Development of UME CRM 1008: Certified Reference Material for C - reactive protein

doi:10.21203/rs.3.rs-3120831/v1

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Development of UME CRM 1008: Certified Reference Material for C - reactive protein

https://doi.org/10.21203/rs.3.rs-3120831/v1

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published 12 Jan, 2024

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A certified reference material (CRM) specifically designed for C-reactive protein (CRP) measurement in clinical laboratories was produced in this study. CRP, a protein synthesized by the liver, serves as an indicator of inflammation when present in elevated concentrations in the bloodstream. The primary goal is to ensure accurate measurement and quality control in accredited clinical laboratories. To achieve this, a recombinant CRP solution was selected as the most suitable candidate material for the CRM due to its exceptional purity. The homogeneity and stability of the developed CRM were thoroughly examined using size exclusion chromatography high performance liquid chromatography (SEC-HPLC). Additionally, amino acid analysis coupled with isotope dilution mass spectrometry (ID-LC/MS-AAA) was employed to determine the precise protein concentration of CRP in the CRM solution. The evaluation of uncertainties originating from factors such as characterization, homogeneity, long-term stability, and short-term stability data was incorporated to assess the uncertainty associated with the certified CRP value. Consequently, the certified value of the CRM, denoted as UME CRM 1008, was determined to be 43.2 ± 2.2 µmol kg-1, with an expanded uncertainty at a coverage factor of k = 2. The resulting CRP-certified reference material from this study will serve as an essential standard for validating CRP measurement methods and fulfilling quality control requirements within clinical laboratories. The ultimate aim is to make this reference material widely accessible to both national and international laboratories, thereby promoting accurate and standardized CRP measurements across the world.

CRM

CRP

C - reactive protein

ID-LC/MS-AAA

C-reactive protein (CRP) is a human serum protein widely used for diagnostic purposes as an acute-phase reactant [1]. It serves as a biomarker, exhibiting a significant increase, up to 100-fold, in response to inflammation, injury, or infection [2]. Additionally, CRP is present in normal human serum at very low concentrations, typically below 1 mg/L, as an acute-phase protein [2]. Abnormal levels of CRP have been associated with cardiovascular and cerebrovascular diseases [3, 4].The measurement of CRP presents challenges due to the lack of standardization in measurement methods and the diversity of reagents used, leading to significant deviations in measurement results [5]. Even the most commonly accepted CRP measurement assays show considerable discrepancies when using the IFCC CRP secondary reference material ERM-DA474/IFCC [6]. Furthermore, the clinical testing procedures for CRP measurements are highly dependent on the instrumentation and procedure used, further contributing to the lack of standardization in metrological procedures [7]. To address these issues and enhance diagnostic accuracy, the establishment of a reference measurement system for CRP is crucial. Such a system would enable the standardization of measurement results and provide traceability for in vitro diagnostics. The development of primary and secondary reference materials is essential for the evaluation and calibration of measurement methods [8]. Primary reference materials can be single compounds or mixtures used to verify and calibrate the primary reference method, with the measurement procedure defined by the Consultative Committee for the Amount of Substance (CCQM). On the other hand, secondary reference materials are employed to validate the calibration of serum-based protein standards and provide control materials for national metrology institutes or clinical diagnostic laboratories during the development of immunoassay measurement methods for CRP. Currently, there are CRP primary reference materials, such as CRM 6201-b and SRM 2924, produced by the National Metrology Institute of Japan and the National Institute of Standards and Technology, respectively [9][10]. However, the traceability of secondary reference materials, including ERM-DA474/IFCC and the WHO International Standard 85/506, is hindered by the lack of sufficient primary reference materials for their valuation [5].

