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Abstract INTRODUCTION:
Beta-globinopathies are among the most prevalent inherited disorders worldwide, affecting approximately 0.27% of conceptions. They result from either reduced β-globin synthesis or structural abnormalities in the protein, caused by variants in the HBB gene (1). Curative interventions are being developed for beta-globinopathies. Since the substitution at position 87 from threonine to glutamine afford a strong antisickling effect, ex vivo gene therapies utilizing lentiviral vectors carrying the therapeutic β-globin gene (βT87Q -globin) has been developed. The safety and efficacy of this approach has been demonstrated in multiple clinical trials for β-thalassemia and sickle cell disease (2).
Measurement of βT87Q -globin has been employed as a quality control to verify the ability of transduced cells to produce the therapeutic βT87Q-globin in the vector production. A reverse phase HPLC method was utilized to distinguish βT87Q-globin chain from the normal proteoform in a semi-quantitative fashion. (2) However, the implementation of the HPLC method remains challenging in the individual lab. Herein, a UPLC-MS/MS method was developed in the Center for Diagnostic Innovation (CDI) at the Children’s Hospital of Philadelphia (CHOP) to quantitatively determine the ratio of transgenic 87Q β-globin to wild type 87T β-globin.
METHODS:
A targeted proteomic approach was adopted in this method. Tryptic peptides (87T: GTFATLSELHCDK and 87Q: GTFAQLSELHCDK) encompassing position 87 in β-globin were selected. Stable isotope labeled internal standard (IS) of these peptides were synthesized. Red blood cell (RBC) or hematopoietic cells after differentiation underwent reduction, alkylation, and tryptic digestion. IS was added to the reaction mixture before centrifugation and then the supernatant was transfer to sample analysis plate. The UPLC-MS/MS system used were Waters Acquity I class PLUS UPLC coupled with Waters Xevo TQ Absolute or Shimadzu LC 40X3 UPLC coupled with Sciex 7500+. The analytical column was Waters UPLC CSH C18 column (1.7μm, 2.1 mm x 50 mm).
Since adult human RBCs contain extremely low or undetectable levels of the 87Q peptide, and cynomolgus monkey RBCs lack the 87T peptide, mixing the two at defined ratios generates a range of 87Q/87T peptide ratios. Standard calibrators and quality controls were prepared by combining human and monkey RBC lysates in specific proportions. Theoretical peptide ratios were inferred from hemoglobin concentrations determined using the standard cyanmethemoglobin method.
RESULTS:
The 87T and 87Q peptides were chromatographically separated and specific MRM transitions were selected, ensuring accurate quantitation of both analytes. The digestion protocol was optimized to maximize the digestion efficiency. The assay validation was conducted according to the FDA M10 validation guidance. In all analytical runs, the calibration curves had coefficient of determinations above 0.998. With four level of QCs, the intra-day accuracy of all QC was between -2.9% to 7.4% bias; the intra-day precision was within 4.7% CV; the inter-day accuracy was between -0.5% to 4.6% bias; and the inter-day precision was within 3.6% CV. The 87Q/87T ratio was stable in the samples for up to 18 hours at 4°C or RT in the short-term; and up to 22 days at -80 °C in the long-term. Furthermore, the 87Q/87T ratio was consistent after 5 freeze/thaw cycles. With six lots of different human and monkey RBC combination, the assay had an accuracy between -13.0% to 14.5% bias; and a precision within 6.9% CV. There were no carryover and interference concern.
CD34⁺ cells transduced with the ALS20 vector (3) were evaluated against wild-type controls upon erythroid differentiation (>90% of cells resulted positive to benzidine staining). The transduced cells exhibited 87Q/87T ratios ranging from 0.64 to 0.93, with higher ratios correlating to increased vector copy number (VCN), whereas the control cells showed a ratio of 0.14.
DISCUSSION:
This quantitative assay described herein measures the ratio of 87T to 87Q peptides in a highly accurate, precise and specific manner. The assay’s specificity was demonstrated by its ability to identify an off-target vector used during the production process, enabled by the combination of chromatographic separation and specific MRM for each peptide. Notably, the use of real biological matrices proved critical, as initial evaluations revealed that extracted hemoglobin or recombinant β-globin yielded peptide levels several orders of magnitude lower than those recovered from hemoglobin in RBCs, for reasons not yet fully understood. Therefore, adult human and cynomolgus monkey RBCs were utilized as β-globin source. This targeted proteomic approach serves as a valuable complementary tool to existing quality control measures, supporting both vector selection and the assessment of transgenic protein expression.
REFERENCES:
(1) Modell B, Darlison, M. Global epidemiology of haemoglobin disorders and derived service indicators. Bull World Health Organ. 2008;86(6):480-487.
(2) Negre O, Eggimann AV, Beuzard Y, et al. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the β(A(T87Q))-Globin Gene. Hum Gene Ther. 2016;27(2):148-165.
(3) Breda L, Ghiaccio V, Tanaka N, et al. Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies. Mol Ther. 2021;29(4):1625-1638.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
