰C.

Phase IIIa: Estimates of ampouled preparations 84/510, 13/146 and 13/146 frozen baseline by immunoassay Serum and urine patient samples and ATD samples of 13/146 were available in limited numbers only, and therefore Phase III of the study was split into two parts. The first, Phase IIIa, in which the ampouled preparations of 13/146 and 13/146 ATD samples (where available) were measured by participants using their in-house immunoassay in order to confirm the immunoreactivity of the standard across a variety of immunoassay methods worldwide. The second, Phase IIIb consisted of a more in-depth analysis of the commutability of the candidate standard with the inclusion of serum and urine samples in immunoassays alongside the ampouled preparations 13/146, a frozen baseline of 13/146 and the 1st IRR 84/510. Estimated concentrations (ng/ml) of reference material dilutions (84/510, 13/146 and 13/146 frozen baseline) from both Phase IIIa and IIIb were provided by participants, as calculated by comparison with kit standards using their in-house method. Estimated ampoule contents (µg/ampoule) have been calculated from these dilutions and these are summarised in Table 11. Assay results by laboratory 24g were excluded from further analysis as they represented anomalously low values for ampoules of the candidate standard. Lab 19 compared reconstitution of 13/146 in either PBS/BSA 0.1% (19a) as suggested in the study protocol or horse serum (19b) as suggested by the kit manufacturer. Results from lab 19a (buffer) were markedly higher for 84/510, 13/16 and 13/146 frozen baseline compared with the manufacturer’s recommended matrix, and these were therefore removed from the overall analysis of laboratory estimates as they were deemed anomalous for that assay system.

Table 11: Immunoassay results for 84/510, 13/146 and 13/146 frozen baseline Lab 10 13 14 15 Mean estimates (µg/ampoule) 84/510 13/146 9.10 9.69 9.89 9.92

13/146 frozen baseline 9.48 9.33 9.55

9.63 9.32 8.55

WHO/BS/2015.2256 Page 17 16 17a 17b 17c 17d 18a 18b 18c 18d 19b 20 21 22a 22b 23 24a 24b 24c 24d 24e 24f 24h 25 Mean 95% Confidence limits 8.90 9.77 9.28 8.27 8.24 9.34 9.39 9.63 9.40 7.39 8.78 9.47 9.05 8.86 8.86 8.73 10.22 9.78 10.72 9.59 8.51 10.64 10.76 10.29 12.21 11.21 10.09 8.79 10.43 13.20 7.89 8.25 9.14 9.78 9.31 – 10.25 8.91 9.00 9.04 9.19 8.83 8.42 9.76 8.16 9.44 8.65 8.33

9.91 9.37 9.04 11.60 8.01 9.66 9.20 8.77 – 9.63

9.00 8.72 – 9.29

Laboratory estimates for ampoule content were in reasonable agreement. The mean of laboratory estimates of 84/510 ampoule content gave 9.20 µg/ampoule (95% confidence limits 8.77 – 9.63), approximately 8% lower than the assigned content of 10 µg/ampoule for 84/510. The mean of laboratory estimates of 13/146 ampoule content gave 9.78 µg/ampoule (95% confidence limits 9.31 – 10.25), which is approximately 13% higher than the estimated content of 8.64 µg/ampoule for 13/146, as assigned by HPLC in Phase II of this study. Interestingly, mean laboratory estimates for the frozen baseline of 13/146 gave 9.00 µg/ampoule (95% confidence limits 8.72 – 9.29), which lies within the expanded uncertainty range (8.21-9.07) of the assigned content of 8.64 µg/ampoule to 13/146. It should be noted that the C-peptide content of 84/510 was assigned by immunoassay estimates in a limited study (5 laboratories with a total of 7 immunoassays) in the late 1980s. The assigned content of 84/510 is 10µg per ampoule, but it is possible this may be an overestimation. Taking the assigned content of 13/146 from phase II (8.64 µg/ampoule) and the ratio of immunoreactivity between 84/510 and 13/146 observed in this study, which is 0.94, it is likely

WHO/BS/2015.2256 Page 18 that the content of 84/510 may be closer to 8.1µg/ampoule. Since the manufacturer’s kit standards in this study are calibrated in terms of 84/510, with its assigned value of 10µg/ampoule, it is not surprising that immunoassay estimates of the content of the candidate standard are significantly higher than would be expected on the basis of the physicochemical assays. Manufacturers should therefore be made aware of the potential impact that replacement of 84/510 with 13/146, which has been assigned a more accurate value using physicochemical methods, may have on their assay system. It is also of interest that the buffer participants were recommended to use for reconstitution of reference materials (PBS/0.1% BSA) may be incompatible with some immunoassay systems. This is highlighted by data provided by laboratory 19, in which ampouled preparations were reconstituted in PBS/0.1% BSA and these further diluted in either PBS/0.1% BSA, or in assay matrix (horse serum), as recommended by the manufacturer. A higher estimate of C-peptide concentration was seen at each reference material dilution in PBS/0.1% BSA buffer compared with assay matrix (data comparing mean ampoule estimates in PBS/0.1% BSA and recommended assay buffer from these laboratories is provided in appendix Table A2). Similarly, in addition to data provided for the study, lab 24 also compared the PBS/0.1% BSA diluent with the recommended assay buffer for five assays, however, only 1 immunoassay system showed significant differences between the manufacturer’s recommended buffer and PBS/0.1% BSA (Table A2). These data highlight the need for users to define the appropriate conditions for their own immunoassay systems.

Phase IIIa: Stability of 13/146 assessed by immunoassay Five laboratories tested 13/146 and ATD samples of 13/146 at +4, +20, +37 and +45⁰C that were stored at elevated temperatures for 18 months. Using 13/146 stored at -20⁰C as a baseline, estimates of the relative ampoule contents of 13/146 ATD samples were calculated (Table 12). Laboratories were in good agreement, giving a mean relative potency of 1.00 for +4⁰C, 1.00 for +20⁰C, 1.02 for +37⁰C and 0.97 for +45⁰C. Compared with HPLC analysis of degradation samples, there appears to be very little effect of thermal degradation on the immunoreactivity of the candidate standard, with no loss in immunoreactivity detected at +4, +20 or +37⁰C elevated temperatures, and a very small percentage loss of 3% +45⁰C. This limited loss in activity did not allow a predicted yearly loss of activity to be determined from immunoassay data.

WHO/BS/2015.2256 Page 19 Table 12: Immunoassay results for 13/146 and ATD samples of 13/146 Lab 10 21 22a 22b 23 25 Mean 95% Confidence limits Mean estimates of ATD sample as percentage content of 13/146 -20⁰C baseline B +4⁰C D +20⁰C E +37⁰C C +45⁰C 1.04 1.11 1.17 1.08 1.00 0.95 ND 0.82 1.00 0.99 1.00 ND 1.00 0.98 0.96 ND 0.99 ND 0.97 0.97 ND 1.02 1.04 1.00 1.00 0.99 – 1.01 1.00 0.98 – 1.03 1.02 0.99 – 1.06 0.97 0.91 – 1.02

Phase IIIb: Commutability of 13/146, 13/146 frozen baseline and 84/510 There was reasonable agreement between laboratories in the C-peptide concentrations reported for serum samples (14 labs) and urine samples (11 labs). Serum samples C, D and K and urine samples T and U were obtained from Type I diabetics for inclusion in the study. As expected, values reported for these samples were described as zero or at/below the limit of detection for all immunoassays. These samples were then removed from further analysis. The statistical analyses used in the assessment of commutability of the ampouled preparations are described above. Similar to the analysis in Phase IIIa, for laboratory 19, only ampoule estimates from laboratory 19b results were further analysed as these represented ampouled preparations reconstituted in assay buffer as recommended by the manufacturer (horse serum matrix). In addition, assay run 2 results for samples AC-AF (13/146 spiked into C-peptide negative urine) performed by laboratory 17 in assays 17a, 17b, 17c and 17d were also excluded from further analysis due to potential technical error in the preparation or dilution of the samples.

Average bias in serum and urine sample results Estimated concentrations (ng/ml) for serum samples, urine samples and reference material dilutions are shown in Tables 13, 14, 15, 16 and 17. The average bias in log10 serum and urine sample results for each laboratory pair is shown in Table 18. The magnitude of the average bias ranged from zero (no difference between labs) to 0.230 (serum samples; lab pair 18a and 20) and 0.136 (urine samples; lab pair 16 and 18b). Commutability criteria were calculated as described in the statistical analysis section above and gave acceptable ranges of ±0.124 and ±0.200 for serum and urine samples respectively.