In this study, a certified reference material of CRP was developed by TÜBİTAK National Metrology Institute of Türkiye (UME). The establishment of traceability in measurement methods played a crucial role in the development of the reference material, ensuring consistent and reliable results. Two reference measurement methods were employed: amino acid analysis coupled with isotope dilution mass spectrometry (ID-LC/MS-AAA) and size exclusion chromatography high-performance liquid chromatography (SEC-HPLC). ID-LC/MS-AAA is a highly preferred technique for protein quantification, providing traceable results aligned with the International System of Units (SI) [11]. The hydrolysis process, a critical step in amino acid analysis [9, 12], was optimized, and vapor-phase hydrolysis was chosen for its efficiency and the purity of the obtained samples. ID-LC/MS-AAA was used for primary reference characterization and concentration measurement, while SEC-HPLC served as a secondary reference measurement method for stability assessment, homogeneity analysis, and sensitivity correction. Statistical analysis, including ANOVA, was performed, and uncertainty contributions were determined to establish an uncertainty budget for the experimental process[11].

2.1 Chemicals and Materials

The reagents used in the study included NIMJ CRMs for the natural amino acid standards or calibration standards: L-alanine (Ala, CRM 6011-a, 99.9 ± 0.2%), L-leucine (Leu, CRM 6012-a, 99.9 ± 0.2%), L-phenylalanine (Phe, CRM 6014-a, 99.9 ± 0.2%), L-proline (Pro, CRM 6016-a, 99.9 ± 0.2%), and L-valine (Val, CRM 6015-a, 99.8 ± 0.2%). For the ID-LC/MS method, internal standards of labeled amino acids were obtained from Cambridge Isotope Laboratories. The internal standards used were as follows: Ala (13C3, 99%; 15N, 99%), Leu (13C6, 99%; 15N, 99%), Phe (13C9, 99%; 15N, 99%), Pro (13C5, 99%; 15N, 99%), and Val (13C5, 99%; 15N, 99%). Furthermore, as quality control in the study, standard reference materials from the National Institute of Japan (NIMJ CRM 6201-c) and the National Institute of Standards and Technology in Gaithersburg, MD (NIST SRM 2924) were employed.

2.2 Preparation of CRP

Recombinant human CRP was purchased from OYC Americas (Andover, MA) in a 1.25-liter bottle containing a buffer solution of 20 mM Tris (pH 7.5), 140 mM sodium chloride, 2 mM calcium chloride, and 0.05% (w/v) sodium azide. The recombinant CRP was produced in Escherichia coli as described by Tanaka et al. [11] and purified using affinity binding to 6-aminohexanoic acid. The 1.250 ml of recombinant CRP was divided into 1 ml aliquots. These aliquots were placed into 1250 polypropylene SRM vials and labeled accordingly, following the order of the vials. The material, which was shipped at 4°C, was subsequently stored at -80°C after being bottled.

2.3 HPLC Parameters

The Thermo ScientificTM UHPLC system with a Dionex Ultimate 3000 RS HPLC detector and RS diode array, Dionex Ultimate 3000 RS autosampler, Dionex Ultimate 3000 RS pump, column compartment, and column heater, along with an Agilent Bio-SEC33µm, 150Å 4.6×300mm HPLC column, was used for size exclusion chromatography analysis of CRP in an aqueous solution. The Thermo ScientificTM Chromeleon 7 software was employed for data analysis, and the peak areas of the analyte were determined. Prior to the analysis, the autosampler was set up with specific parameters, including a column temperature of 30°C, an injection volume of 10 µl, a loop volume of 100 µl, and the partial injection mode. The autosampler washing solution consisted of a 50:50% mixture of isopropanol and water (v/v). For the analysis, the reference material bottles were thawed at room temperature (20°C − 25°C) for one hour. A 10 µl injection of the CRP sample, derived from a 40 µM CRP concentrated stock solution, was introduced onto the system column. The elution mobile phase contained 0.1 M Tris HCl (pH 7.5) with 0.15 M NaCl and 1 mM EDTA. A gradient elution method was employed over 20 minutes with a flow rate of 0.3 ml.min^− 1 at a temperature of 30°C. The absorption peaks of the proteins were monitored at 225 nm and 280 nm by adjusting the parameters of detector A and detector B, respectively.

