Challenges in preclinical bioanalytical method development of the deoxypyridinoline (DPD) bone resorption biomarker in rat urine

G.A. Tremblay, M. Hada, T. Gruetzner, S. Lavalle, G. Pinard Charles River Laboratories Preclinical Services Montreal Inc., 22022 Transcanadienne, Senneville, Quebec, Canada H9X 3R3

Abstract
Purpose. Biomarkers for preclinical drug development can be determined with immunoassays using kits available on the market. The discrepancy between research-purpose kits and the regulatory requirements for preclinical bioanalysis may cause difficulties in the course of GLP method R&D and validation. One such example was found with the validation of a commercial kit for Deoxypyridinoline (DPD), a biomarker of bone resorption. Using the ELISA kit meant to analyze human urine samples, a method was developed for the determination of DPD in rat urine. Methods. The kit is a competitive enzymatic immunoassay, where DPD conjugated to alkaline phosphatase competes with DPD from the samples. The kit comprises a standard curve and quality control (QC) samples prepared in assay buffer. The reference material of the kit is purified bovine DPD which serves to quantify human test samples. The standard curve and QCs were prepared with buffer, urine, reference material or endogenous material in order to better represent test samples and document the assay performance. Results. Upon assessing precision and accuracy in rat urine using a standard curve in buffer and QC samples in urine, it was found that the higher QC concentration levels over-recovered at values 150-200%; there was no parallelism at the higher end of the curve. However, when preparing the standard curve with the endogenous material, parallelism was reestablished.

Introduction (continued)
DPD is a useful biomarker for bone resorption and may be measured in urine tests in patients when osteoporosis is suspected. We looked at validating a Ligand-Binding Assay (LBA) in the form of an enzymatic immunoassay (EIA) for DPD in rat urine, but also in other similar matrices such as monkey urine and rat serum. Most commercially available kits typically are developed for research purpose only, and although they can be used in GLP-compliant work, most often these assay kits cannot be used right out of the box and may need to be optimized in order to validate them. Thus, research purpose kits may be at times difficult to validate because they were not intended for GLP-compliant work. That was the case with a commercial EIA kit for the quantitation of DPD. This poster tells the tale of a validation leading to a method that was considered fit-for-purpose.

Results (continued)
Table I. Intra-assay precision and accuracy for Quality Control (QC) samples made with rat urine. Recovery is above 25% for the ULOQ, QC3 and QC2 levels. Standard curve is made with purified DPD in buffer whereas QCs consist of the endogenous rat DPD. Red=out of acceptance results.
DPD Concentration in rat urine (nmol/L) Intra-Assay Precision and Accuracy (n=3)

Results (continued)
For comparison purposes, in Figure 3, a successful parallelism experiment for urea in monkey bronchoalveolar (BAL) fluid is shown, where the endogenous urea concentration is compared with urea-spiked assay diluent. The parallelism is wellwithin recovery acceptance along the entire curve range, which was not the case with DPD. In order to solve the problem of over-recovery at high concentrationgranted the opportunity of a large endogenous DPD concentration in rat urine, we tried to formulate the standard curve using solely the endogenous DPD in the biological matrix, as opposed to using the purified bovine DPD from the kit in assay diluent. The endogenous concentration of DPD in rat urine is required to be determined in the first place using the purified bovine DPD in the kit. An appropriate dilution of the biological matrix was taken in order to obtain a reading around the inflexion point of the standard curvei.e. at mid curve range, where the assay is most reliable. Table II. Intra-assay precision and accuracy for Quality Control (QC) samples in rat urine. Recovery is above 25% for the LLOQ. Both the standard curve and QCs are prepared with the endogenous DPD in rat urine. Red=out of acceptance results.
DPD Concentration in rat urine (nmol/L) Intra-Assay Precision and Accuracy (n=3)

Discussion
In a recent paper, Valentin and colleagues emphasize the critical importance of parallelism for biomarker validation8, as it serves several purposes and is often missing from immunoassay validation or mistaken with selectivity. In the present case, the main issue was the lack of proportionality between the reference standard and the endogenous value at high DPD concentration. How is it possible to explain the over-recovery at high concentration in rat urine? Quidels DPD EIA kit is designed to analyze Human urine. DPD exists both in free and peptide-bound form in urine. Rat urine has a ratio of free-to-total DPD of approximately 20%6, whereas that ratio is about 40% in Human urine7. There is a chance that the over-recovery is due to cross-reactivity of the anti-DPD antibody with peptide-bound DPD in rat, which would have gone unnoticed in Human; however the kit insert claims that there should be less than 2.5% reactivity of the anti-DPD antibody with the DPD peptide. Alternatively, a simpler explanation could be that because there are generally high CVs with this kit, it is possible that there is a large variability in the replicates due to the design of and limitations inherent with this kit, and this weakness shows at low optical density. The solution to this problem was to determine the endogenous value for DPD where parallelism was effective, i.e. at mid curve range, then to formulate the standard curve with the endogenous DPD and to readjust the curve range according to the new sensitivity of the assay. There are limitations in validating a commercial kit. Claims of a kit supplier should not be taken at face value, and unforeseen inter-species variability may arise. For instance, many kits are designed for Human samples, whereas preclinical samples will require various animal species. Even for FDA-approved kits, their use in preclinical studies will require optimization to abide with the GLP Fortunately, simpler adjustments may be . more common when developing/validating a biomarker method from a commercial kit. In conclusion, a biomarker method for DPD in rat urine was validated after adjusting various parameters and this may serve as lesson learned in adapting a commercial kit to a GLP-compliant assay.