WHO/BS/2015.2256 Page 20 Table 13: Estimated concentrations for serum samples (ng/ml) Serum Sample Lab 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 A 5.54 3.52 4.67 4.90 5.97 6.19 4.84 4.46 4.83 5.13 4.28 5.04 5.78 7.64 B 1.44 1.44 1.24 1.50 1.54 1.51 1.40 1.42 1.17 1.35 1.24 1.47 1.79 2.26 E . 1.55 1.35 1.60 1.62 1.56 1.29 1.27 1.33 1.55 1.39 1.65 1.91 2.73 F 0.92 0.89 0.76 1.00 0.93 0.91 0.78 0.79 0.73 0.90 0.84 0.99 1.24 1.88 G . 1.03 1.12 1.30 1.42 1.38 1.23 1.16 1.18 1.38 1.24 1.44 1.76 2.10 H 3.15 2.55 2.98 3.00 3.80 3.59 2.97 2.83 3.11 3.38 3.04 3.53 3.83 4.40 I 1.97 1.57 1.66 1.90 1.94 1.93 1.60 1.56 1.68 1.93 1.72 2.00 2.34 2.76 J 2.27 2.39 2.19 2.50 2.67 2.67 2.19 2.13 2.29 2.50 2.17 2.50 2.94 4.13 L . 0.98 0.90 1.00 0.97 0.95 0.86 0.85 0.78 0.95 0.86 1.01 1.27 1.52 M 4.07 4.34 4.84 4.70 4.31 4.29 3.98 4.01 4.29 4.43 3.70 4.24 4.82 4.54 N 1.93 2.40 2.33 2.40 2.01 2.02 2.06 2.02 1.93 2.18 1.81 2.07 2.50 2.37 O 1.05 1.32 1.17 1.30 0.98 0.96 0.98 1.06 0.92 1.10 0.95 1.08 1.40 1.33 P 0.50 0.64 0.57 0.70 0.45 0.45 0.48 0.54 0.37 0.50 0.45 0.53 0.73 0.83

Table 14: Estimated concentrations for urine samples (ng/ml) Urine Sample Lab AA 13 15 16 17a 17b 17c 17d 18a 18b 18c 18d 28.19 26.18 23.90 26.25 27.70 24.05 24.33 29.25 31.00 28.98 31.65 AB . 21.83 20.40 21.45 21.10 20.48 20.39 23.75 25.50 24.37 27.33 AC 4.61 4.76 3.58 4.07 4.06 4.21 4.16 5.12 5.40 4.90 5.40 AD 2.27 2.30 2.28 1.86 2.07 2.15 2.33 1.30 2.60 1.36 1.54 AE 1.11 1.04 1.21 0.92 0.89 1.07 1.20 1.42 . 1.48 1.66 AF 0.64 0.55 0.63 0.47 0.27 0.54 0.59 0.66 . 0.70 0.82 Q 4.69 3.88 4.10 4.10 4.35 3.95 4.40 5.00 7.00 5.69 6.90 R 99.32 80.47 74.80 92.35 91.35 80.60 82.83 94.78 94.50 88.32 97.75 S 32.93 27.36 33.80 28.45 32.45 21.12 20.33 42.28 43.50 37.82 42.88 V 44.99 41.17 34.30 38.65 43.40 38.58 38.04 48.43 51.00 49.96 51.53 W . 14.16 17.10 15.10 15.70 14.38 14.94 13.68 15.00 12.23 14.60 X 8.74 6.60 7.10 7.25 7.45 6.87 7.47 9.38 10.00 10.27 12.05 Y 24.17 21.47 20.00 21.30 21.70 19.10 21.03 24.53 27.00 24.95 27.30 Z . 4.16 4.50 7.00 7.40 9.58 10.11 8.08 10.00 6.76 7.73

WHO/BS/2015.2256 Page 21 Table 15: Estimated concentrations for 13/146 (ng/ml) Dilution Lab 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 1 . . . . . . . . 24.09 24.60 25.48 26.49 . . 2 12.45 9.70 11.21 10.80 12.95 12.89 10.70 10.14 . . 11.49 12.37 10.96 9.23 3 5.76 5.64 5.68 5.20 5.83 5.17 5.46 5.12 5.86 6.11 5.52 6.35 5.33 5.14 4 2.68 2.95 2.73 2.60 2.68 2.62 2.45 2.57 2.77 3.01 2.71 3.03 2.70 2.44 5 1.36 1.50 1.31 1.20 1.28 1.20 1.24 1.29 1.18 1.44 1.37 1.48 1.41 1.24 6 0.66 0.75 0.68 0.60 0.58 0.57 0.62 0.64 0.52 0.67 0.70 0.77 0.74 0.63 7 . 0.37 0.34 . . . . . 0.25 0.38 0.34 0.39 . . 8 . . 0.23 . . . . . . . . . . .

Table 16. Estimated concentrations for 13/146FB (ng/ml) Dilution Lab 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 1 . . . . . . . . 22.79 23.10 22.21 23.97 . . 2 11.99 8.39 11.08 10.50 12.55 12.72 11.50 11.08 11.42 11.58 10.02 11.10 9.52 8.76 3 5.68 5.01 5.78 5.00 5.68 5.74 5.64 5.45 5.59 5.76 4.71 5.44 4.59 4.73 4 2.70 2.77 2.69 2.50 2.58 2.62 2.70 2.40 2.53 2.81 2.33 2.75 2.31 2.45 5 1.30 1.56 1.29 1.30 1.17 1.15 1.27 1.22 1.13 1.37 1.11 1.32 1.18 1.33 6 0.65 0.75 0.65 0.70 0.53 0.53 0.59 0.61 0.50 0.67 0.54 0.67 0.79 0.60 7 . . 0.36 . . . . . 0.22 0.36 0.27 0.32 . . 8 . . 0.18 . . . . . . . . . . .

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Table 17. Estimated concentrations for 84/510 (ng/ml) Dilution Lab 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 1 . . . . . . . . 20.84 . 21.58 21.88 . . 2 10.61 7.93 9.05 9.00 11.60 11.65 8.82 8.29 10.34 . 9.64 9.28 9.01 7.33 3 4.89 4.56 4.35 4.30 5.21 5.15 4.34 3.96 5.02 . 4.55 4.96 4.56 3.72 4 2.36 2.29 2.13 2.20 2.39 2.28 2.07 2.01 2.33 . 2.29 2.42 2.30 1.84 5 1.15 1.15 0.98 1.10 1.12 0.98 0.95 1.01 1.06 . 1.07 1.13 1.18 0.90 6 0.57 0.59 0.52 0.60 0.52 0.47 0.50 0.55 0.46 . 0.57 0.57 0.64 0.48 7 . 0.30 0.27 . . . . . . . . 0.29 . .

Table 18. Average bias in log10 serum and urine sample results for each laboratory pair 13 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 0.005 0.008 -0.040 -0.018 -0.014 0.030 0.029 0.049 -0.010 0.047 -0.018 -0.099 -0.166 0.007 -0.044 -0.029 -0.024 0.024 0.026 0.042 -0.020 0.034 -0.032 -0.113 -0.189 -0.051 -0.036 -0.030 0.017 0.019 0.035 -0.027 0.027 -0.039 -0.120 -0.195 0.015 0.021 0.068 0.070 0.086 0.024 0.078 0.012 -0.069 -0.144 0.006 0.053 0.055 0.071 0.009 0.063 -0.003 -0.084 -0.159 0.048 0.050 0.065 0.003 0.058 -0.009 -0.090 -0.165 0.002 0.018 -0.045 0.010 -0.056 -0.138 -0.213 0.016 -0.046 0.008 -0.058 -0.139 -0.215 -0.062 -0.008 -0.074 -0.155 -0.230 0.055 -0.012 -0.093 -0.168 -0.066 -0.148 -0.223 -0.081 -0.156 -0.075 14 . 15 0.052 . 16 0.055 . -0.003 17a 0.068 . -0.005 -0.001 17b 0.072 . -0.004 -0.001 0.000 17c 0.078 . -0.004 -0.001 0.001 0.000 17d 0.056 . -0.024 -0.020 -0.019 -0.020 -0.020 18a -0.011 . -0.072 -0.068 -0.067 -0.067 -0.068 -0.048 18b -0.067 . -0.130 -0.136 -0.113 -0.092 -0.125 -0.109 -0.063 18c -0.018 . -0.069 -0.066 -0.064 -0.065 -0.065 -0.045 0.003 0.070 18d -0.069 . -0.122 -0.119 -0.117 -0.118 -0.118 -0.098 -0.050 0.018 -0.053 19 . . . . . . . . . . . . 20 . . . . . . . . . . . . .