2.4 Homogeneity Analysis

Homogeneity analysis of the candidate reference material was conducted using the size exclusion chromatography (SEC) method. The analysis was performed was performed in triplicates using 12 randomly selected bottles from the pre-prepared 1250 vials, each containing 1 ml of the CRP solution. The concentration values of the 12 samples were determined based on their UV absorption at 225 nm and by analyzing the peak areas of their main peaks. The resulting data were then subjected to further statistical analysis.

2.5 Stability Testing

In the long-term stability studies for UME CRM 1008, two temperature conditions, -20°C and + 4°C, were selected, and the time points for testing were set at the 1st, 3rd, 6th, 9th, and 12th months. For each time interval at the designated temperatures, two units were placed in the respective temperature chambers, resulting in a total of 10 units for each temperature condition. As for the reference point in the stability test, two units were allocated and stored directly at the reference temperature of -80°C. At the end of each test period, two units were transferred from each test temperature environment to the reference temperature. After completing the 12-month test period, all units transferred to the reference temperature were analyzed together with the units designated as reference.

Similarly, in the short-term stability studies for UME CRM 1008, two temperature conditions, + 4°C and − 20°C, were selected, and the time points for testing were set at the 0th, 1st, 2nd, 3rd, and 4th weeks. For each time interval at the designated temperatures, two units were placed in the respective temperature chambers, resulting in a total of eight units for each temperature condition. As for the reference point in the stability test, two units were allocated and stored directly at the reference temperature of -80°C. At the end of each test period, two units were transferred from each test temperature environment to the reference temperature. After completing the 4-week test period, all units transferred to the reference temperature were analyzed together with the units designated as reference. For testing, the UV absorption values or peak areas at 225 nm in HPLC-UV were measured for each sample using size exclusion chromatography (SEC). Also, as part of the stability testing, the freeze and thaw cycle was repeated 5 times in duplicates to be able to observe the degradation level.

2.6 Molecular Weight Determination

To determine the approximate molecular weight of the pentameric candidate reference material, a calibration curve was constructed using the isocratic SEC-HPLC method and the Sigma Molecular Weight Kit (Sigma MWGF1000). The analysis involved injecting 3 µl of the candidate reference material solution into an Agilent BioSec-3 column with dimensions of 4.6x300 mm and a particle size of 3 µm (PN: 5190 − 2513 3 µM). The mobile phase used was phosphate buffer with a pH of 7.6. Elution was performed isocratically at a flow rate of 0.25 ml.min^− 1, and the column temperature was maintained at room temperature.

2.7 Amino Acid Analysis

The natural amino acid mixture was prepared gravimetrically in a ratio that closely matched the concentration of each amino acid in CRP. All these preparations were made fresh and independently for each hydrolysis reaction. Amino acid concentrations corresponding to 1.25 µM, 2.5 µM, 5.0 µM, 10 µM, 20 µM, and 40 µM CRP were used for each amino acid calibration standard, and each solution was prepared accordingly. The same procedure was applied for the preparation of labeled amino acid solutions. A six-point calibration curve was prepared gravimetrically for each hydrolysis method by mixing the natural and labeled amino acid mixtures. The final concentration of each natural and labeled amino acid in the calibration mixtures was adjusted to be nearly the same as in the sample blends.

2.7.1 Vapor-Phase Acid Hydrolysis

Six calibration standards were prepared, consisting of a mixture of natural and labeled amino acid solutions, and five candidate reference material solutions were prepared, containing isotopically labeled amino acids. After weighing the solutions in the glass tubes, they were placed into separate Pico-Tag vials along with the calibration mixtures. The calibration and sample mixtures were completely dried using a Pico-Tag Workstation (Waters, Milford, MA, USA). Then, they were hydrolyzed under inert atmosphere conditions at 130°C for 24 hours in hydrolysis tubes containing 200 µl of 6 M constant boiling HCl and 1% phenol. After the completion of hydrolysis, the samples were evaporated under vacuum until completely dry and then reconstituted with 200 µl of 20 mM HCl for the derivatization process of amino acids.