LLOQ QC1 QC2 QC3 ULOQ

Theoretical 0.30 0.90 7.87 17.97 29.10

Determined 0.32 1.05 6.15 12.68 19.39

CV (%) 5.8 1.0 3.8 3.0 3.0

Recovery (%) 87.1 85.0 128.0 141.7 150.1

High recovery above 25% was observed (Table I) at high QC concentrations, i.e. at a low response since this is a competitive assay. In some instances, there was a high recovery at low concentrations. Variability was also seen when testing dog or monkey urine formulated QCs. There were high CVs leading to a non-robust assay in rat urine. A wider range of QC concentration was formulated along the working curve range to test parallelism between purified (kit) DPD and endogenous (matrix) DPD. The experiment demonstrates that there was no parallelism at high DPD concentration; weak parallelism was also observed at lower concentration. Figure 2. DPD Standard Curve in buffer (triangles) superimposed on Quality Control samples in rat urine (circles) at concentrations along the calibration curve range showing the lack of robustness at higher concentration. The curve fit is a four-parameter logistic regression.
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Material and Methods

The MicroVue DPD assay kit (Quidel, San Diego) is a competitive Enzyme ImmunoAssay (EIA) in a micro-titer strip-well format utilizing a monoclonal anti-DPD antibody coated on the strips in order to capture DPD. In this competitive EIA, the DPD analyte in the sample competes with a DPD-Alkaline Phosphatase conjugate (DPD-AP) for binding to the coated antibody and the reaction is detected with a pNPP substrate. The standard curve therefore displays an inverse relationship connecting colorimetric detection and analyte concentration. The standards and quality control samples (QCs) are prepared either with the purified free bovine DPD provided with the kit or using the endogenous concentration of DPD found in a biological matrix. The concentration and matrix identity are indicated in the text or on the figures, where applicable. All samples are analyzed in duplicate. For comparing parallelism assessment with different kits and matrices, a Urea Assay Kit was used (BioVision, Mountain View, CA).

AAPS, Washington, D.C.

Conclusion. Qualification data demonstrate that the procedure is suitable for bioanalysis of DPD in rat urine. Considerations for developing a GLP method with a research-purpose kit are discussed. This work represents a lesson learned in adapting a commercial kit to regulatory bioanalysis.

LLOQ QC1 QC2 QC3 ULOQ

Theoretical 0.29 0.88 5.86 11.72 19.70

Determined 0.50 1.00 5.19 10.70 18.95

CV (%) 6.2 4.8 0.8 1.8 1.9

Recovery (%) 170.4 113.7 88.5 91.3 96.2

Standard Curve

Introduction
Bone is a dynamic tissue where production and resorption regulate bone homeostasis via osteoblast and osteoclast functions, respectively. Osteoporosis is a metabolic bone disease characterized by an imbalance in this homeostasis. In osteoporosis, bone resorption is favored over production leading to bone porosity and increased bone fractures. The most common cause of osteoporosis is menopause as a consequence of oestrogen depletion. Cessation of oestrogen production by the ovaries results in at least 10% to 15% bone loss over the next 10 to 15 years post menopause1. Figure 1. Structure of the two major collagen cross-linking molecules PYD and DPD along with a schematized representation of intermolecular crosslinks at amino- and carboxy termini in bone type I collagen3.

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As shown in Table II, although the problem of over-recovery at high concentration was eliminated by using a standard curve formulated with the endogenous DPD in rat urine, there was now over-recovery at the LLOQ level. The decision was taken to increase the LLOQ from the kits recommended 0.3 nmol/L up to 1 nmol/L, giving an overall range from 1 to 20 nmol/L, which is an acceptable curve range. Figure 4. Representative calibration curve where endogenous DPD in rat urine is used to prepare the standard curve.
Standard Curve
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Results
Although there are no regulatory requirements defining the bioanalytical method validation parameters, the FDA and EMEA issued bioanalytical method validation guidelines, and there is a fairly clear scientific consensus on fit-for-purpose GLP validation parameters for biomarkers4,5. In order to comply with agreeable validation parameters, a kit for the quantitation of DPD in rat urine required several adjustments in order to be validated. Guidelines state that a minimum of six non-zero standard points should be defined, whereas there were only five non-zero standards found with Quidels DPD EIA kit. A high concentration standard from the kit was used to generate a greater number of data points. There were only two QC sample concentration levels, whereas the FDA guidelines state that three concentrations representing the entire range of the standard curve should be studied, namely, low, middle and high QC sample concentrations. Endogenous DPD concentration was to be determined in order to formulate the QC samples with the biological matrix and for parallelism assessment, as the method should demonstrate that biological rat urine samples can be reliably analyzed. The endogenous concentration of DPD in rat urine was determined around the middle of the standard curve range provided with the kit (0.3 up to 20 nmol/L) using the purified bovine DPD reference standard of the kit. The concentration of DPD was high in rat urine (approximately 1500 nmol/L) and thus the QC samples were generated by preparing the biological matrix solely with the endogenous DPD in rat urine. QC samples corresponding to the lower limit of quantitation (LLOQ), low QC (QC1), mid QC (QC2) and high QC (QC3) and the upper limit of quantitation (ULOQ) were thus prepared by serial dilution.

