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. 2022 Mar;39(3):553-562.
doi: 10.1007/s11095-022-03213-1. Epub 2022 Mar 15.

A Reference Standard for Analytical Testing of Erythropoietin

Huiping Tu  1 , Kevin Carrick  1 , Rebecca Potts  1 , Mark Hasselberg  1 , Mark Verdecia  1 , Chris Burns  2 , Ben Cowper  2 , Fouad Atouf  3
Affiliations

A Reference Standard for Analytical Testing of Erythropoietin

Huiping Tu et al. Pharm Res. 2022 Mar.

Abstract

Purpose: Erythropoietin (EPO) is a 165 amino acid protein that promotes the proliferation of erythrocytic progenitors. A decrease in endogenous EPO production causes anemia that can be treated with recombinant Human EPO (rHuEPO).

Objective: To ensure the safety and efficacy of the rHuEPO, manufacturers must use analytical methods to demonstrate similarity across batches and between different products. To do this they need reference standards to validate their equipment and methods.

Method: We used peptide mapping, size-exclusion chromatography, glycoprofiling, and isoelectric focusing to analyze a rHuEPO reference standard.

Results: Characterization demonstrates that our rHuEPO reference standard meets the criteria for quality.

Conclusion: The rHuEPO reference standard is fit for purpose as a tool for validating system suitability and methods.

Keywords: Biologics; erythropoietin; lyophilization; manufacturing; reference standards.

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Figures

Fig. 1
Fig. 1
Peptide mapping. 100 μg of rHuEPO-DS (grey) and USP rHuEPO-RS (orange) were digested with the enzyme Lys-C and separated by UPLC. (A) rHuEPO-DS prepared with water, (B) rHuEPO-DS prepared with PBS; (C) USP rHuEPO-RS resuspended with water, (D) USP rHuEPO-RS resuspended with PBS, (E) USP rHuEPO-RS buffer exchanged with dialysis, (F) USP rHuEPO-RS buffer exchanged with a spin concentrator. Expected peaks are labeled 1 through 10. Unknown peaks are denoted with an asterisk (*).
Fig. 2
Fig. 2
Analysis of rHuEPO monomer. 80 μg of rHuEPO-DS (grey) and USP rHuEPO-RS (orange) and an equivalent amount of excipient to that in 80 μg of USP rHuEPO-RS (orange) were analyzed by size-exclusion chromatography. (A) rHuEPO-DS, (B) USP rHuEPO-RS resuspended in water. (C) Equivalent amounts of excipients to (B) but without USP rHuEPO-RS. Monomer and Tween®20 peaks are labeled. AU is absorbance units at 230 nm.
Fig. 3
Fig. 3
Analysis of aggregates after lyophilization. 25 μg of USP rHuEPO-RS (orange) was analyzed by size-exclusion chromatography. (A) no lyophilization, (B) lyophilization without Tween®20, (C) lyophilization with 0.001% Tween®20. Monomer and aggregate peaks are labeled. AU is absorbance units at 230 nm.
Fig. 4
Fig. 4
N-glycan analysis. 100 μg of rHuEPO-DS (grey) and USP rHuEPO-RS (orange) were treated with PNGase to remove the N-glycans. The free N-glycans were separated by HPLC. (A) rHuEPO-DS, (B) USP rHuEPO-RS buffer exchanged, (C) close-up view (3x) of rHuEPO-DS, (D) close-up view (3x) of USP rHuEPO-RS. Bi-sialylated (2 N), tri-sialylated (3 N), and tetra-sialylated (4 N) glycans are bracketed.
Fig. 5
Fig. 5
Isoelectric focusing. 20 μg of rHuEPO-DS and USP rHuEPO-RS were separated by IEF (pH 3 to 10). Each sample separates into 5 isoforms (isoforms 10–14) separated by the number of attached sialic acids. rHuEPO-DS (lane 1), USP rHuEPO-RS reformulated in water (lane 2), and USP rHuEPO-RS buffer exchanged (lane 3).
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