2.7.2 Derivatization for LC-MS

The dissolved samples (200 µl each) were mixed with 200 µl of Propanol:Pyridine (7:1) reagent and vortexed. Then, they were mixed with 200 µl of Isooctane:PCF (5:1) and incubated for 1 minute. Subsequently, 100 µl of CHCl3:Isooctane:PCF (24:16:1) reagent was added, vortexed, and incubated for 1 minute. Finally, 200 µl of 5% HCl was added, vortexed, and centrifuged. The upper phase of each sample was separated and transferred to clean tubes. The samples were then evaporated using the Caliper TurboVap LV evaporator under nitrogen. Lastly, each sample and calibrants in the tubes were dissolved by adding 50 µl of H₂O:acetonitrile (95:5) – 0.1% FA sample solution and transferred to suitable vials for LC-MS analysis.

2.7.3 LC-MS Analysis

LC analysis was carried out using a Dionex UPLC™ system (Thermo Scientific, Bremen, Germany) coupled with a Q Exactive HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific, Bremen, Germany) equipped with an electrospray ion source. Amino acids were injected and separated on Phenomenex EZ:faast 4 µ AAA column. The mobile phase was composed of, A: 10 mM ammonium formate, 50:50 methanol:water in water, B: 10 mM ammonium formate, and methanol. The autosampler wash solvent included a mixture of water, acetonitrile, and NH₄OH in a ratio of 40:50:10 (v:v:v) for both weak and strong washes. The injection volume was 10 µl. The gradient program used involved an increase in the B phase from 35–55% over 12 minutes, followed by an increase to 100% B. The flow rate was maintained at 0.250 µl.min^− 1 during a 5-minute wash with 100% B. Finally, reequilibration was achieved by washing with a 35% B solution for 5 minutes. The mass spectrometry analysis was performed in Full MS mode, and the MS parameters for amino acids following PCF derivatization are presented in Table 1

Table 1

MS Parameters for Amino Acids after PCF Derivatization
Natural Amino Acids PCF MS		Labeled Amino Acids PCF MS
Alanine	218.000	Alanine IS	221.980
Proline	244.154	Proline IS	250.168
Valine	246.169	Valine IS	252.183
Phenylalanine	294.169	Phenylalanine IS	304.196
Leucine	260.185	Leucine IS	267.202

All data are acquired in positive ion mode. The following acquisition parameters were set for the instrument: The spray voltage was set to 3.5 kV, sheath gas flow rate to 45, sweep gas flow rate to 2 and capillary temperature to 250°C. Full MS resolution was set to 120.000 for m/z 200 with maximum injection time of 200 ms. The Full MS AGC target was set to 3E6 and the mass range was defined as 100 − 500. Additionally, mass data extraction and analysis were performed using QuanBrowser™ Software. Following the MS measurements, the mass fractions of peptide samples were calculated for Leu, Pro, Val, Gly, and Ala amino acids based on the integral values of signal peak areas using calibration curves. The assignment process was completed by considering the gravimetric dilution factors.

3.1 The Recombinant Protein Structure

The study utilized recombinant human C-reactive protein (rhCRP) obtained from Oriental Yeast Co., Ltd. as the reference material due to its higher purity compared to CRP purified from human serum [12]. Extensive analysis confirmed that rhCRP shares the same protein structure as native CRP. This confirmation was based on factors such as molecular weight, ability to bind to phosphorylcholine in a calcium-dependent manner, N-terminal amino acid analysis, electrophoretic behavior, and immunochemical properties[12]. To ensure that the recombinant CRP maintained its pentameric structure similar to the native CRP, purification was conducted based on its calcium-dependent binding ability. Consequently, using recombinant CRP in immunochemical assays, such as the prediction of cardiovascular diseases, offers increased sensitivity due to its resemblance to native CRP and improved homogeneity [13].

