Overexpression of recombinant human antithrombin III in Chinese hamster ovary cells results in malformation and decreased secretion of recombinant protein

Overexpression of recombinant proteins in animal cells is commonly achieved by using gene amplification techniques. Gene amplified cells possess up to several thousand genes coding for the target protein. Constitutive expression of these genes leads to high levels of the corresponding mRNA species and the immature protein in the cell. Inefficient processing of these precursors may result from their great abundance in the cell. To study the influence of elevated intracellular levels of a recombinant protein on its maturation and secretion, we examined the maturation and secretion of human antithrombin III (hATIII) in Chinese hamster ovary (CHO) cells at different levels of gene amplification. No loss of vitality was caused by elevated secretion of hATIII. As the intracellular hATIII content increased, the efficiency of hATIII secretion decreased steadily. The state of intracellular hATIII from the different cell lines was studied by determining the specific heparin cofactor activity of hATIII. Intracellular hATIII from the highest amplified cell line displayed a lowered specific heparin cofactor activity indicating the presence of malfolded, only partially folded, or incompletely or incorrectly posttranslationally modified hATIII in this cell line. Thus, the ability of CHO cells to fold and/or introduce posttranslational modifications and subsequently to secrete the recombinant protein becomes saturated, and therefore these processes may become limiting for protein secretion at highly elevated expression levels. This limitation was not due to a general exhaustion of the secretory capacity of the cells because hATIII constituted only a minor fraction of the secreted proteins, even at high expression levels. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 547-559, 1997.     

Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells

Physicochemical properties of recombinant human erythropoietin were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human urine when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Sedimentation equilibrium experiments showed the recombinant erythropoietin preparation to be essentially a single macromolecular component with a molecular weight of 30,400 and a carbohydrate content of 39%. The Stokes radius of recombinant erythropoietin was estimated to be 32 A from gel filtration, much larger than the 20-A radius calculated for a sphere of the observed molecular weight. This difference may be ascribed to the extensive glycosylation. The fluorescence and phosphorescence spectra showed that the luminescent tryptophan(s) is (are) solvent-exposed and can be quenched by I- and acrylamide but not by Cs+. On acid titration, the recombinant erythropoietin showed a conformational transition with a midpoint of pH 4.1. This suggests that the net charges on the protein moiety rather than on the whole molecule play a role in protein structure stability.     

Purification and characterization of recombinant human antithrombin containing the prelatent form in Chinese hamster ovary cells

Antithrombin (AT) is a serine proteinase inhibitor and a major regulator of the blood coagulation cascade. AT in human plasma has two isoforms, a predominant alpha-isoform and a minor beta-isoform; the latter lacks N-glycosylation at Asn 135 and has a higher heparin affinity. From the difference in its folding states, the AT molecule can be separated into three forms: a native form, a denatured and inactive form known as the latent form, and a partially denatured form called the prelatent form. In this study, we purified and characterized recombinant human AT (rAT) containing the prelatent form produced by Chinese hamster ovary (CHO) cells. When rAT was purified at physiological pH, its specific activity was lower than that of plasma-derived human AT (pAT). The latent and prelatent forms were detected in rAT by using hydrophobic interaction chromatography analysis. However, when rAT was purified at alkaline pH, the prelatent form was reversibly folded to the native form and the inhibitory activity of rAT increased to a value similar to that of pAT. Highly purified rAT was analyzed and compared with pAT by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, amino acid composition, N-terminal sequence, monosaccharide composition, peptide mapping, and heparin-binding affinity. From these analyses, rAT was found to be structurally identical to pAT, except for carbohydrate side-chains. rAT in CHO cells had a high beta-isoform content and it caused a higher heparin affinity than by pAT and also pH-dependent reversible inhibitory activity.     

