Design and validation of a new method to detect and quantify residual host cell DNA in human recombinant erythropoietin (rEPO).

During the purification of human recombinant erythropoietin (rEPO) from host cells, residual DNA may remain in final products. This contamination is a risk factor for patients and may result in the inactivation of some tumor suppressor genes or activation of oncogenes if its concentration is more than the standard defined by WHO. Based on WHO's criteria, acceptable level of residual DNA in biopharmaceuticals is less than 10-100 pg/dose. In this study, we have designed a sensitive and specific quantitative real-time polymerase chain reaction (PCR) assay for the detection of residual DNA in human rEPO products. All reported sequences of CHO's GAPDH gene were retrieved from GenBank, and a multiple alignment was performed using Mega 6 software to find conserved regions of the gene. Primers and probe were designed by AlleleID7 software for the highly conserved region. Quantitative real-time PCR showed an R2 value more than 0.99 and the efficiency equal to 101% indicating a highly accurate and efficiency of the reaction, respectively. Based on the standard curve, the limit of detection of the assay was determined to be 10 copies/µL (0.00967 fg/µL). In addition, the inter- and intra-assay of the test were determined to be 1.14% and 0.65%, respectively, which are in acceptable range according to the WHO's guidelines.

Detection of de-N-glycosylated EPO with SDS-PAGE: A complementary confirmation procedure for recombinant EPO in blood samples.

Frameshift variant c.577del in the EPO gene can result in the extension of the amino acid sequence of EPO by invalidating the termination codon. As the molecular weight of its encoded protein EPO (VAR-EPO) is similar to that of rEPO, the World Anti-Doping Agency has published Annex B to the TD2022EPO in order to protect athletes with variant c.577del from the suspicion of rEPO administrations. However, it is still necessary to develop a confirmation method for rEPO that can discriminate rEPO from VAR-EPO. Based on the glycosylated characteristic of EPO, we selected the detection of de-N-glycosylated EPO as a complementary confirmation method for rEPO in blood samples. All samples were analyzed for both intact EPO and de-N-glycosylated EPO with SDS-PAGE, including rEPO spiked samples and blank samples. The results showed that, after de-N-glycosylation, a single-band was detected in samples collected from non-variant carriers, no matter whether the sample was spiked with rEPO. In samples collected from variant carriers, a double-band was detected. The ratio of lower band to upper band increased significantly corresponding to the concentration of rEPO. We calculated a series of cut-off values by normality distribution function to identify the presence of rEPO. Neither false positive results in blank samples nor false negative results in spiked samples at the applicable Minimum Required Performance Levels were found. This indicates that this method could be adopted as a complementary confirmation method for rEPO in blood samples. A revised testing strategy was also proposed, which would discriminate rEPO directly without further investigation.

UPLC-MS assessment on the structural similarity of recombinant human erythropoietin (rhEPO) analogues from manufacturers in China for attribute monitoring.

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy. Since the expiration of the patent of the innovator epoetin alfa, numerous rhEPO products have emerged in global markets. As described here, multiple complementary analytical approaches are utilized for the extensive characterization of rhEPO molecules, and more importantly for the structural comparison of the rhEPO analogues on the Chinese market. The focus of this study is placed on the overall glycosylation profiling, O-glycan profiling, and N-glycan mapping by UPLC-MS with an aim to develop an effective analytical methodology to monitor the product quality attributes of rhEPO analogues. Two rhEPO analogues manufactured in China were analyzed to demonstrate the principle of the developed methods. Each rhEPO product showed a characteristic glycoform profile with respect to the distribution of sialic acids across multi-antennary structures, the occurrence of O-glycosylation, O-acetylation on sialic acids, and the extension of N-glycan antennae with N-acetyllactosamine units. The study demonstrates that UPLC-MS is an effective analytical tool to characterize and monitor the glycosylation profiles among rhEPO analogues in order to detect and account for the divergence between rhEPO products, as well as the presence of unusual or unexpected glycans.

Biphasic cultivation strategy to avoid Epo-Fc aggregation and optimize protein expression.

In biphasic cultivations, the culture conditions are initially kept at an optimum for rapid cell growth and biomass accumulation. In the second phase, the culture is shifted to conditions ensuring maximum specific protein production and the protein quality required. The influence of specific culture parameters is cell line dependent and their impact on product quality needs to be investigated. In this study, a biphasic cultivation strategy for a Chinese hamster ovary (CHO) cell line expressing an erythropoietin fusion protein (Epo-Fc) was developed. Cultures were run in batch mode and after an initial growth phase, cultivation temperature and pH were shifted. Applying a DoE (Design of Experiments) approach, a fractional factorial design was used to systematically evaluate the influence of cultivation temperature and pH as well as their synergistic effect on cell growth as well as on recombinant protein production and aggregation. All three responses were influenced by the cultivation temperature. Additionally, an interaction between pH and temperature was found to be related to protein aggregation. Compared with the initial standard conditions of 37°C and pH 7.05, a parameter shift to low temperature and acidic pH resulted in a decrease in the aggregate fraction from 75% to less than 1%. Furthermore, the synergistic effect of temperature and pH substantially lowered the cell-specific rates of glucose and glutamine consumption as well as lactate and ammonium production. The optimized culture conditions also led to an increase of the cell-specific rates of recombinant Epo-Fc production, thus resulting in a more economic bioprocess.

Effects of Recombinant EPO on Death of Cortical Neuron in Chronic Hypoxia / 체질인류학회지

Chronic hypoxia has been associated with change in neurovascular behavior, mediated, in part, by erythropoietin (EPO). EPO, a hematopoietic growth factor, could act as a neurotrophic factor. In the present study, we investigated the characteristics of EPO and erythropoietin receptor (EPOR) expressions by cortical neuron in vivo and in vitro and tested the hypothesis that EPO serves protective functions under chronic hypoxia. E18, P5 and P7 mice for 3 days and primary cultured neurons for 6 days were incubated in hypoxic conditions consisted of a mixture of 10% O2, 5% CO2, 85% N2. To study expressions of EPO, EPOR, caspases, pAKT, pERK, and PARP, immunohistochemical stainning and western blotting were carried out. In addition to expressing EPO and EPOR under normoxic conditions, neurons increased their expression of EPO and EPOR under hypoxia. The effects of recombinant EPO appeared to be mediated via the phosphatidylinositol (PI) 3- kinase-AKT pathway, correlated directly with activation of caspase 3. Also recombinant EPO decreased expression of caspase 8, but not caspase 9. Finally, recombinant EPO decreased apoptosis of cultured neurons as evaluated by expression of PARP. These data support a role for EPO in maintenance of cortical neuron under chronic hypoxia.