3.2 The Structural Analysis and the Homogeneity Study

Mass spectrometry analysis was conducted using an Orbitrap Q-Exactive HF-X (Thermo Scientifc, USA) instrument operating in the High Mass Range Mode to determine the molecular weight of the CRP monomer. The acquired data was processed using BioPharma Finder (Thermo Scientifİc, USA) software to perform deconvolution and resolve the molecular masses. The results of the analysis revealed that the average relative molecular weight of CRP is 23,029 gr/mol. The findings are presented in Fig. 1, which includes two components: (a) the Mass Spectrum of the Candidate CRM, illustrating multiple charges, and (b) the Deconvoluted Mass Spectrum of the Candidate CRM, demonstrating the resolved molecular masses.The findings are presented in Fig. 1, which consists of two parts: (a) the mass spectrum of the candidate CRM, displaying multiple charges, and (b) the deconvoluted mass spectrum of the andidate CRM, showing the resolved molecular masses. This value is in good agreement with the theoretical molecular mass of human CRP, which is 23,028 gr/mol. The purity analysis using SEC-HPLC method revealed that CRP displayed a distinct and well-separated single peak with no impurities detected at 280 nm (Fig. 2a). At 220 nm, a major peak corresponding to the absorption of the CRP was observed, along with a minor peak attributed to the absorption of sodium azide (Fig. 2b). Furthermore, an approximate molecular weight calculation was performed using SEC molecular weight marker proteins. The estimated molecular weight of CRP was approximately 120 kDa. Native CRP consists of five identical subunits that are non-covalently linked, resulting in a total molecular weight of approximately 115 kDa. Therefore, the molecular weight of the tested material as measured by SEC-HPLC closely matched the theoretical molecular weight of the CRP pentameric structure, with no impurities detected.

A homogeneity study is conducted to evaluate if the values determined for CRP are within a certain confidence interval across all units, aiming to represent the entire production. According to ISO Guide 35 [14], the number of units for homogeneity study should be at least 10 units, with the production size being approximately the cube root of that number, ensuring representation of the entire material. For the production of UME-CRM 1008, a homogeneity study was performed using a stratified random sample selection program called TRaNS produced in TUBITAK UME, including 12 units along with their backups. This program is designed to assist in selecting representative samples from a population for analysis or testing purposes. By utilizing stratified random sampling, the program ensures that the selected samples are proportionally representative of different strata or subgroups within the population. Homogeneity analyses were carried out by measuring three subsamples from each unit under repeatability conditions. The homogeneity analysis was performed using size exclusion chromatography (SEC-HPLC) method. The homogeneity study results are shown in Fig. 3. The data obtained for UME CRM 1008 were evaluated, and the relative standard deviation was found to be 1.07%, indicating that the material possesses sufficient homogeneity suitable for its intended use in the inter-unit homogeneity test. Subsequently, the data obtained from the results were used for further statistical analysis. The Shapiro-Wilk test yielded a p-value of 0.056, it was concluded that the data followed a normal distribution.

3.3 Stability Study

Stability studies for UME CRM 1008 were conducted to assess the effects of environmental conditions during the shipment of the reference material (short-term stability - STS) and storage conditions (long-term stability - LTS) by replicating similar conditions in the laboratory. Stability studies were performed using an isochronous design, where measurements were taken simultaneously.

3.3.1 Short Term Stability Testing

For UME CRM 1008, short-term stability studies were conducted at temperatures of + 4°C and − 20°C, with time intervals set at 0, 1, 2, 3 and 4 weeks. The simultaneously conducted measurements for UME CRM 1008 were first grouped according to the same temperature conditions, and evaluation was performed for each time point. After analyzing the data, the standard uncertainty contributions were determined to be 0.09% at -20°C and 0.14% at + 4°C after one week. Taking into account the maximum shipping duration of two weeks, the expiration date for short-term storage conditions was determined to be two weeks, considering the uncertainty contributions of 0.18% at -20°C and 0.29% at + 4°C after two weeks. In summary, both − 20 ºC and + 4 ºC storage conditions showed good short-term stability, with no significant degradation observed during the test period. The short-term stability results are also shown in Fig. 4.