Serum Urine

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Commutability of 13/146, 13/146FB and 84/510 Reference material dilutions 3-6 only were used for the commutability assessment with respect to serum samples as these corresponded to the range of contents in serum samples tested. Similarly, only dilutions 2-6 were used for the commutability assessment with respect to urine samples. Tables 19, 20 and 21 summarise the proportion of reference material dilutions concluded to be non-commutable for each laboratory pair. For example, where there are four dilutions of 13/146, a value of 0.50 represents 50% dilutions that are non-commutable (coloured green) in that laboratory pair. Table 19. Proportion of 13/146 dilutions concluded to be non-commutable for each laboratory pair Lab 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 0 0 0 0 0 0 0 0 0 0 0 0.25 1.00 0 0.50 0.25 0.25 0 0 0 0 0 0 0.75 1.00 Serum Urine 0 0 0 0 0 0 0 0 0 0.50 1.00 0 0 0 0 0.25 0 0.50 0 0 0.75 0 0 0 0 0 0.25 0 0 0.75 0 0 0 0 0.25 0 0 0.75 0 0 0 0 0 0.25 1.00 0 0 0 0 0 1.00 0 0 0 0.50 1.00 0 0 0.50 1.00 0 1.00 1.00 0.50 1.00 0.50 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 13 14 15 0 16 0 17a 0 17b 0 17c 0 17d 0 18a 0 18b 0 18c 0 18d 0 19 . . . . . . . . . . . . 20 . . . . . . . . . . . . .

WHO/BS/2015.2256 Page 24 Table 20. Proportion of 13/146FB dilutions concluded to be non-commutable for each laboratory pair Lab 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 0 0 0 0 0 0 0 0 0 0 0 0.75 1.00 0 0.00 0.50 0.50 0 0 0.25 0 0 0 0.75 1.00 Serum Urine 0 0.25 0 0 0 0 0 0 0 0.75 1.00 0 0 0 0 0.25 0 0 0 0 1.00 0 0 0 0 0 0 0 0.50 0.50 0 0 0 0 0 0 0.50 0.50 0 0 0 0 0 0.75 1.00 0 0 0 0 0.75 1.00 0 0 0 0.75 1.00 0 0 0.75 1.00 0 0.50 1.00 0.75 1.00 0.25 13 14 . 15 0 . 16 0 . 0 17a 0 . 0 0 17b 0 . 0 0 0 17c 0 . 0 0 0 0 17d 0 . 0 0 0 0 0 18a 0 . 0 0.20 0 0 0 0 18b 0 . 0 0 0 0 0 0 0 18c 0 . 0 0 0 0 0 0 0 0 18d 0 . 0 0 0 0 0 0 0 0 0 19 . . . . . . . . . . . . 20 . . . . . . . . . . . . .

Table 21. Proportion of 84/510 dilutions concluded to be non-commutable for each laboratory pair Lab 13 14 15 16 17a 17b 17c 17d 18a 18b 18c 18d 19 20 0 0 0 0 0 0 0 0 . 0 0 0.25 1.00 0 0 0 0 0 0 0 . 0 0 0 1.00 Serum Urine 0 0 0 0 0 0 . 0 0 0 1.00 0 0 0 0 0.25 . 0 0 0 1.00 0 0 0 0 . 0 0 0.25 1.00 0 0 0 . 0.25 0 0.25 1.00 0 0 . 0 0 0 1.00 0 . 0 0 0 1.00 . 0 0 0.50 1.00 . . . . 0 0.50 1.00 0 1.00 1.00 13 14 . 15 0 . 16 0 . 0 17a 0 . 0 0 17b 0 . 0 0 0 17c 0 . 0 0 0 0 17d 0 . 0 0 0 0 0 18a 0 . 0 0 0 0 0 0 18b . . . . . . . . . 18c 0 . 0 0 0 0 0 0 0 . 18d 0 . 0 0 0.20 0.20 0 0 0 . 0 19 . . . . . . . . . . . . 20 . . . . . . . . . . . . .

WHO/BS/2015.2256 Page 25 The differences in bias between reference material dilutions and the average bias for each laboratory pair are shown graphically in Figures 1, 2, 3, 4, 5 and 6. In these graphs, each column on the x-axis represents a different lab or method pair (not specified) and each individual point is a different dilution of the reference material. Where these different reference material dilutions lie on the y-axis represents how different the lab bias for each reference material dilution is from the average bias for serum or urine samples within that lab pair. The line at zero shows where observed bias for reference material is the same as that observed for serum samples. The commutability criteria as calculated above for serum, ±0.124, and urine, ±0.200, are also depicted. With the exception of labs 19 and 20, the majority of points sit within the commutability criteria limits.

Figure 1. Difference in bias between 13/146 and serum samples

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Figure 2. Difference in bias between 13/146 and urine samples

WHO/BS/2015.2256 Page 27 Figure 3. Difference in bias between 13/146FB and serum samples

Figure 4. Difference in bias between 13/146FB and urine samples

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Figure 5. Difference in bias between 84/510 and serum samples

Figure 6. Difference in bias between 84/510 and urine samples

WHO/BS/2015.2256 Page 29 Table 22 shows the overall percentages of each reference material dilution concluded to be commutable. The Tables and Figures clearly indicate that, with the exception of labs 19 and 20, the 1st IRR 84/510, the candidate 1st IS 13/146 and the frozen baseline of 13/146, demonstrate commutability with respect to serum and urine samples.

Table 22. Percentage of cases with reference material dilution concluded to be commutable Standard 13/146 13/146 13/146FB 13/146FB 84/510 84/510 Sample type Serum Urine Serum Urine Serum Urine Dilution 2 100 100 96 3 77 100 74 100 77 100 4 80 100 75 100 82 100 5 81 100 78 100 85 100 6 81 100 84 98 83 100

Tables 23 and 24 show between-lab GCV values for each serum and urine sample determined from reported results or results expressed relative to the corresponding lab mean for 13/146 or 84/510. Similar values (pooled GCV ~20%) were obtained regardless of the standard (in-house calibrators, 13/146 or 84/510) used. As a subset of this data, the between-laboratory variability of 13/146 spiked in C-peptide negative serum samples (M-P) or urine samples (AC-AF) also showed no difference in %GCV whether calibrated to in-house standards, 13/146 or 84/510. These spiked in samples were included to assess the performance of dilutions of standard in different matrices, and it can be concluded that the candidate standard behaves in a similar manner, regardless of matrix. These data indicate that the introduction of the candidate standard, 13/146, for the calibration of immunoassays for C-peptide does not alter the spread of the data.

Table 23. Summary of between-laboratory variability (%GCV) for serum samples Serum Sample Data used Reported Relative to 13/146 Relative to 84/510 A 20% 25% 26% B 18% 21% 23% C 23% 24% 27% F 27% 29% 32% G 21% 24% 26% H 15% 18% 19% I 17% 19% 21% J 19% 22% 24% L 19% 21% 24% M 8% 11% 11% N 11% 12% 14% O 15% 15% 18% P 25% 26% 29% Pooled 19% 21% 23%

WHO/BS/2015.2256 Page 30 Table 24. Summary of between-laboratory variability (%GCV) for urine samples Urine Sample Data used Reported Relative to 13/146 Relative to 84/510 AA 10% 7% 8% AB 11% 7% 9% AC 14% 11% 14% AD 27% 28% 29% AE 23% 21% 23% AF 36% 34% 37% Q 24% 19% 18% R 10% 8% 7% S 30% 27% 25% V 15% 11% 13% W 9% 13% 11% X 22% 18% 19% Y 13% 9% 10% Z 36% 36% 37% Pooled 21% 19% 20%

Conclusions Measurement of C-peptide by immunoassay contributes to the diagnosis of a number of disorders related to beta cell function. Stocks of the current IRR for C-peptide, used to calibrate these immunoassays, are exhausted, and this report summarises the international collaborative study to establish a replacement 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 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 determine the C-peptide concentration of 16 serum and 16 urine samples in order to assess the impact of the introduction of the candidate preparation on the routine measurement of C-peptide in native samples. Laboratories were in good agreement in phase I and II, and calibration of the candidate standard by comparison with PC01 in RP-HPLC gave a final estimate of the content of the candidate standard of 8.64 µg/ampoule with expanded uncertainty of 8.21-9.07 (95% confidence; k=2.45). The candidate standard 13/146 appears to be sufficiently stable to serve as an international standard, based on HPLC analysis of accelerated thermal degradation samples of 13/146 that gave an acceptable predicted yearly loss of activity at -20⁰C of 0.07%. These results indicate that 13/146 is likely to be highly stable under long term storage conditions at -20⁰C. In addition, analysis of accelerated thermal degradation samples of 13/146 by immunoassay indicates that any degradation at elevated temperatures is still immunoreactive. The candidate standard has also been shown to have appropriate immunological activity, and there was reasonable agreement in the mean estimates of the immunoreactivity of 13/146 and the 1st IRR 84/510. Compared to the assigned value for 13/146 of 8.64 µg/ampoule, which was assigned by HPLC calibration, estimates of the content of 13/146 appear to be higher when measured using immunoassays. This is most likely to be a result of the overestimation in the assigned content of 84/510. Users should be aware of the potential impact of introducing the candidate standard for calibration of their immunoassays. Assessment of the commutability of the candidate standard was an important part of this study, and serum and urine samples were provided to participants to measure C-peptide concentrations alongside the candidate standard, 13/146, and the 1st IRR for C-peptide, 84/510. A frozen baseline for 13/146 was also included to analyse if there was any effect of standard processing (freeze drying) on the behaviour of the standard in these assays. With the exception of two