Characterization of recombinant human adipocyte-derived leucine aminopeptidase expressed in Chinese hamster ovary cells

Adipocyte-derived leucine aminopeptidase (A-LAP) is a recently identified novel member of the M1 family of zinc-metallopeptidases. Transfection of the A-LAP cDNA into COS-7 cells resulted in the secretion of the enzyme. In this study, recombinant A-LAP was expressed in Chinese hamster ovary cells, purified to homogeneity and its enzymatic properties were characterized. The purified enzyme was active towards a synthetic substrate, L-leucyl-p-nitroanilide, yielding a V(max) of 3.55 micromol/min/mg and a K(m) of 1.28 mM, and was shown to be a monomeric protein with molecular mass of 120 kDa in solution. By monitoring the sequential N-terminal amino acid liberation, it was found that the enzyme hydrolyzes a variety of bioactive peptides, including angiotensin II and kallidin. Immunohistochemical analysis indicated that the enzyme is expressed in the cortex of the human kidney, where tissue kallikrein is localized. Taken together, these results indicate that A-LAP possesses a broad substrate specificity towards naturally occurring peptide hormones and suggest that it plays a role in the regulation of blood pressure through the inactivation of angiotensin II and/or the generation of bradykinin in the kidney.     

Characterization of the oligosaccharide structures on recombinant human prorenin expressed in Chinese hamster ovary cells.

Prorenin was isolated by immunoprecipitation from the culture medium of Chinese hamster ovary cells transfected with a human prorenin cDNA. The N-linked oligosaccharide structures on the in vivo [3H]mannose-labeled, purified protein were characterized using a combination of serial lectin affinity chromatography, high-pressure liquid chromatography, ion-exchange chromatography, and size-exclusion chromatography and treatment with specific glycosidases and methylation analysis. Approximately 61% of the oligosaccharides on the molecule are complex type, in the form of tetraantennary (2%), 2,6-branched triantennary (13%), 2,4-branched triantennary (3%), and biantennary (43%) structures. The majority of all complex type structures are core-fucosylated. Sialic acids are linked at the C-3 position of terminal galactose, and the degree of sialylation of the bi- and triantennary structures varies between nonsialylated and fully sialylated; no tetraatennary structure contains more than three sialic acid residues. Recombinant prorenin contains 4% hybrid-type structures, all of which carry a terminal sialic acid residue. The remaining 35% of the structures on the molecule are high mannose type, composed of 5, 6, or 7 mannose residues. Approximately 6% of the high mannose type structures and 10% of the hybrid structures are phosphorylated, as judged by their susceptibility to treatment with alkaline phosphatase. Compositional analysis of an unlabeled preparation of the protein suggested the presence of approximately 1.4 oligosaccharide units per molecule.     

Characterization of recombinant murine interleukin 5 expressed in Chinese hamster ovary cells.

We have purified recombinant murine interleukin 5 (rmIL-5) from the supernatant of Chinese hamster ovary cells. Each peptide fragment of the purified rmIL-5 generated by Achromobacter protease I digestion was characterized and glycosylation sites were determined. Although rmIL-5 contains three potential sites of N-linked glycosylation (Asn-26, Asn-55 and Asn-69), Asn-69 is not glycosylated. The oligosaccharides released from the protein by hydrazinolysis were fractionated by paper electrophoresis, lectin column chromatography and gel permeation chromatography, and their structures were analysed by sequential exoglycosidase digestion in combination with methylation analysis. The results indicated that they are a mixture of bi-, tri- and tetraantennary complex-type sugar chains with and without a fucose at the C-6 position of the proximal N-acetylglucosamine residue and high-mannose-type sugar chains. Although > 80% of the sugar chains are neutral oligosaccharides similar to recombinant human IL-5 (rhIL-5; Kodama, S., Endo, T., Tsuroka, N., Tsujimoto, M. and Kobata, A. (1991) J. Biochem., 110, 693-701), rmIL-5 has more tetraantennary oligosaccharides than rhIL-5. A site differential study revealed that Asn-55 has more tetraantennary oligosaccharides than Asn-26.     