3.3.2 Long Term Stability Testing

For UME CRM 1008, long-term stability studies were conducted at temperatures of -20°C and + 4°C, with time intervals set at 1, 3, 6, 9, and 12 months. The material's stability under storage conditions was tested for 12 months at both − 20°C and + 4°C, and no signs of degradation were observed (Fig. 5). The linear regression analysis conducted for the long-term study showed significant results at a 1% significance level. No outliers or trends were detected in either of the long-term studies. Additionally, for the long-term study, the standard uncertainty contribution was determined to be 0.37% at -20°C and 0.97% at 4°C after 12 months. Based on the evaluation of the results from the long-term stability studies, it can be concluded that UME CRM 1008 can be stored for one year at temperatures below − 20°C.

3.4 Value Assignment and ID-LC/MS-AAA

For the characterization of UME CRM 1008, the ID-LC/MS Amino Acid Analysis (AAA) method was employed. AAA provides an absolute quantitative measure of the protein content of a sample based on the content of amino acids comprising the test product, independent of an external protein reference standard. In the AAA method used to measure the absolute concentration of CRP, vapor-phase acid hydrolysis conditions were set at 100 µL, 130 ºC, and 24 hours, which had been previously optimized by Kato et al. [9]. Five amino acids (Pro, Val, Leu, Phe, and Ala) were selected as analytes, and analysis was performed in duplicate after the hydrolysis process conducted for three days. Calibration curves for amino acid measurements were established using calculated standard mixtures of selected natural and labeled amino acids, prepared according to the known amino acid sequence of CRP. Additionally, contributions from impurities arising from hydrolysis or derivatization processes of peptide molecules were determined by analyzing peptide/protein-related impurities with the PICAA analysis approach and subtracted from the overall value. The uncertainty evaluations of amino acid analysis used in this study were conducted following the ISO GUIDE Reference materials-Guidance for characterization and assessment of homogeneity and stability 4th edition [13] and the EURACHEM/CITAC Guide Quantifying Uncertainty in Analytical Measurement [14]. Table 2 presents the total protein concentrations along with the contributions of relative uncertainty.

Table 2

Total Protein Concentrations and Relative Uncertainty Contributions (%)
	Pro	Val	Leu	Phe	Ala
Quantitative Value (µmol/kg)	43.0	43.4	43.0	42.9	43.6
Uncertainty Components (Relative, %)
Intra-Day Variation (AA)	0.00	0.00	0.00	0.00	0.10
Inter-Day Variation (AA)	1.46	1.34	1.59	1.58	2.73
Inter-Amino Acid Variation	0.31	0.310	0.31	0.31	0.31
Calibration Curve	1.56	1.29	2.42	0.43	0.99
Amino Acid Purity	0.10	0.10	0.10	0.10	0.10
Balance	8x10³	8x10³	8x10³	8x10³	8x103
Combined Relative Standard Uncertainty, (%)	2.16	1.89	2.92	1.67	2.93
Combined Standard Uncertainty (µmol/kg)	0.9	0.8	1.4	0.7	1.3
Expanded Standard Uncertainty (µmol/kg)	1.9	1.6	2.6	1.4	2.6

The validity of the measurements in this study was confirmed through the analysis of NIST SRM 927d and NIMJ CRM 6201-c CERTİFİED reference materials. The same method and analysts were used to quantitatively analyze and determine the certified CRP concentration in these materials. The assigned value for NIST was 20.37 µmol/L with an uncertainty of ± 1.56 mol/L, while for NIMJ it was 39.79 µmol/kg with an uncertainty of ± 1.94 µmol/kg. These values were compared to the certified values of the reference materials, which were reported as 20.6 µmol/L with an uncertainty of ± 1.2 µmol/L (k = 2) for NIST, and 40.2 µmol/kg with an uncertainty of ± 1.7 µmol/kg (k = 2) for NIMJ [11]. The results of this comparison showed that the concentration values obtained using the same measurement method had minimal deviations from the certified values of the reference materials. This provides strong evidence for the validity and reliability of the measurement method employed in this study.