WHO/BS/2015.2256 Page 31 laboratories, the 1st IRR 84/510, the candidate standard 13/146 and the 13/146 frozen baseline, all demonstrated commutability with respect to serum and urine samples. In addition, the between-lab GCV values for each serum and urine sample from both participant-reported results and also results expressed relative to the corresponding lab mean for 13/146 or 84/510 showed that, regardless of the choice of standard, similar values of pooled GCV of ~20% were obtained, indicating that the introduction of the candidate standard, 13/146, for the calibration of immunoassays for C-peptide would not alter the spread of data. It should be noted that the analysis of commutability using bias, as described in this study, is only one of a number of methods that may be used to assess this parameter. Here, the limits of commutability have been assigned based on the statistical analysis of the bias, rather than any clinical limits of detection.

Proposal It is recommended that the preparation in ampoules coded 13/146 is established as the First International Standard for human C-peptide with an assigned content of 8.64 µg/ampoule with expanded uncertainty of 8.21-9.07 (95% confidence; k=2.45). A draft instructions for use has been included in Appendix 6.

Acknowledgements We gratefully acknowledge the important contributions of all the participants, the volunteer donors who donated blood and urine for inclusion in this study, Mr Hirofumi Koide of the Japanese Committee for Clinical Laboratory Standards for organising participating laboratories from Japan, and finally the Centre for Biological Reference Materials, NIBSC, for preparation of the ampouled materials.

References [1] Jones, A.G. and Hattersley, A.T. (2013) The clinical utility of C-peptide measurement in the care of patients with diabetes. Diabet Med, 30: 803-817. [2] Oram, R.A., Jones, A.G., Besser, R.E.J., Knight, B.A., Shields, B.M., Brown, R.J., Hattersley, A.T. and McDonald, T.J. (2014) The majority of patients with long-duration type 1 diabetes are insulin microsecretors and have functioning beta cells, Diabetologia, 57 (1):187-191 [3] Steffes, M.W., Sibley, S., Jackson, M. and Thomas, W. (2003) Beta-cell function and the development of diabetes-related complications in the Diabetes Control and Complications Trial. Diabetes Care, 26:832-836. [4] Wang, L., Lovejoy, N. and Faustman D.L. (2012) Persistence of prolonged C-

WHO/BS/2015.2256 Page 32 peptide production in type 1 diabetes with an ultrasensitive C-peptide assay. Diabetes Care, 35(3):465-470 [5] Cryer, P.E., Axelrod, L., Grossman A.B., Heller, S.R., Montori, V.M., Seaquist, E.R., et al (2009) Evaluation and management of adult hypoglycemic disorders: an Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab, 94:709-728 [6] Ryan E.A., Paty, B.W., Senior, P.A., Lakey, J.R., Bigam, D., Shapiro, A.M. (2005) Betascore: an assessment of beta-cell function after islet transplantation. Diabetes Care, 28:343-347. [7] Bristow, A.F. and Gaines Das, R.E. WHO International reference reagents for human proinsulin and human insulin C-peptide. J Biol Standardisation (1986) 16, 179-186 [8] WHO Tech Rep Ser No 800, 1990 181-214 [9] CombiStats v5.0, EDQM – Council of Europe, www.combistats.eu. [10] Kirkwood TBL (1977). Predicting the stability of biological standards and products. Biometrics, 33: 736-742.

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Appendix 1 Appendix Table A1: HPLC results for 13/146 Lab Ampoule A-1 A-2 A-3 5 L M N P A-1 A-2 A-3 7 L M N P A-1 A-2 A-3 8 L M N P A-1 9 A-2 8.67 Injection Mean (μg) 8.62 8.46 8.70 9.10 8.16 8.71 7.78 8.83 8.77 8.72 8.54 7.91 8.77 7.72 8.54 8.49 8.51 8.38 8.06 8.58 7.65 8.63 8.61 8.38 8.06 8.58 7.65 8.51 8.54 7.91 8.77 7.72 8.77 9.10 8.16 8.71 7.78 8.59 Sample mean (μg)

WHO/BS/2015.2256 Page 34 A-3 L M N P A-1 A-2 A-3 10 L M N P A-1 A-2 A-3 11 L (without shoulder peak) M N P A-1 A-2 A-3 12 L M N P / / / 8.82 8.90 8.95 8.80 8.10 9.05 7.76 8.80 8.10 9.05 7.76 8.89 / / / / / 8.54 8.42 7.80 8.57 7.37 8.45 / / 7.75 8.15 8.15 7.25 8.61 8.59 8.73 8.64 7.75 8.15 8.15 7.25 8.45 8.42 7.80 8.57 7.37

WHO/BS/2015.2256 Page 35 Appendix Table A2: Effect of different buffers for reconstitution of ampouled preparations (mean ampoule estimates from Lab 19 and 24) Lab 19a 19b 24a 24b 24c 24d 24h Mean ampoule estimates (µg/ampoule) reconstituted in PBS/0.1% BSA 11.53 12.12 12.16 11.21 10.09 8.79 10.43 8.25 Mean ampoule estimates (µg/ampoule) reconstituted in assay recommended buffer 9.40 9.91 9.37 9.04 9.66 9.99 9.76 6.78 9.59 11.92 10.43 9.67 8.94 8.77 8.65

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Appendix 2 Study Protocol Replacement of the WHO International Reference Reagent for human C-peptide (84/510) Phase I

INTRODUCTION C-peptide is a single chain peptide (MW 3020) of 31 amino acids synthesised in pancreatic beta cells as the connecting peptide for the A and B chains of insulin in the proinsulin molecule. Proinsulin is converted to insulin and C-peptide by a process of enzymatic cleavage, and these are then secreted into the portal circulation, by the beta cells, in equimolar amounts. C-peptide has a longer half-life than insulin and so higher concentrations persist in the peripheral circulation. In addition, there is less fluctuation in C-peptide levels compared to insulin levels. Because of this, measurement of plasma C-peptide levels may be a more reliable indication of pancreatic insulin secretion than the level of insulin itself and these measurements are used in the assessment of residual beta cell function, diagnosis of fatuous hypoglycaemia or insulinoma and as a marker of residual pancreatic tissue after pancreatectomy.

Stocks of the current International Reference Reagent (IRR) for human C-peptide, 84/510, are almost exhausted and a new International Standard is required for the calibration of C-peptide immunoassays. A new preparation of synthetic human C-peptide, has been filled into ampoules, coded 13/146, 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 C-peptide immunoassay.

AIMS OF THE STUDY C-peptide 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 84/510, 10µg/amp, is a consensus estimate derived from immunoassay. The candidate standard, 13/146, contains approximately 7 µg of formulated synthetic C-peptide. 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 13/146 in mass units and characterise its immunoreactivity.

WHO/BS/2015.2256 Page 37 Phase I involves the establishment of a primary calibrant (PC01), consisting of a limited number of ampoules containing approximately 175 µg C-peptide. 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 13/146, 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 C-peptide standard in immunoassay systems.

The aims of the study are therefore:

1. To calibrate the new preparation, 13/146, by physiochemical methods in mass units, relative to the primary calibrant, PC01. 2. To demonstrate the suitability of the preparation 13/146 to serve as the International Standard for human C-peptide 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 13/146 by examining its relationship with patient samples by immunoassay. 5. To determine the stability of the preparation 13/146 by comparison with ampoules stored at elevated temperatures.

MATERIALS Preparations supplied to participants in collaborative study, Phase I. Chemically synthesised C-peptide was formulated in a buffer of 10mM sodium phosphate, pH 7.0 and 0.5% trehalose. This solution was dispensed into 3ml glass crimped top vials (at 0.5 ml per vial), lyophilised and sealed.

The materials for Phase I of this study are listed in Table 1.

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Table 1.

C-peptide preparation Primary calibrant PC01

Vial content Nominally 175 µg C-peptide

TESTS REQUESTED Handling of material On receipt, vials should be stored at -20°C until use. It is recommended that the contents of each vial are reconstituted in double distilled water for physicochemical analyses.