Isolation and characterization of recombinant human prorenin in Chinese hamster ovary cells

Recombinant human prorenin (rh-prorenin) was purified from supernatants of Chinese hamster ovary (CHO) cell line transfected with the cDNA for rh-prorenin by employing a simple two-step procedure which consisted of ammonium sulfate precipitation and immunoaffinity chromatography using a monoclonal antibody specific for the profragment of human prorenin. About 100-fold purification with 35% recovery was achieved after the two steps. Purified rh-prorenin migrated as a single protein band with apparent molecular weights of 46,000-47,000 and about 50,000 on SDS-PAGE and gel filtration (HPLC), respectively, although it consisted of multiple components (pI values, 5.6-6.4) that could be resolved by isoelectric focusing (IEF). The treatment of rh-prorenin with endo-beta-N-acetylglucosaminidase converted the rather broad protein band to a sharp band on SDS-PAGE and reduced the number of multiple pI peaks on IEF. Amino-terminal sequence analysis of both the purified rh-prorenin and rh-renin revealed Leu-Pro-Thr-Asp- and Leu-Thr-Leu-Gly-, respectively, which agreed with those predicted from the base sequences of their cDNA. These data suggested that microheterogeneity of rh-prorenin is due to the carbohydrate moiety, but not to the protein moiety. Purified rh-prorenin was almost inactive, but was cleaved at the carboxyl end of a dibasic pair Lys-2-Arg-1 by trypsin and converted to active renin. However, at the early stage during trypsin activation, new intermediate forms between rh-prorenin and rh-renin were formed, suggesting multiple activation steps of rh-prorenin in addition to the one step activation.     

Characterization of latent recombinant TGF-beta 2 produced by Chinese hamster ovary cells.

Latent recombinant transforming growth factor-beta 2 (LrTGF-beta 2) complex has been purified from serum-free media conditioned by Chinese hamster ovary cells transfected with a plasmid encoding the TGF-beta 2 (414) precursor. Under neutral conditions, LrTGF-beta 2 had an apparent molecular weight of 130 kDa. The complex contained both mature and pro-region sequences. Acidification of LrTGF-beta 2 resulted in the release of mature 24 kDa TGF-beta 2 from the high molecular weight pro-region-containing complex, suggesting that TGF-beta 2 was non-covalently associated with this complex. These results were confirmed by crosslinking experiments performed on partially purified LrTGF-beta 2. Protein sequence analysis of the purified TGF-beta 2 pro-region indicated that signal peptide cleavage occurred between ser(20) and leu(21). The pro-region, which previously was found to contain mannose-6-phosphate, bound to the mannose-6-phosphate receptor. Proteolytic cleavage of mature TGF-beta 2 from pro-TGF-beta 2 was inhibited by monensin and chloroquine suggesting that binding to this receptor and subsequent transport to acidic vesicles may be involved in the processing of rTGF-beta 2 precursor.     

Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.     

Structure of recombinant human interleukin 5 produced by Chinese hamster ovary cells

The complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.     

Heterogeneity within populations of recombinant Chinese hamster ovary cells expressing human interferon-gamma

The Chinese hamster ovary (CHO) cell line has great commercial importance in the production of recombinant human proteins, especially those for therapeutic use. Much attention has been paid to CHO cell population physiology in order to define factors affecting product fidelity and yield. Such studies have revealed that recombinant proteins, including human interferon-gamma (IFN-gamma), can be heterogeneous both in glycosylation and in proteolytic processing. The type of heterogeneity observed depends on the growth physiology of the cell population, although the relationship between them is complex. In this article we report results of a cytological study of the CHO320 line which expresses recombinant human IFN-gamma. When grown in suspension culture, this cell line exhibited three types of heterogeneity: (1) heterogeneity of the production of IFN-gamma within the cell population, (2) heterogeneity of the number of nuclei and mitotic spindles in dividing cells, and (3) heterogeneity of cellular environment. The last of these arises from cell aggregates which form in suspension culture: Some cells are exposed to the culture medium; others are fully enclosed within the mass with little or no direct access to the medium. Thus, live cells producing IFN-gamma are heterogeneous in their environment, with variable access to O(2) and nutrients. Within the aggregates, it appears that live cells proliferate on a dead cell mass. The layer of live cells can be several cells deep. Specific cell-cell attachments are observed between the living cells in these aggregates. Two proteins, known to be required for the formation of certain types of intercellular junctions, spectrin and vinculin, have been localized to the regions of cell-cell contact. The aggregation of the cells appears to be an active process requiring protein synthesis. (c) 1995 John Wiley & Sons, Inc.     