3.5 Uncertainty Evaluation

The combined standard uncertainty (uc) of the property value for the candidate CRM was determined by combining the contributions from value assignment (uchar), material inhomogeneity (sbb), uncertainty due to long-term instability (ults), and uncertainty due to short-term instability (usts). The calculation of uc was performed using Eq. 1 [28].

(1)\({u}_{c}^{2}={u}_{char}^{2}+{s}_{bb}^{2}+{u}_{lts}^{2}+{u}_{sts}^{2}\)

Table 3 presents the relative and combined results of the uncertainty contributions, which were utilized in assigning a value to the candidate reference material. Ultimately, the certified concentration of the reference material was determined as 43.170 µmol/kg ± 2.148 µmol/kg (k = 2)

Table 3

Uncertainty Contributions for Analysis Results (Relative and Combined Values)
Relative Combined Measurement Uncertainty	2.15%
Contribution of Homogeneity to Uncertainty (Relative)	1.07%
Contribution of Long-Term Stability to Uncertainty (Relative)	0.37%
Contribution of Short-Term Stability to Uncertainty (Relative)	0.14%
Relative Combined Uncertainty Contribution	2.43%
Standard Uncertainty, u	1.1 µmol.kg^− 1
Expanded Uncertainty, k:2	2.2 µmol.kg^− 1

In conclusion, the development of a certified reference material (CRM) for C-reactive protein (CRP) measurement is a significant step towards enhancing diagnostic accuracy and standardizing CRP measurements in clinical laboratories. This study successfully produced a CRM, UME CRM 1008, specifically designed for CRP measurement, ensuring accurate and reliable results in accredited laboratories. The selection of a recombinant CRP solution as the CRM material was based on its exceptional purity, making it a suitable candidate for accurate measurement and quality control purposes. The characterization of the CRM involved the use of advanced analytical techniques such as SEC-HPLC and and ID-LC/MS-AAA. Through the evaluation of uncertainties originating from factors of characterization, homogeneity, long-term stability, and short-term stability, an uncertainty budget was established to assess the uncertainty associated with the certified CRP value. The certified value of the CRM, 43.2 ± 2.2 µmol.kg^− 1, with an expanded uncertainty at a coverage factor of k = 2, provides a reliable reference for CRP measurements. The availability of this CRP-certified reference material will contribute to the validation of CRP measurement methods and fulfill quality control requirements in clinical laboratories. By promoting accurate and standardized CRP measurements, the aim is to facilitate improved diagnosis and management of inflammatory conditions, as well as associated cardiovascular and cerebrovascular diseases. Ultimately, the widespread accessibility of this reference material to both national and international laboratories will support the harmonization of CRP measurements and foster global consistency in clinical practice. The establishment of traceability in measurement methods and the use of reference materials are vital for ensuring reliable and comparable CRP results, leading to enhanced patient care and improved healthcare outcomes.

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Development of UME CRM 1008: Certified Reference Material for C - reactive protein

Status:

Journal Publication

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Chemicals and Materials

2.2 Preparation of CRP

2.3 HPLC Parameters

2.4 Homogeneity Analysis

2.5 Stability Testing

2.6 Molecular Weight Determination

2.7 Amino Acid Analysis

2.7.1 Vapor-Phase Acid Hydrolysis

2.7.2 Derivatization for LC-MS

2.7.3 LC-MS Analysis

3. Results and Discussion

3.1 The Recombinant Protein Structure

3.2 The Structural Analysis and the Homogeneity Study

3.3 Stability Study

3.3.1 Short Term Stability Testing

3.3.2 Long Term Stability Testing

3.4 Value Assignment and ID-LC/MS-AAA

3.5 Uncertainty Evaluation

4. Conclusion

References

Additional Declarations

Status:

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