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. 2020 Oct;48(10):925-933.
doi: 10.1124/dmd.120.000075. Epub 2020 Jul 28.

Quantification of Neonatal Fc Receptor and Beta-2 Microglobulin in Human Liver Tissues by Ultraperformance Liquid Chromatography-Multiple Reaction Monitoring-based Targeted Quantitative Proteomics for Applications in Biotherapeutic Physiologically-based Pharmacokinetic Models

Xiazi Qiu  1 , Michael Zhuo Wang  2
Affiliations

Quantification of Neonatal Fc Receptor and Beta-2 Microglobulin in Human Liver Tissues by Ultraperformance Liquid Chromatography-Multiple Reaction Monitoring-based Targeted Quantitative Proteomics for Applications in Biotherapeutic Physiologically-based Pharmacokinetic Models

Xiazi Qiu et al. Drug Metab Dispos. 2020 Oct.

Abstract

Neonatal Fc receptor (FcRn) and beta-2 microglobulin (β2M) play an important role in transporting maternal IgG to fetuses, maintaining the homeostasis of IgG and albumin in human body, and prolonging the half-life of IgG- or albumin-based biotherapeutics. Little is known about the influence of age, gender and race, and interindividual variability of human FcRn and β2M on the protein level. In this study, an ultraperformance liquid chromatography-multiple reaction monitoring mass spectrometry-based targeted quantitative proteomic method was developed and optimized for the quantification of human FcRn and β2M. Among the 39 human livers studied (age 13-80 years), the mean (±S.D.) concentrations of FcRn and β2M were 147 (±39) and 1250 (±460) pmol/g of liver tissue, respectively. A four-fold interindividual variability (63-243 pmol/g of liver tissue) was observed for the hepatic FcRn concentration. A moderate correlation was found between the hepatic β2M and FcRn expression levels. Influences of age, gender, and race on the hepatic expression of FcRn and β2M were evaluated. The findings from this study may aid the development of physiologically-based pharmacokinetic models that incorporate empirical FcRn tissue concentrations and interindividual variabilities, and the development of personalized dosing of biopharmaceuticals. SIGNIFICANCE STATEMENT: This is the first study to evaluate the influence of age, gender, and race on the expression of neonatal Fc receptor (FcRn) and beta-2 microglobulin (β2M) and their interindividual variability in human livers. This study describes a validated ultraperformance liquid chromatography-multiple reaction monitoring-based targeted quantitative proteomic method for quantifying human FcRn and β2M in biological tissues. Results from this study may aid current development of physiologically-based pharmacokinetic models for biotherapeutics, where FcRn plays a significant role in clearance mechanism, and its expression level and interindividual variability are largely unknown.

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Figures

Fig. 1.
Fig. 1.
UPLC-MRM chromatograms of FcRn and β2M heavy stable isotope–labeled signature peptides. Three transitions of each peptide were monitored, and the highest MS intensity was used as quantification trace in the analysis.
Fig. 2.
Fig. 2.
Influences of trypsin digestion time (A) and trypsin:protein ratio (B) on the UPLC-MRM signals of both signature peptides. The relative MRM peak area was determined by normalizing light peptide area from PNS digest to the spiked-in isotope-labeled signature peptides. Symbols and error bars represent the average and S.D. of triplicate digestions, and the dotted lines in (B) denotes the best-fit lines determined using least-square linear regression analysis.
Fig. 3.
Fig. 3.
Recovery of FcRn and β2M in isolated PNS. The normalized relative MRM peak area was determined by normalizing the ratio of light peptide area and the spiked-in heavy peptides area from 10 μg digest to the total amount of protein in isolated PNS. Lines and error bars represent the average and S.D. of triplicate digestions. Symbols represent the normalized relative MRM peak area from each of the triplicate digestion. Unpaired two-tailed t test was used to compare the values from P1, P2, and PNS. Quantitative recovery percentages are listed in Table 2.
Fig. 4.
Fig. 4.
Standard curves of FcRn and β2M quantification. The relative MRM peak area was determined by normalizing light peptide area to the spiked-in heavy peptides. Symbols and error bars represent the average and S.D. of triplicate injections of the same standard digest, and the dotted lines denotes the best-fit lines determined using quadratic fitting with 1/x weighting. The insert shows a blow-up of the lower standard concentration region.
Fig. 5.
Fig. 5.
Absolute quantification of FcRn and β2M in individual human livers (n = 39). Concentration of both proteins are expressed in picomole protein per gram of liver tissue. Lines and error bars represent the average and S.D. of all human liver analyzed. Symbols represent the average concentration determined from three separate digestions for each individual donor. The y-axis for FcRn quantification is plotted on the left, and the y-axis for β2M is plotted on the right.
Fig. 6.
Fig. 6.
FcRn (A) and β2M (B) concentrations based on gender. Lines and error bars represent the average and S.D. of all human liver analyzed in each gender group. Symbols represent the average concentration determined from three separate digestions for each individual donor. Unpaired two-tailed t test found no significant statistical differences between two groups for both FcRn and β2M.
Fig. 7.
Fig. 7.
FcRn (A and C) and β2M (B and D) concentrations based on age. (A and B) Symbols and error bars represent the average and S.D. of triplicates of digestion for each human liver donor. (C and D) Lines and error bars represent the average and S.D. of all human liver analyzed in each age group. Symbols represent the average concentration determined from three separate digestions for each individual donor. One-way ANOVA followed by Tukey’s multiple comparisons test with a single pooled variance found no significant statistical differences among age groups for both FcRn and β2M.
Fig. 8.
Fig. 8.
FcRn (A) and β2M (B) concentrations based on race. Lines and error bars represent the average and S.D. of all human liver analyzed in each race group. Symbols represent the average concentration determined from three separate digestions for each individual donor. Unpaired two-tailed t test found no significant statistical differences between Caucasian and African American donors for FcRn expressions and found significant statistical differences (P = 0.0218) for β2M expressions including the statistical outlier determined by Grubb’s test.
Fig. 9.
Fig. 9.
Correlation analysis of FcRn concentration and β2M concentration (n = 39). Symbols and error bars represent the average and S.D. of triplicates of digestion for each human liver donor. The dotted line represents the best-fit lines determined using least-square linear regression analysis.
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