In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity

Hammerling, Ulf; Kroon, Richard; Wilhelmsen, Tore W; Sjödin, Lars

doi:10.1016/0731-7085(96)01799-2

Cited by 30 publications

(20 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The bioactivity of modified Epo proteins was measured in a cell proliferation assay using the human erythroleukemic cell line, designated TF-1, which is dependent on GM-CSF, IL-3, and Epo for growth (40,41). The TF-1 cells were cultured in 10 ng/ml recombinant human GM-CSF.…”

Section: Bioactivity Of Modified Epo Proteinsmentioning

confidence: 99%

Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Tangri¹,

Mothé

Eisenbraun³

et al. 2005

The Journal of Immunology

160

169

View full text Add to dashboard Cite

Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. Our hypothesis is that the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes, and that reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity. To test this hypothesis, we studied the protein therapeutic erythropoietin (Epo). Two regions within Epo, designated Epo 91–120 and Epo 126–155, contained HTL epitopes that were recognized by individuals with numerous HLA-DR types, a property common to immunodominant HTL epitopes. We then engineered analog epitopes with reduced HLA binding affinity. These analog epitopes were associated with reduced in vitro immunogenicity. Two modified forms of Epo containing these substitutions were shown to be bioactive and nonimmunogenic in vitro. These findings support our hypothesis and demonstrate that immunogenicity of protein drugs can be reduced in a systematic and predictable manner.

show abstract

Section: Bioactivity Of Modified Epo Proteinsmentioning

confidence: 99%

Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Tangri¹,

Mothé

Eisenbraun³

et al. 2005

The Journal of Immunology

160

169

View full text Add to dashboard Cite

show abstract

“…It has been reported that in vitro proliferation assay of EPO in TF-1 cells show high accuracy (Hammerling et al, 1996). Treatment of TF-1 cells with EPO in 24h down-regulates the EpoR and reach a peak in 6h.…”

Section: Analysis Of Rhuepo In Vitro Proliferation Activitymentioning

confidence: 99%

“…(methylthiazol tetrazolium) proliferation assay of rHuEPO in TF-1 was done based on Hammerling et al (1996).TF-1 cells were cultured in RPMI 1640 media (Sigma) supplemented with 2mM L-glutamin (Sigma), 10% of FBS (Gibco), 4mM 100U benzyl penicillin-100µg streptomycin (Gibco), 5ng/mL GM-CSF (Invitrogen) in CO2 incubator with 5% CO2 at 37C. One day before harvesting, the cells were washed with PBS and changed with medium without GM-CSF.…”

Section: Mttmentioning

confidence: 99%

Expression of Modified Recombinant Human Erythropoietin in Cho-K1 Cells and Its in Vitro Proliferation Assay in Tf-1 Cells

Santoso

2014

Indonesian J. Pharm.

View full text Add to dashboard Cite

Erythropoietin (EPO) is a 30 kDa glycoprotein hormone which is important for red blood cells maturation. EPO consists of 165 amino acids and possesses 3N-linked carbohydrate chains. Recombinant human erythropoietin (rHuEPO), such as epoetin-α and epoetin-β, have been used for many years to treat anemia in patients with chronic renal failure, systolic heart failure, HIV-AIDS, or cancer. In vivo stability of rHuEPOs were low due to rapid metabolisms by galactosyl receptor of the hepatocytes. Previously, a novel erythropoiesis stimulating protein (NESP) called darbapoetin-α (DARB) which possesses two additional Nlinked glycosylation had been studied. It was observed that NESP showed better in vivo stability and biological activity compared to the unmodified form (containing only 3N-linked carbohydrate chains). Based on the above study, we attempted to synthesize recombinant human EPO (rHuEPO) by generating CHO-K1 cell lines expressing codon-optimized human epo open reading frame (ORF) in CHO-K1 cells. The ORF was modified to contain 5 Nlinked carbohydrate chains. The media obtained from CHO-K1 cell culture was collected and diafiltrated with the use of tangential flow filtration. The rHuEPO protein containing polyhistidine tag was purified using affinity chromatography. An SDS/PAGE and Western blotting analyses confirmed that the purified protein was the modified rHuEPO. MTT based proliferation assay was conducted in TF-1 bone marrow cell line as a model. The result showed that the modified rHuEPO was able to enhance TF-1 cells proliferation.

show abstract

“…Bone marrow samples may be available only in limited quantities, and the erythroid cultures need 7 d of incubation. Cell lines that have been used to demonstrate epoetin-neutralizing activity are TF-1 and UT-7, derived from patients with erythroleukemia, and 32D-EPO, a murine hematopoietic cell line transfected with the human erythropoietin receptor (19,20). Different erythropoietin-sensitive cell lines have different inherent sensitivities to the neutralizing effects of anti-erythropoietin antibodies in the bioassay; however, cell lines are sensitive to growth factors other than erythropoietin, and tight controls must be included in the bioassays.…”

Section: Identification Of Anti-erythropoietin Antibodiesmentioning

confidence: 99%

Pure Red Cell Aplasia Induced by Erythropoiesis-Stimulating Agents

Pollock¹,

Johnson²,

Hörl³

et al. 2008

Clinical Journal of the American Society of Nephrology

View full text Add to dashboard Cite

Pure red cell aplasia in patients who are treated for anemia of chronic kidney disease with erythropoiesis-stimulating agents such as epoetin was first reported in 1998. Although the incidence of pure red cell aplasia peaked in 2002, it remains important for nephrologists to know how to investigate a suspected case of pure red cell aplasia and how to identify other causes of hyporesponsiveness to erythropoiesis-stimulating agents, which account for the vast majority of such cases. The authors reviewed the current status of information in the literature and drew on their personal experiences with patients regarding the diagnosis and management of epoetin-induced pure red cell aplasia. The mechanism for development of epoetin-induced pure red cell aplasia remains unconfirmed. It generally occurs after the production of neutralizing anti-erythropoietin antibodies. Elucidation of a suspected pure red cell aplasia case requires a systematic approach, beginning with simple measurements such as blood cell counts, because most cases of erythropoiesis-stimulating agent hyporesponsiveness are attributable to other causes. If these criteria indicate that the patient's response to erythropoiesis-stimulating agent therapy is very poor, then bone marrow examination and measurement of anti-erythropoietin antibodies is justified. If pure red cell aplasia is confirmed, then cessation of erythropoiesis-stimulating agent therapy and initiation of immunosuppressive therapy are recommended. Continued study of epoetin-induced pure red cell aplasia is needed to help nephrologists prevent or manage future cases and will have implications for the use of other protein-based therapeutic agents.

show abstract

In vitro bioassay for human erythropoietin based on proliferative stimulation of an erythroid cell line and analysis of carbohydrate-dependent microheterogeneity

Cited by 30 publications

References 44 publications

Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Rationally Engineered Therapeutic Proteins with Reduced Immunogenicity

Expression of Modified Recombinant Human Erythropoietin in Cho-K1 Cells and Its in Vitro Proliferation Assay in Tf-1 Cells

Pure Red Cell Aplasia Induced by Erythropoiesis-Stimulating Agents

Contact Info

Product

Resources

About