Impact of host cell line choice on glycan profile

Protein glycosylation is post-translational modification (PTM) which is important for pharmacokinetics and immunogenicity of recombinant glycoprotein therapeutics. As a result of variations in monosaccharide composition, glycosidic linkages and glycan branching, glycosylation introduces considerable complexity and heterogeneity to therapeutics. The host cell line used to produce the glycoprotein has a strong influence on the glycosylation because different host systems may express varying repertoire of glycosylation enzymes and transporters that contributes to specificity and heterogeneity in glycosylation profiles. In this review, we discuss the types of host cell lines currently used for recombinant therapeutic production, their glycosylation potential and the resultant impact on glycoprotein properties. In addition, we compare the reported glycosylation profiles of four recombinant glycoproteins: immunoglobulin G (IgG), coagulation factor VII (FVII), erythropoietin (EPO) and alpha-1 antitrypsin (A1AT) produced in different mammalian cells to establish the influence of mammalian host cell lines on glycosylation.     

用三步法纯化重组人促红细胞生成素

在生物反应器中培养中国仓鼠卵巢细胞—促红细胞生成素(CHO-EPO)C2细胞株,培养上清液中重组人促红细胞生成素(rHuEPO)表达水平达2000～3000U/ml。培养上清经过三步纯化:第一步为反相柱色谱,可将样品浓缩约96.7％,其收集液经充分透析后进行第二步的DEAE-离子交换色谱,最后进行分子筛色谱,总回收率为30％以上,经纯化的rHuEPO比活性为1.5×10~8U/g蛋白,SDSPAGE一条带,扫描测试纯度达98％以上。     

Coordinated effects of electromagnetic field exposure on erythropoietin-induced activities of phosphatidylinositol-phospholipase C and phosphatidylinositol 3-kinase

Initial studies with the erythropoietin-sensitive human hematopoietic cell line, TF1, demonstrated both multifarious effects of pulsed electromagnetic field (EMF) exposure on lipid signal transduction and antiproliferative effects of EMF. Stimulation of TF1 cells with erythropoietin resulted in increased phosphatidylinositol 3-kinase activity within 2 min. Addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, produced a decrease in cell proliferation as measured by accumulation of cells in the G₀/G₁ phase of the cell cycle and suppression of erythropoietin-induced DNA synthesis. Similar effects on cell proliferation were seen under EMF treatment. Phosphatidylinositol 3-kinase activity in erythropoietin-stimulated TF1 cells, measured in whole-cell extracts, increased 34% within 2 min and remained above basal levels for at least 20 min. EMF decreased erythropoietin-stimulated phosphatidylinositol 3-kinase activity to lower than basal levels. Additionally, translocation of the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase to the membrane was prevented by EMF. Phosphatidylinositol-specific phospholipase C was activated, as reflected by increases in diacylglycerol and inositol trisphosphate at 15–60 s after EMF treatment. These results provide the first evidence of subtle coordinated changes by EMF associated with loss of phosphatidylinositol 3-kinase activity, inhibition of the translocation of p85 to the membrane, and activation of phosphatidylinositol-phospholipase C.     

An efficient translational termination of human erythropoietin in Escherichia coli by altering the base following the stop codon

Each of the four nucleotides (A, C, G and T) was introduced as the base following the stop codon to investigate the effect of this fourth base on translational termination efficiency during the heterologous expression of human erythropoietin (hEPO) in E. coli. The efficiency of peptide chain termination in E. coli was markedly dependent on the fourth base. The choice of the fourth base was crucial to prevent the expression of undesirable proteins due to the translational infidelity such as frameshifting and stop codon read-through, and translational termination efficiency could be improved with adenosine as the fourth base.     

Macromolecular docking of a three-body system: the recognition of human growth hormone by its receptor.

Human growth hormone (hGH) binds to its receptor (hGHr) in a three-body interaction: one molecule of the hormone and two identical monomers of the receptor form a trimer. Curiously, the hormone-receptor interactions in the trimer are not equivalent and the formation of the complex occurs in a specific kinetic order (Cunningham BC, Ultsch M, De Vos AM, Mulkerrin MG, Clauser KR, Wells JA, 1991, Science 254:821-825). In this paper, we model the recognition of hGH to the hGHr using shape complementarity of the three-dimensional structures and macromolecular docking to explore possible binding modes between the receptor and hormone. The method, reported previously (Hendrix DK, Kuntz ID, 1998, Pacific symposium on biocomputing 1998, pp 1234-1244), is based upon matching complementary-shaped strategic sites on the molecular surface. We modify the procedure to examine three-body systems. We find that the order of binding seen experimentally is also essential to our model. We explore the use of mutational data available for hGH to guide our model. In addition to docking hGH to the hGHr, we further test our methodology by successfully reproducing 16 macromolecular complexes from X-ray crystal structures, including enzyme-inhibitor, antibody-antigen, protein dimer, and protein-DNA complexes.     