Phase I Participants are requested to derive estimates of the C-peptide 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. C-peptide is a polypeptide of 3020 Da and 31 amino acids. The C-peptide amino acid sequence is detailed below:

EAED LQVGQVELGG GPGAGSLQPL ALEGSLQ

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

WHO/BS/2015.2256 Page 39

For further information, please contact: Dr Chris Burns Head, Endocrinology Section 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

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Appendix 3 Study Protocol Replacement of the WHO International Reference Reagent for human C-peptide (84/510) Phase II

AIMS OF THE STUDY C-peptide 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 84/510, 10µg/amp, is a consensus estimate derived from immunoassay. The candidate standard, 13/146, contains approximately 8 µg of formulated synthetic C-peptide. 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 13/146 in mass units and characterise its immunoreactivity.

WHO/BS/2015.2256 Page 41 Phase I involved the establishment of a primary calibrant (PC01), consisting of a limited number of ampoules containing 209 µg C-peptide. This value has been assigned to the primary calibrant by amino acid analysis.

Phase II will involve calibration of the candidate standard 13/146, in terms of the primary calibrant by HPLC.

The aims of the study are therefore:

1. To calibrate the new preparation, 13/146, by physiochemical methods in mass units, relative to the primary calibrant, PC01. 2. To demonstrate the suitability of the preparation 13/146 to serve as the International Standard for human C-peptide by examining its behaviour in immunoassays. 3. To evaluate the likely commutability of the proposed IS 13/146 by examining its relationship with patient samples by immunoassay. 4. To determine the stability of the preparation 13/146 by comparison with ampoules stored at elevated temperatures.

MATERIALS Preparations supplied to participants in collaborative study, Phase II. Chemically synthesised C-peptide was formulated in a buffer of 10mM sodium phosphate, pH 7.0 and 0.5% trehalose. This solution was dispensed into 2ml crimped top vials (at 0.5 ml per vial) for PC01 and into 3ml glass ampoules (at 0.5 ml per ampoule) for the candidate standard, 13/146. Both PC01 and 13/146 were then lyophilised and sealed.

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.

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Table 1.

C-peptide preparation Primary calibrant PC01 Candidate standard, 13/146, stored at -20°C Accelerated thermal degradation (ATD) samples of 13/146 stored at +4°C, +20°C, +37°C and +45°C

Vial content 209 µg C-peptide Nominally 8.5 µg C-peptide Content assumed to be identical to 13/146

TESTS REQUESTED Handling of material On receipt, vials and ampoules should be stored at -20°C until use. It is recommended that the contents of each vial are reconstituted in double distilled water for physicochemical analyses.

Phase II The primary calibrant, PC01, was assigned the consensus value of 209 µg per ampoule in Phase I of the study. Using this value, participants are asked to provide, in triplicate, estimates of the Cpeptide content of the three ampoules of the candidate standard (13/146) provided, and its accelerated thermal degradation samples (coded and provided in singlicate), by comparison with PC01 in a single HPLC run, using the HPLC method used in house. An outline HPLC 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 C-peptide content per ampoule.

REFERENCES

WHO/BS/2015.2256 Page 43

1. WHO Tech Rep Ser No 800, 1990 181-214

For further information, please contact:

Dr Chris Burns Head, Endocrinology Section 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

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Appendix 1 – Example protocol for HPLC assay of C-peptide content

NB. Participants are provided with an extra vial of primary calibrant to optimize HPLC conditions and sample reconstitution and injection volumes in line with the requirements of their system. Participants are also provided with an extra ampoule of the candidate standard in case of breakage.



HPLC column: C8 or C18, pore size 150-300 Å, particle size 5-7 µm Examples: Waters Symmetry 300 C18, 5 µm, 300 Å ,4.6 x 250 mm; Brownlee Aquapore octyl RP-300, C8, 7µm, 300 Å, 2.1 x 220 mm.

 

If available, a chilled autosampler should be used to maintain samples at 4°C. If available, a column oven should be used to maintain a constant column temperature. For the Waters Symmetry 300 C18 column, a temperature of 30°C was used. Mobile phase conditions: All aqueous buffers should be prepared in double distilled, or HPLC grade water (eg. Sigma Chromasolv®water for HPLC (Sigma-Aldrich, Cat. No. 270733), filtered and degassed. Mobile phase A: 0.1M NaH2PO4.2H2O (eg. Sodium Phosphate monobasic dihydrate, BioUltra grade, Sigma-Aldrich Cat. No. 71505) 0.5 % (v/v) H3PO4 (eg. Phosphoric acid for HPLC, 85-90 % (Fluka), Sigma-Aldrich Cat. no. 79606; final concentration in mobile phase A, approx. 0.425 – 0.45 % (v/v))



Mobile phase B: Acetonitrile (eg. Acetonitrile HiPerSolv CHROMANORM® for HPLC, VWR Cat. No. 83639.320)  Preparation of the standard curve of the primary calibrant Reconstitute vial contents in double distilled or HPLC grade water. All additions of water can be weighed to determine the exact mass of water added. A standard curve of the peak areas resulting from duplicate injections of a minimum of four quantities of C-peptide ranging from 1-5 µg should be prepared. Actual injection volumes and the concentration of the stock primary calibrant will depend on the loop volume and injection accuracy of

WHO/BS/2015.2256 Page 45 the HPLC system. Please provide full details of reconstitution and injection volumes used. For the Waters Symmetry 300 C18 column, the primary calibrant was diluted in HPLC grade water to a concentration of 40 µg/ml and injection volumes of 100 µl, 75 µl, 50 µl, 25 µl and 12.5 µl provided a standard curve with determinations of 4µg, 3 µg, 2µg, 1 µg and 0.5 µg C-peptide primary calibrant.



Preparation of coded ampoules Reconstitute ampoule contents in 0.5 ml double distilled or HPLC grade water. All additions of water can be weighed to determine the exact mass of water added. Injections of 100 µl, in triplicate, should provide sufficient material (approximately 1.8 µg Cpeptide) for the assay. Reconstitution in a smaller volume may be required if a smaller injection volume is used. A glass vial should be used for storage of the reconstituted samples in order to reduce non-specific adsorption and reconstituted ampoules should be used within 24 h. Please provide full details of reconstitution and injection volumes used. Sample Gradients: C peptide is eluted using a linear gradient of 5 % - 50 % mobile phase B in mobile phase A. A sample gradient for use with a flow rate of 1 ml/min is outlined in Table 1 below. The column is pre-equilibrated in 95 % mobile phase A, 5 % mobile phase B. Using the Waters Symmetry 300 C18 at a flow rate of 1 ml/min, a peak was seen between 29 and 31 minutes, but this may vary depending on the type of column and conditions used. Time (min) 0-5 5 - 45 45 - 50 50 - 52.5 52.5 – 62.5 Mobile phase A 95 % 95→50 % 50 % 50→95 % 95 % Mobile phase B 5% 5→50 % 50 % 50→5 % 5%



Table 1: Sample gradient for the elution of C-peptide from a Waters Symmetry 300 C18 column NB. Analysis of the candidate standard, 13/146, using the above protocol resulted in a minor peak detected at 39 min which absorbs at both 214 and 280 nm (C-

WHO/BS/2015.2256 Page 46 peptide does not absorb at 280nm). This peak was not present in the primary calibrant. Further analysis of this peak by MALDI-MS did not detect C-peptide or other protein and analysis by ultrasensitive C-peptide ELISA demonstrated no immunoreactivity. This suggests that the minor peak is not C-peptide related and can be disregarded from the HPLC analysis.  Absorbance Absorbance is monitored at 214 nm. Data analysis Using the chromatograms from the triplicate injections of each ampouled preparation, calculate the C-peptide content of each ampouled preparation from the standard curve of the primary calibrant by linear regression analysis. Please attach all corresponding chromatograms.



WHO/BS/2015.2256 Page 47

WHO/BS/2015.2256 Page 48

Appendix 4 Study Protocol Replacement of the WHO International Reference Reagent for human C-peptide (84/510) Phase IIIa

AIMS OF THE STUDY C-peptide 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 84/510, 10 µg/amp, is a consensus estimate derived from immunoassay. The candidate standard, 13/146, contains approximately 8 µg of formulated synthetic C-peptide. 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 13/146 in mass units and characterise its immunoreactivity.

WHO/BS/2015.2256 Page 49 Phase I involved the establishment of a primary calibrant (PC01), consisting of a limited number of ampoules containing 209 µg C-peptide. This value has been assigned to the primary calibrant by amino acid analysis.