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References
(1) Yu SL, Ho LM, Lim BC, Sim ML. Urinary deoxypyridinoline is a useful biochemical bone marker for the management of postmenopausal osteoporosis. Ann Acad Med Singapore. 1998 Jul;27(4):527-9. (2) Gerrits MI, Thijssen JH, van Rijn HJ. Determination of pyridinoline and deoxypyridinoline in urine, with special attention to retaining their stability. Clin Chem. 1995 Apr;41(4):571-4. (3) Urea P De Vernejoul MC. Circulating biochemical markers of bone remodeling in uremic , patients. Kidney Int. 1999 Jun;55(6):2141-56. Review. (4) Lee JW, Devanarayan V, Barrett YC, Weiner R, Allinson J, Fountain S, Keller S, Weinryb I, Green M, Duan L, Rogers JA, Millham R, OBrien PJ, Sailstad J, Khan M, Ray C, Wagner JA. Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res. 2006 Feb;23(2):312-28. (5) Lee JW, Hall M. Method validation of protein biomarkers in support of drug development or clinical diagnosis/prognosis. J Chromatogr B Analyt Technol Biomed Life Sci. 2009 May 1;877(13):1259-71. Review. (6) Preedy VR, Sherwood RA, Akpoguma CI, Black D. The urinary excretion of the collagen degradation markers pyridinoline and deoxypyridinoline in an experimental rat model of alcoholic bone disease. Alcohol Alcohol. 1991;26(2):191-8. (7) Gerrits MI, Thijssen JH, van Rijn HJ. Determination of pyridinoline and deoxypyridinoline in urine, with special attention to retaining their stability. Clin Chem. 1995 Apr;41(4):571-4. (8) Valentin M-A, Ma S, Zhao A, Legay F, Avrameas A. Validation of immunoassay for protein biomarkers: bioanalytical study plan implementation to support pre-clinical and clinical studies. J Pharm Biomed Anal. 2011 Jul 15;55(5):869-77.

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As shown on Figure 2, the high concentration is prone to error in recovery. When drawing a line between the two curves at high concentration, for the same OD, there will be a large discrepancy in concentration between a QC sample and a standard data point. Thus QC concentrations will not be accurately determined at high DPD concentration. Figure 3. Standard Curve in buffer spiked with urea (triangles) superimposed on Quality Control samples for urea in monkey BAL fluid (circles) at concentrations along the calibration curve range showing the recovery of the samples in a matrix read off from the pure analyte in buffer. The curve fit is a four-parameter logistic regression.

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The final standard curve for DPD in rat urine is shown in Figure 4. The R2 is of 0.999. Note that two accessory standards, around 0.7 and 30 nmol/L, are used below the LLOQ (1 nmol/L) and above the ULOQ (20 nmol/L), respectively, in order to anchor the standard curve. Table III. Inter-assay precision and accuracy for Quality Control (QC) samples in rat urine. Recovery is within 25% for all QCs. Both the standard curve and QCs are prepared with the endogenous DPD in rat urine.
DPD Concentration in rat urine (nmol/L) Inter-Assay Precision and Accuracy (n=21)

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Standard Curve

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Type I collagen represents around 90% of the proteins in bones. Deoxypyridinoline (DPD) and Pyridinoline (PYD) are formed by the enzyme-catalyzed intermolecular condensation of lysine residues 103 and 1043 of type I collagen 1-chain (Figure 1). DPD and PYD cross-links are major contributors to the tensile strength of bone. PYD is predominant in cartilage whereas DPD is predominant in bone. Upon bone degradation by osteoclasts, DPD and PYD are released either free or in peptidebound form2, 3.

LLOQ QC1 QC2 QC3 ULOQ

Theoretical 1.00 3.05 5.95 11.90 20.00

Determined 1.14 3.05 5.90 12.25 19.98

CV (%) 7.7 5.9 4.9 8.2 6.6

Recovery (%) 114.0 100.1 99.1 102.9 99.9

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Upon achieving the new curve range from 1 to 20 nmol/L, the subsequent interassay precision and accuracy assessment was within acceptance (Table III). All other parameters determined in the course of the validation were within acceptance, including selectivity in various lots of rat urine, parallelism, prozone and short-term stability at 4C.