Label-free quantification of host cell protein impurity in recombinant hemoglobin materials.

Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA2) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U15N-labeled HbA2. For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA2. To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA2 mass fractions (or purities) of 923 and 928 mg(HbA2)/g(total protein) were found with expanded uncertainties (U) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA2 quantification when these materials are used as calibrators. Further purification of the natural HbA2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty (U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.

Determination of HbA2 by quantitative bottom-up proteomics and isotope dilution mass spectrometry.

BACKGROUND: Poor comparability between laboratories is often observed in the measurement of HbA2. A measurement procedure of higher metrological order is needed for value assignment to a reference material that shall be used as primary calibrator. METHOD: A reference measurement procedure has been developed based on isotope dilution mass spectrometry (IDMS). The α- and δ- subunits are quantified by signature peptides released by tryptic digestion of a 25 µL-blood sample. Full length U-15N-labeled HbA0 and HbA2 are used as internal standards and added to the sample at concentrations closely matching the levels of the natural forms in blood. By this, an improvement in precision could be achieved with respect to previous mass-spectrometry based methods. RESULTS: Recovery of HbA2 added to a blood sample was within 102.6-105.2%. Repeatability and within-laboratory imprecision was <2.0% for two blood samples containing HbA2 at a low and a high fraction. Total combined measurement uncertainty is estimated as 5.5%. Good agreement (r = 0.998) of results was obtained in a comparison of two laboratories using the described IDMS procedure. There is good correlation between commercial analytical systems and IDMS (r = 0.975-0.989). Some of the platforms provide significantly biased results, however, which potentially could be mitigated by reference to IDMS. CONCLUSION: IDMS holds a promise to be suitable as a reference measurement procedure for standardization of HbA2-measurements in laboratory medicine.

Developing a reference system for the IFCC standardization of HbA2.

The importance of hemoglobin A2 (HbA2) as an indicator of the presence of ß-thalassemia was established many years ago. However, clinical application of recommended HbA2 cut off values is often hampered due to poor equivalence of HbA2 results among methods and laboratories. Thus, the IFCC standardization program for HbA2 was initiated in 2004 with the goal of achieving a complete reference system for this measurand. HbA2 standardization efforts are still in progress, including the development of a higher-order HbA2 reference measurement procedure and the preparation of a certified reference material in collaboration with the IRMM. Here, we review the past, present and future of HbA2 standardization and describe the current status of HbA2 testing.

Gaps in the traceability chain of human growth hormone measurements.

BACKGROUND: Human growth hormone (hGH) is measured for the diagnosis of secretion disorders. These measurements fall under the EU Directive 98/79/EC on in vitro diagnostic medical devices requiring traceability of commercial calibrator values to higher-order reference materials or procedures (Off J Eur Communities 1998 Dec 7;L 331:1-37). External quality assessment schemes show large discrepancies between results from different methods, even though most methods provide results traceable to the recommended International Standard (IS 98/574). The aim of this study was to investigate possible causes for these discrepancies. METHODS: We investigated the commutability and recovery of hGH in reconstituted IS 98/574. We tested different reconstitution protocols and used 4 different serum matrices for spiking. These IS preparations were measured together with serum samples. We quantified hGH by 5 different methods in 4 different laboratories. RESULTS: Results from the different methods correlated well for the serum samples. Mean discrepancies between results from different methods were ≤20%. None of the IS preparations was commutable for all the method comparisons. The recovery of hGH in preparations of IS 98/574 depended on the reconstitution protocol (>10-fold differences) and background matrix (relative differences ≤17% for different serum matrices). CONCLUSIONS: The use of different protocols for reconstitution and spiking of hGH reference preparations affects quantification by immunoassays, potentially leading to a bias between commercial methods, despite the use of calibrators with values claimed to be traceable to the same higher-order reference material.