Phase II involved calibration of the candidate standard 13/146, in terms of the primary calibrant by HPLC. HPLC estimates of C-peptide content of 13/146 from laboratories in Phase II assigned a value of 8.6 µg per ampoule. Accelerated thermal degradation samples were included in this phase of the study to provide data on the predicted stability of the candidate standard in storage at -20⁰C.

The purpose of Phase III is to provide confirmatory data of the candidate standard’s immunoreactivity by immunoassay. In addition, clinical samples will be incorporated into this phase to provide information on the potential commutability of the candidate C-peptide standard in immunoassay systems.

The purpose of Phase IIIa is to assess the performance of the candidate standard by immunoassay alongside accelerated thermal degradation samples of the candidate standard, to provide confirmatory immunoassay data of the predicted stability of the candidate standard.

The aims of Phase IIIa of the study are therefore:

7. To demonstrate the suitability of the preparation 13/146 to serve as the International Standard for human C-peptide by examining its behaviour in immunoassays. 8. To assess the relationships among existing local standards and the proposed IS 13/146. 9. To evaluate the stability of the proposed IS 13/146 by examining its accelerated thermal degradation samples by immunoassay.

MATERIALS Preparations supplied to participants in collaborative study, Phase IIIa. The candidate standard, 13/146, was prepared from chemically synthesized C-peptide and was formulated 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. Accelerated thermal degradation samples of the candidate standard, stored at +4⁰C,

WHO/BS/2015.2256 Page 50 +20⁰C, +37⁰C and +45⁰C. The materials for Phase IIIa of this study are listed in Table 1. All thermal degradation samples will be coded by letter.

Table 1. C-peptide preparation Candidate standard 13/146 Accelerated thermal degradation samples of 13/146 at +4⁰C, +20⁰C, +37⁰C and +45⁰C Ampoule content 8.6 µg C-peptide per ampoule. Content assumed identical to above.

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.

Phase IIIa 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. These two assays should measure each sample provided in duplicate or triplicate, using fresh ampoules where provided. Each assay should include the candidate standard plus all coded ampoules provided, each at a minimum of five dose levels in the linear part of the dose-response curve. A detailed protocol and a plate map for the suggested assay set up are provided in Appendix 1.

WHO/BS/2015.2256 Page 51 Estimates of the C-peptide content of each ampoule 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 e.g. counts for each sample, in electronic format if possible. Participants’ own calculated estimates of C-peptide 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.

REFERENCES 1. WHO Tech Rep Ser No 800, 1990 181-214

For further information, please contact:

Dr Melanie Moore Senior Scientist, Endocrinology Section National Institute for Biological Standards and Control Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG UK Tel: 44 (0) 1707 641246 Fax: 44 (0) 1707 641057

WHO/BS/2015.2256 Page 52 Appendix 1 Standard and sample processing A. Reconstitution and dilution of the candidate standard 13/146 and accelerated thermal degradation samples 1. Before opening, ampoules should be brought to room temperature to minimize moisture uptake. 2. Reconstitute each ampoule in 1 ml PBS plus 0.1% BSA or appropriate assay buffer. 3. Add solution in step 2 to 7.64ml PBS plus 0.1% BSA or appropriate assay buffer to provide a 1 µg/ml stock solution. 4. Dilute solution in step 3 1:50. E.g., add 40 µl to 1960 µl PBS plus 0.1% BSA or appropriate assay buffer to provide a 20 ng/ml working stock solution. This will form dilution 1 and the solution from which serial dilutions should be made.  Dilute the solution in Step 4 1:2 into PBS plus 0.1% BSA or appropriate assay buffer.  Prepare a further 8 1:2 dilutions as described above. Table A1 below provides the full details of the dilutions and their expected concentrations.  To enable comparison across different immunoassays at the same dilution point, participants are asked to include the 5 core concentrations highlighted in bold which should be included in all assays. If assay space permits, additional concentrations should be included. Table A1 List of dilution steps and corresponding standard concentrations Ampoule dilution Concentration (ng/ml)

Step 2. Step 3. Step 4./Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6 Dilution 7 Dilution 8

8640 1000 20 10 5 2.5 1.25 0.625 0.3125 0.156

WHO/BS/2015.2256 Page 53 Dilution 9 Dilution 10 0.078 0.039

WHO/BS/2015.2256 Page 54

B. Assay design and plate layout Alongside local standards and controls, each assay should include the candidate standard 13/146 and the provided coded degradation samples, each at a minimum of five dose levels in the linear part of the dose-response curve as described in section A and B above. Each dilution should be tested in duplicate or triplicate according to the in house method. To enable us to gather data regarding inter and intra-assay variability within each laboratory, participants are requested, where possible, to perform at least two independent assays with the samples provided. A plate map is provided below as an example of the suggested assay set up for a 96 well manual ELISA plate. Here an 8 point standard curve in duplicate for each ampoule has been used, leaving 2 rows for in-house kit calibrators. Please adapt the layout to suit the assay format required, leaving space for in-house calibrators and controls. All coded samples provided should be included on all plates alongside the candidate standard and local standards. Example plate maps Assay 1 Using Assay 1 ampoules: 13/146 dil 1 13/146 dil 2 13/146 dil 3 13/146 dil 4 13/146 dil 5 13/146 dil 6 13/146 dil 7 13/146 dil 8 13/146 dil 1 13/146 dil 2 13/146 dil 3 13/146 dil 4 13/146 dil 5 13/146 dil 6 13/146 dil 7 13/146 dil 8 B dil 1 B dil 1 C dil 1 C dil 1 D dil 1 D dil 1 E dil 1 E dil 1

B dil 2

C dil 2

D dil 2

E dil 2

B dil 3

C dil 3

D dil 3

E dil 3

B dil 4

C dil 4

D dil 4

E dil 4

B dil 5

C dil 5

D dil 5

E dil 5

B dil 6

C dil 6

D dil 6

E dil 6

B dil 7

C dil 7

D dil 7

E dil 7

B dil 8

C dil 8

D dil 8

E dil 8

The area shaded blue should be used for in-house calibrators and controls.

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Assay 2 As above using Assay 2 ampoules

C. Data reporting Estimates of the C-peptide content of all coded ampoules of the candidate standard 13/146 and its accelerated degradation samples should be calculated in comparison with the in-house assay kit standard. Participants are requested to provide details of the assay method used, including dilution steps, together with all raw data e.g. counts for each sample in electronic format if possible. Participants’ own calculated estimates of C-peptide concentration are also requested. A sample table for data reporting is provided below in Table A2.

Table A2 Sample data reporting table Assay 1 2 C-peptide concentration (ng/ml or ug/ampoule) 2 Avg

Sample replicates Baselines Kit standard 1 Kit standard 2 Kit standard 3 Kit standard 4 Kit standard 5

Avg

WHO/BS/2015.2256 Page 56 13/146 dil 1 13/146 dil 2 13/146 dil 3 13/146 dil 4 13/146 dil 5 13/146 dil 6 13/146 dil 7 13/146 dil 8 B dil 1 B dil 2 B dil 3 B dil 4 B dil 5 B dil 6 B dil 7 B dil 8 C dil 1 C dil 2 C dil 3 C dil 4 C dil 5 C dil 6 C dil 7 C dil 8 D dil 1 D dil 2

WHO/BS/2015.2256 Page 57

D dil 3 D dil 4 D dil 5 D dil 6 D dil 7 E dil 1 E dil 2 E dil 3 E dil 4 E dil 5 E dil 6 E dil 7 E dil 8

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Appendix 5 Study Protocol Replacement of the WHO International Reference Reagent for human C-peptide (84/510) Phase IIIb

AIMS OF THE STUDY C-peptide 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 84/510, 10 µg/amp, is a consensus estimate derived from immunoassay. The candidate standard, 13/146, contains approximately 8 µg of formulated synthetic C-peptide. 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 13/146 in mass units and characterise its immunoreactivity.

WHO/BS/2015.2256 Page 59

Phase I involved the establishment of a primary calibrant (PC01), consisting of a limited number of ampoules containing 209 µg C-peptide. This value has been assigned to the primary calibrant by amino acid analysis.

The aims of Phase III of the study are therefore:

10. To demonstrate the suitability of the preparation 13/146 to serve as the International Standard for human C-peptide by examining its behaviour in immunoassays. 11. To assess the relationships among existing local standards, the 1st IRR 84/510 and the proposed IS 13/146. 12. To evaluate the likely commutability of the proposed IS 13/146 by examining its relationship with patient samples by immunoassay.