Gene therapy for rat renal anemia with implantation of erythropoietin-transgenic myoblasts

To investigate whether an erythropoietin (EPO) gene-based therapy could serve as an alternative to the repeated injection of rhEPO in treatment to renal anemia, the genetically modified myoblasts of rats, named Myo/ EPO, were implanted through intramuscular injection to model rats with renal anemia. The hemoglobin (Hb) and hematocrit (HCT) of the rats increased from (92. 5±3.0) g/L and 0.29 ±0.04 to the peak values of (103.8 ±5.0) g/L and 0. 32 ±0. 04 respectively 14 d after implantation, and sustained the pre-implantation level for 90 d. Otherwise, the control rats implanted with Myo/X, which carried the parent retroviral vector, gradually became severe in anemia. The PCR detection for hEPO cDNA in the rat muscle adjacent to injection sites indicated that the Myo/EPO cells survived for a long period in the muscle of rats. The results primarily demonstrate that myoblast gene transfer of EPO is effective for the treatment of rat renal anemia.     

人红细胞生成素乳腺特异性表达载体构建及其可行性检验方法的建立

将大鼠乳清酸蛋白（ＷＡＰ）基因的上游调控序列与人红细胞生成素基因组ＤＮＡ融合,构建了ｐＷＡＰ－ＥＰＯ－ＷＡＰ３乳腺特异性表达载体,该载体经脂质体包裹后,通过显微外科技术,经乳腺导管注入妊娠中后期大鼠乳腺中进行表达。经ＥＬＩＳＡ检测,大鼠乳汁中有ＥＰＯ表达,外周血网织红细胞计数法测定表达的ＥＰＯ具有体内生物活性,说明乳腺导管注入法是一种较理想的评价乳腺特异性表达载体可行性方法     

In vitro neuroprotective action of recombinant rat erythropoietin produced by astrocyte cell lines and comparative studies with erythropoietin produced by Chinese hamster ovary cells

In the central nervous system, astrocytes produce erythropoietin (Epo) and neurons express its receptor. To examine whether or not the brain Epo protects the in vitro cultured neurons from glutamate-induced cell death, we established rat astrocyte cell lines containing the plasmid for production of recombinant rat Epo. Epo partially purified from the culture medium showed a neuroprotective effect similar to that of rat Epo produced by Chinese hamster ovary (CHO) cells. Comparison was made in some other properties between Epo produced by these astrocyte cell lines and that by CHO cells. Digestion of Epo with glycosidases indicated that there was a little difference in glycosylation of Epo produced by two types of the cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Decreased DMT1 and increased ferroportin 1 expression is the mechanisms of reduced iron retention in macrophages by erythropoietin in rats

Recycled iron from reticuloendothelial macrophages to erythroid precursors is important to maintain the iron homeostasis. However, the molecular mechanisms underlying iron homeostasis in macrophages are poorly understood. In this study, male Sprague-Dawley rats were treated with recombinant human erythropoietin (rHuEpo, 500 IU/day, s.c.) for 3 days. At the fifth day, peritoneal exudate macrophages were harvested, and then (55)Fe uptake and release were measured by liquid scintillation counting method. The expression of divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in peritoneal exudate macrophages was detected by RT-PCR and Western blot. In order to exclude the direct effect of rHuEpo on macrophages, the parallel experiments were performed with incubation normal peritoneal exudate macrophages with rHuEpo (2 IU/ml). Our results showed rHuEpo injection reduced the peritoneal exudate macrophages iron retention. The uptake of Fe(II) was decreased via the suppression of DMT1 (+IRE) expression and the release of Fe(II) was increased with increasing the expression of FPN1 in macrophages. Moreover, the expression of HAMP mRNA was four times lower in rHuEpo-treated liver of rats than control group (CG). HAMP mRNA expression was increased; the synthesis of DMT1 had no significant change, whereas the FPN1 was decreased in normal peritoneal exudate macrophages after treatment with rHuEpo in vitro. We conclude that hepcidin may play a major, causative role in the change of FPN1 synthesis and that decreased the iron retention in macrophages of rHuEpo-treated rats.