(T1530-01-03) The Development and the Applications of a Multiple Reaction Monitoring (MRM) Method for Bile Salt Export Pump (BSEP) Measurements in In Vivo and In Vitro Models for Early-Phase Drug Development

Purpose: Progressive familial intrahepatic cholestasis type 2 (PFIC2), is an autosomal recessive monogenic disorder affecting approximately 1:100,000 newborns (Selvakumar PK, 2019). PFIC2 is caused by a dysfunctional or absent bile salt export pump (BSEP) protein, leading to cholestasis in infancy, for which there are no approved pharmacologic treatment options (Baker A, 2019). The present investigation describes a targeted proteomics approach to quantify BSEP protein expression in the development of a lipid nanoparticle (LNP)-based BSEP mRNA delivery drug system, with the goal of achieving healthy bile acid movements in hepatocytes.

Methods: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using multiple reaction monitoring (MRM) was established for its flexibility in measuring both total and species-specific BSEP in diverse in vitro and in vivo models to assess drug pharmacokinetics. Two BSEP proteotypic peptides were identified, ⁶²²AADTIIGFEHGTAVER⁶³⁷ and ¹¹²⁰STSIQLLER¹¹²⁸_, representing human BSEP and multi-species pan-BSEP, respectively. Synthetic ¹³C₆,¹⁵N₄ isotope-labeled peptides, purchased from BioSynth (Gardner, MA), were spiked into RIPA lysis buffer (Thermo Fisher, Waltham, MA) as internal standards (IS). For cell culture experiments, the input protein was normalized to 50 µg. For in vivo experiments, liver tissue lysates (100 mg/mL) were first diluted 10 times with 50 mM tetraethyl ammonium bicarbonate (TEAB), and 20 µL of the diluted lysate was then used for the analysis. The samples were then processed with the PreOmics sample preparation kit based on the manufacturer’s protocol. BSEP calibrator stock material was developed by value assignment of BSEP in commercially available inverted vesicles (BSEP-Hi5, Sigma-Aldrich). The amount of BSEP in vesicle preparations was determined by isotope peptide titration. The standard lysate was serially diluted into a fixed, known amount of IS peptide. The resulting samples were then alkylated and digested with the PreOmics protocol and analyzed with the optimized MRM method. A dose-response curve was constructed based on the peptide response ratio. The BSEP protein standard produced a signal equivalent to the IS peptide at the dilution factor ranged from 2.6 to 3.3 (Figure 1A, B). By taking account into the dilution factors on multiple BSEP peptides, the amount of BSEP in total protein was assigned to the inverted vesicle stock. The calibration curve and the quality control (QC) samples were prepared by spiking the BSEP protein standard into the blank matrix at designated levels. Varying amounts of BSEP KO liver tissue lysate were added to BSEP protein standard to complement the matrix protein contributed by the protein standard in the preparation of calibrators and QCs.

Results: The MRM assay was qualified based on the endogenous and the projected BSEP expression in the experimental models. The upper limit of quantitation (ULOQ) was 29.2 fmol/µg of total protein. The lower limits of quantitation (LLOQ) were found to be 0.04 and 0.1 fmol/µg for the pan- and human-BSEP peptides, respectively (Figure 1C, D). Using the assay, endogenous BSEP expression in the wild-type mouse liver tissue was estimated to be 2.2 ± 0.3 fmol/µg, within the range of the assay. QC samples used in assay qualification were prepared by spiking BSEP protein into the matrix at 0.5 (QCL), 2 (QCM), and 5 (QCH) fmol/µg of total protein. In a 3x3 performance characterization experiment, the assay demonstrated good accuracy (85.1 – 99.3%), intra-assay imprecision (0.7 – 9.6%), and inter-assay imprecision (20.0 – 26.6%; Table 1). Based on the expected 5 – 10-fold BSEP induction upon LNP delivery in primary hepatocytes (data not shown), the assay was deemed fit for exploratory studies. The assay provided high-quality pharmacokinetics data when it was used to characterize the induced BSEP expression in different systems. The BSEP mRNA construct was transfected into mouse and human primary hepatocyte cultures to determine if the pharmacokinetic profiles were similar across species. In Figure 2A, BSEP t_max was observed at 48 h post-transfection in both mouse and human hepatocytes with the pan-BSEP peptide. When the drug substance was transfected into BSEP null iHeps (human stem cell-derived induced hepatocytes) (Hayashi H, 2021, protein expressions were observed 7 days post-transfection with the pan-BSEP peptide (Figure 2B). In the in vivo pharmacokinetics experiment, WT mice dosed with LNP-encapsulated BSEP mRNA saw hBSEP expression peak at 48 h post-dose with no observable hBSEP protein by Day 7 (Figure 2C). Interestingly, there was a transient spike in total BSEP at 12 h post-dose, which could possibly be attributed to a response to the cholesterol in the LNP formulation, suggesting the existence of additional protein regulatory mechanisms yet to be explored. This finding also demonstrates the utility in differentiating the endogenous and induced expression of BSEP by multiple reaction monitoring (MRM).

Conclusion: The BSEP MRM assay demonstrates a highly specific and adaptable assay format monitoring target protein expression. The data generated can be used in the dose schedule modeling and allometric scaling in the later stage of drug development.

References: Baker A, K. N. (2019). Systematic review of progressive familial intrahepatic cholestasis. Clin Res Hepatol Gastroenterol, 20-36.
Hayashi H, O. S. (2021). Modeling Human Bile Acid Transport and Synthesis in Stem Cell-Derived Hepatocytes with a Patient-Specific Mutation. Stem Cell Reports, 309-323.
Selvakumar PK, H. V. (2019, Jul). Pediatric cholestatic liver disease: Successful transition of care. Cleve Clin J Med, 454-464.

Acknowledgements: We thank Dr. Cameron Ellis for the helpful discussions on the sample preparation procedure optimization with the PreOmics sample preparation kit.

Funding: This study was funded by Ultragenyx. Conflict of Interest Statements: The authors declare no conflicts of interest.


(A, B) the peptide response ratio versus the dilution factor of BSEP-Hi5 standard; (C, D) calibration curves of the pan- and human-BSEP peptides.


(A) The BSEP expression normalized by Cmax in mouse and human hepatocytes; (B) BSEP expression in iHeps 7 days post-dose; (C) the pharmacokinetics profile of the BSEP LNP drug product over 7 days post-dose.


Accuracy, intra- and inter-assay CV of the pan-BSEP peptide in the assay.