MATERIALS Preparations supplied to participants in collaborative study, Phase III. The 1st IRR for human C-peptide, 84/510, consists of recombinant human C-peptide 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 C-peptide content of 10 µg per ampoule by immunoassay. The candidate standard, 13/146, was prepared from chemically synthesized C-peptide and was formulated 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

WHO/BS/2015.2256 Page 60 frozen baseline. Although it has not been possible to source a large number of clinical samples in order to perform a comprehensive commutability study, we are able to provide 16 serum samples and 16 urine samples for inclusion in immunoassays alongside the candidate standard 13/146, the 1st IRR 84/510 and local standards, which we hope will provide sufficient information on the relationship of the candidate standard 13/146 with patient samples and its likely commutability with these samples by immunoassay. Patient serum samples were kindly collected by Dr Gwen Wark (UK NEQAS), purchased from FirstLink (UK) Ltd and obtained from NIBSC in-house volunteer blood donors. Serum from UK NEQAS and FirstLink (UK) Ltd was frozen and sent to NIBSC on dry ice, where it was thawed, dispensed into 0.5 ml or 1 ml aliquots and stored at -80⁰C. Blood from in-house NIBSC blood donors was allowed to clot at room temperature for 1 hour, before transferring to 4⁰C overnight. The sample was then centrifuged at 2000/3000xg for 15 mins at 4⁰C and the serum removed, dispensed into 0.5 ml or 1 ml aliquots and stored at -80⁰C. Patient urine samples were purchased from Sera Laboratories International Ltd and obtained from NIBSC in-house volunteer donors. Urine samples from Sera Laboratories were frozen and sent to NIBSC on dry ice, where it was thawed, dispensed into 50 µl aliquots and stored at 80⁰C. Urine from NIBSC in-house donors was dispensed into 50 µl aliquots on the day of donation and stored at -80⁰C. C-peptide 13/146 spiked negative serum and C-peptide 13/146 spiked negative urine samples across the measuring range of assays have also been included. The materials for Phase III of this study are listed in Table 1. All patient samples and C-peptide 13/146 spiked negative patient samples will be coded by letter.

Table 1. C-peptide preparation 1st International Reference Reagent for human C-peptide, 84/510 Candidate standard 13/146 Frozen baseline Serum samples A-P Urine samples Q-AF Ampoule content 10 µg C-peptide per ampoule

8.6 µg C-peptide per ampoule. 8.6 µg in 0.5 ml 0.5 ml human serum 50 µl human urine

All serum samples have been tested negative for HIV-1/2, HbsAg and HCV NAT. Urine samples have not been tested for disease markers (with exception of Seralab purchased urine). As with all

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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 as described in the protocol in Appendix 1. 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. It is important that upon delivery, serum and urine samples are stored at -80⁰C until they are required. Serum samples should be thawed at room temperature and mixed well prior to assay. Urine samples should be thawed at room temperature and mixed well, then centrifuged briefly to remove particulate prior to assay. A detailed protocol for reconstitution and dilution of the standards is provided in Appendix 1.

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. These two assays should measure each sample provided in duplicate or triplicate, using fresh ampoules where provided. Each assay should include the current IRR for C-peptide, 84/510, the candidate standard 13/146, and the candidate standard 13/146 frozen baseline, each at a minimum of five dose levels in the linear part of the dose-response curve. This is described in detail in Appendix 1. It is essential that we are able to compare different immunoassays at the same dilution point and therefore a detailed protocol and a plate map for the suggested assay set up are provided in Appendix 1. Each assay should also include the provided serum and/or urine samples, which should be tested in duplicate according to the in house method using a fresh aliquot of serum or urine. Estimates of the C-peptide content of the current IRR for C-peptide, 84/510, the candidate standard 13/146, the candidate standard 13/146 frozen baseline, and the serum and urine samples should be

WHO/BS/2015.2256 Page 62 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 e.g. counts for each sample, in electronic format if possible. Participants’ own calculated estimates of C-peptide concentration are also requested. A sample table for data reporting is provided in Appendix 1.

REFERENCES

1. WHO Tech Rep Ser No 800, 1990 181-214

For further information, please contact:

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Appendix 1 Standard and sample processing

B. Reconstitution and dilution of candidate standard 13/146 5. Before opening, ampoules should be brought to room temperature to minimize moisture uptake. 6. Reconstitute each ampoule in 1 ml PBS plus 0.1% BSA or appropriate assay buffer. 7. Add solution in step 2 to 7.64ml PBS plus 0.1% BSA or appropriate assay buffer to provide a 1 µg/ml stock solution. 8. Dilute solution in step 3 1:50. E.g., add 40 µl to 1960 µl PBS plus 0.1% BSA or appropriate assay buffer to provide a 20 ng/ml working stock solution. This will form dilution 1 and the solution from which serial dilutions should be made.  Dilute the solution in Step 4 1:2 into PBS plus 0.1% BSA or appropriate assay buffer.  Prepare a further 8 1:2 dilutions as described above. Table A1 below provides the full details of the dilutions and their expected concentrations.  To enable comparison across different immunoassays at the same dilution point, participants are asked to include the 5 core concentrations highlighted in bold which should be included in all assays. If assay space permits, additional concentrations should be included. B. Reconstitution and dilution of 1st IRR 84/510

1. Before opening, ampoules should be brought to room temperature to minimize moisture uptake. 2. Reconstitute the ampoule in 1 ml PBS plus 0.1% BSA or appropriate assay buffer. 3. Add solution in step 2 to 9 ml PBS plus 0.1% BSA or appropriate assay buffer to provide a 1 µg/ml stock solution. 4. Dilute solution in step 3 1:50. E.g., add 40 µl to 1960 µl PBS plus 0.1% BSA or appropriate assay buffer to provide a 20 ng/ml working stock solution. This will form dilution 1 and the solution from which serial dilutions should be made.  Dilute the solution in Step 4 1:2 into PBS plus 0.1% BSA or appropriate assay buffer.  Prepare a further 8 1:2 dilutions as described above. Table A1 below provides the full details of the dilutions and their expected concentrations.  The concentrations highlighted in bold are the 5 core concentrations to

WHO/BS/2015.2256 Page 64 be included in all assays. If assay space permits, all concentrations should be included.

C. Dilution of the candidate standard frozen baseline 1. Thaw ampoule at room temperature, and gently tap to ensure contents are at the bottom of the ampoule before opening. 2. Add 0.5 ml PBS plus 0.1% BSA or appropriate assay buffer to give a volume of 1 ml. 3. Add solution in step 2 to 7.64ml PBS plus 0.1% BSA or appropriate assay buffer to provide a 1 µg/ml stock solution. 4. Dilute solution in step 3 1:50. E.g., add 40 µl to 1960 µl PBS plus 0.1% BSA or appropriate assay buffer to provide a 20 ng/ml working stock solution. This will form dilution 1 and the solution from which serial dilutions should be made.  Dilute the solution in Step 4 1:2 into PBS plus 0.1% BSA or appropriate assay buffer.  Prepare a further 8 1:2 dilutions as described above. Table A1 below provides the full details of the dilutions and their expected concentrations.  The concentrations highlighted in bold are the 5 core concentrations to be included in all assays. If assay space permits, additional concentrations should be included.

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Table A1 List of dilution steps and corresponding standard concentrations 13/146 dilution Step 2. Step 3. Step 4./Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6 Dilution 7 Dilution 8 Dilution 9 Dilution 10 Concentration (ng/ml) 8640 1000 20 84/510 dilution Step 2. Step 3. Step 4./Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6 Dilution 7 Dilution 8 Dilution 9 Dilution 10 Concentration (ng/ml) 10000 1000 20 13/146 frozen baseline Step 2. Step 3. Step 4./Dilution 1 Dilution 2 Dilution 3 Dilution 4 Dilution 5 Dilution 6 Dilution 7 Dilution 8 Dilution 9 Dilution 10 Concentration (ng/ml) 8640 1000 20

10 5 2.5 1.25 0.625 0.3125 0.156 0.078 0.039

WHO/BS/2015.2256 Page 66 D. Preparation of clinical samples A-AF Serum samples should be thawed at room temperature and mixed well prior to assay. Urine samples should be thawed at room temperature and mixed well, then centrifuged briefly to remove particulate prior to assay. Urine should be diluted prior to assay in accordance with participants’ in-house assay protocol.

E. Assay design and plate layout Alongside local standards and controls, each assay should include the current IRR for C-peptide, 84/510 and the candidate standard 13/146, each at a minimum of five dose levels in the linear part of the dose-response curve as described in section A and B above. Each assay should also include the patient serum samples and/or the patient urine samples, which should be tested in duplicate according to the in house method. To enable us to gather data regarding inter and intra-assay variability within each laboratory, participants are requested, where possible, to perform at least two independent assays with the samples provided. To aid this, each ampoule/patient sample (with exception of 84/510 1st IRR and 13/146 frozen baseline) will be labelled either Assay 1 or Assay 2. Due to limited stocks of 1st IRR 84/510 this standard and the 13/146 frozen baseline, only 1 ampoule of these can be provided. In this case, perform reconstitution as described in B) above, making 1ml aliquots of the 1 µg/ml stock solution and freezing at -20⁰C for use in Assay 2. A plate map is provided below as an example of the suggested assay set up for a 96 well manual ELISA plate. Here, serum and urine samples have been split into two separate plates, and a 5 point standard curve used due space limitations in the 96 well assay set up. Standards 13/146, 84/510 and 13/146 frozen baseline (FB) should be included on all plates alongside local standards.

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Example plate maps Assay 1 Plate 1: Serum samples 13/146 dil 2 13/146 dil 2 13/146 FB dil 5 13/146 FB dil 6 84/510 dil 2 84/510 dil 3 84/510 dil 4 84/510 dil 5 13/146 FB dil 5 13/146 FB dil 6 84/510 dil 2 84/510 dil 3 84/510 dil 4 84/510 dil 5 Serum B Serum B Serum J Serum J

13/146 dil 3 13/146 dil 4 13/146 dil 5 13/146 dil 6 13/146 FB dil 2 13/146 FB dil 3 13/146 FB dil4

Serum C Serum D Serum E Serum F Serum G

Serum K Serum L Serum M Serum N Serum O

84/510 dil 6 Serum A

Serum H

Serum P

Serum I

The area shaded blue should be used for in-house calibrators and controls.

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Plate 2: Urine Samples 13/146 dil 2 13/146 dil 2 13/146 FB dil 5 13/146 FB dil 6 84/510 dil 2 84/510 dil 3 84/510 dil 4 84/510 dil 5 13/146 FB dil 5 13/146 FB dil 6 84/510 dil 2 84/510 dil 3 84/510 dil 4 84/510 dil 5 Urine R Urine R Urine Z Urine Z

13/146 dil 3 13/146 dil 4 13/146 dil 5 13/146 dil 6 13/146 FB dil 2 13/146 FB dil 3 13/146 FB dil 4

Urine S

Urine AA Urine AB Urine AC Urine AD Urine AE

Urine T Urine U Urine V Urine W

84/510 dil 6

Urine X

Urine AF

Urine Q

Urine Y

Assay 2 Plate 1: Serum samples as above using Assay 2 ampoules Plate 2: Urine samples as above using Assay 2 ampoules

F. Data reporting Estimates of the C-peptide content of the 1st IRR for C-peptide, 84/510, the candidate standard 13/146, and the patient serum and urine samples should be calculated in comparison with the inhouse assay kit standard. Participants are requested to provide details of the assay method used, including dilution steps, together with all raw data e.g. counts for each sample in electronic format if possible. Participants’ own calculated estimates of C-peptide concentration are also requested. A sample table for data reporting is provided below in Table A2.

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Table A2 Sample data reporting table Assay 1 2 C-peptide concentration (ng/ml or ug/ampoule) 2 Avg

Sample replicates Baselines Kit standard 1 Kit standard 2 Kit standard 3 Kit standard 4 Kit standard 5 84/510 dil 2 84/510 dil 3 84/510 dil 4 84/510 dil 5 84/510 dil 6 13/146 dil 2 13/146 dil 3 13/146 dil 4

Avg

WHO/BS/2015.2256 Page 70 13/146 dil 5 13/146 dil 6 13/146 FB dil 2 13/146 FB dil 3 13/146 FB dil 4 13/146 FB dil 5 13/146 FB dil 6 Serum A Serum B Serum C Serum D Serum E Serum F Serum G Serum H Serum I Serum J Serum K Serum L Serum M Serum N Serum O Serum P

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Urine Q Urine R Urine S Urine T Urine U Urine V Urine W Urine X Urine Y Urine Z Urine AA Urine AB Urine AC Urine AD Urine AE Urine AF

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Appendix 6 Draft Instructions for use WHO International Standard 1st International Standard for human C-peptide NIBSC Code: 13/146 Instructions for use (Version 1.0, Dated 12/06/2015) 1. INTENDED USE The World Health Organisation (WHO) Expert Committee on Biological Standardisation (ECBS) has recognised (2010) the need for a replacement for the International Reference Reagent (IRR) for human C-peptide, coded 84/510. A preparation of C-peptide, coded 13/146, 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 C-peptide by the Expert Committee on Biological Standardisation of the WHO in October 2015. This replaces the International Reference Reagent, coded 84/510, as the primary reference material for the calibration of immunoassays of human C-peptide. 2. CAUTION This preparation is not for administration to humans 3. UNITAGE 8.64 µg/ampoule with expanded uncertainty of 8.21-9.07 (95% confidence; k=2.45). 4. CONTENTS Each ampoule contains the residue, after freeze-drying, of 0.5ml of a solution which contained: Synthetic C-peptide di-Sodium hydrogen phosphate anhydrous Sodium di-hydrogen phosphate monohydrate Trehalose 5. STORAGE Unopened ampoules should be stored at -20⁰C. 8.64 µg 0.41 mg 0.29 mg 2.5 mg

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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 (labelled) end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the disposable ampoule breaker covering the ampoule stem between the thumb and first finger of the other hand. Apply a bending force to open the ampoule at the coloured stress point, primarily using the hand holding the plastic collar. Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for example, by the use of suitable gloves and an eye shield. Take care that no material is lost from the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas at slightly less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN ampoule. 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 C-peptide. Depending on the intended use, dissolve the total contents of the ampoule in a known amount of a suitable diluent. Users should make their own investigations into the type of diluent suitable for their own use. 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 13/146 containing a nominal 8.5 µg lyophilized C-peptide, sodium phosphate and trehalose were prepared according to the methods recommended for international biological standards. A weighed portion of the C-peptide was dissolved in a sterile solution containing 10mM sodium phosphate pH7.0, 0.5% (w/v) trehalose and diluted to a final volume of 1500 ml with the same buffer. This solution was passed through a filter (mean pore diameter 0.45 µm) 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 -20⁰C in the dark. An international collaborative study was carried out by 24 laboratories in 10 countries in three phases. Phase I of the study involved the assignment of a value to a primary calibrant in mass units by amino acid analysis and phase II applied this value to the calibration of the candidate standard, 13/146, by RP-HPLC. Laboratory estimates for 13/146 were in good agreement, and the value of 8.64 µg/ampoule, with expanded uncertainty of 8.21-9.07, was assigned from results of phase II. The results of phase II also indicated the candidate standard 13/146 was sufficiently stable, on the basis of a thermally accelerated degradation study, to serve as an International

WHO/BS/2015.2256 Page 74 Standard. In phase III, the candidate standard was assessed by immunoassay to determine its suitability to serve as an International Standard. The results of phase III of the collaborative study indicated that the candidate standard showed appropriate immunological activity, and results of an assessment of the commutability of the candidate standard with a small cohort of patient samples indicated that the candidate standard 13/146 is commutable with serum and urine patient samples as measured by current immunoassays. The immunoassay estimates of 13/146 were some 13% higher than the assigned content based on physicochemical assays in phases I and II. However, given that the manufacturer’s assays used in this study are calibrated in terms of the IRR 84/510, this higher estimate is almost certainly a result of the limited dataset used in the original value assignment of the IRR 84/510. Manufacturers should therefore be aware of the potential impact on their assay calibration, of replacement of 84/510 with 13/146, which has been assigned a more accurate value using physicochemical methods. 9. STABILITY Stability based on HPLC analysis of thermally accelerated degradation samples of the candidate standard showed a predicted yearly loss of activity when stored at -20⁰C of 0.07% and a predicted yearly loss of C-peptide content of 3.2% at 20⁰C. These results indicate that 13/146 is likely to be highly stable under long term storage conditions at -20⁰C 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 all the participants in the collaborative study. 12. FURTHER INFORMATION Further information can be obtained as follows; This material: enquiries@nibsc.org 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:

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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 enquiries@nibsc.org 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: Stable: Yes Oxidising: Irritant: Handling: No No See caution, Section 2 No

Hygroscopic: Yes Flammable: No

Other (specify) Toxicological properties Effects of inhalation: Effects of ingestion: Effects of skin absorption: Suggested First Aid Inhalation: Ingestion: Contact with eyes: Contact with skin: Seek medical advice Seek medical advice Wash with copious amounts of water. Seek medical advice. Wash thoroughly with water. Not established, avoid inhalation Not established, avoid ingestion Not established, avoid contact with skin

WHO/BS/2015.2256 Page 76 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 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 freezedrying. 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.

Informations clés

Organisation Organisation mondiale de la santé (OMS)

Type de document Technical Documents

Date d'adoption 25 novembre 2015

Source Organisation mondiale de la santé