Expression and characterization of human antithrombin III synthesized in mammalian cells

Antithrombin III (ATIII) has been expressed in transiently transfected COS-1 monkey cells and in stably transformed Chinese hamster ovary cells, and the resultant protein has been characterized for biological activity. Both cell types efficiently secrete high levels of heterogeneous molecular weight forms of ATIII antigen. The heterogeneity results from differences in post-translational modifications. However, only a small percentage (5-10%) of the total antigen expressed is biologically active. The fraction of biologically active ATIII has been purified from total ATIII by affinity fractionation on heparin-Sepharose. This fractionation indicates that the differences in the active and inactive forms of expressed ATIII result from differences in their ability to bind heparin. Purified ATIII has a specific activity very similar to that of plasma-derived ATIII and exhibits typical heparin-accelerated ATIII activity. The biologically active fraction of ATIII appears to represent the higher molecular weight forms of the ATIII expressed and is likely not a result of altered asparagine-linked glycosylation; however, the nature of the post-translational modification required for ATIII activity remains unclear. The ability to express biologically active ATIII at such high levels should allow further investigations of the structural requirements for ATIII activity.     

Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells

Factor IX has been expressed to high levels within a recombinant host cell and the biologically active fraction subsequently purified to homogeneity for characterization. The coding sequence for Factor IX was inserted into a mammalian cell expression vector and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells. The integrated DNA was amplified to a high copy number by selection for increasingly higher expression levels of the marker gene, dihydrofolate reductase, contained within a co-transfected plasmid. Cloned cell lines secreting over 100 micrograms/ml Factor IX antigen and up to 1.5 microgram/ml native Factor IX antigen have been obtained. Expression of biologically active Factor IX was dependent on the presence of vitamin K in the culture media. The gamma-carboxylated Factor IX was isolated from cell culture fluid by immunoaffinity chromatography using antibodies conformation-specific for the metal-stabilized conformer of Factor IX. This conformation is dependent upon metal ions and gamma-carboxyglutamic acid. Purified recombinant Factor IX migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an electrophoretic mobility equivalent to plasma-derived Factor IX. The purified recombinant Factor IX demonstrated Factor IX coagulant activity, measured in Factor IX-deficient plasma, of 35-75 units/mg. Amino acid analysis of the alkaline hydrolysate of recombinant Factor IX demonstrated an average of 6-7 mol of gamma-carboxyglutamic acid per mol of Factor IX. NH2-terminal sequence analysis of the first 17 residues revealed equivalent amino acid sequences for both purified recombinant and plasma-derived Factor IX. The results represent the first purification and characterization of a biologically active, gamma-carboxylated vitamin K-dependent protein expressed in a recombinant DNA system.     

Purification and characterization of recombinant human alpha-N-acetylglucosaminidase secreted by Chinese hamster ovary cells

alpha-N-Acetylglucosaminidase (EC 3.2.1.50) is a lysosomal enzyme that is deficient in the genetic disorder Sanfilippo syndrome type B. To study the human enzyme, we expressed its cDNA in Lec1 mutant Chinese hamster ovary (CHO) cells, which do not synthesize complex oligosaccharides. The enzyme was purified to apparent homogeneity from culture medium by chromatography on concanavalin A-Sepharose, Poros 20-heparin, and aminooctyl-agarose. The purified enzyme migrated as a single band of 83 kDa on SDS-PAGE and as two peaks corresponding to monomeric and dimeric forms on Sephacryl-300. It had an apparent K(m) of 0.22 mM toward 4-methylumbelliferyl-alpha-N-acetylglucosaminide and was competitively inhibited by two potential transition analogs, 2-acetamido-1,2-dideoxynojirimycin (K(i) = 0.45 microM) and 6-acetamido-6-deoxycastanospermine (K(i) = 0.087 microM). Activity was also inhibited by mercurials but not by N-ethylmaleimide or iodoacetamide, suggesting the presence of essential sulfhydryl residues that are buried. The purified enzyme preparation corrected the abnormal [(35)S]glycosaminoglycan catabolism of Sanfilippo B fibroblasts in a mannose 6-phosphate-inhibitable manner, but its effectiveness was surprisingly low. Metabolic labeling experiments showed that the recombinant alpha-N-acetylglucosaminidase secreted by CHO cells had only a trace of mannose 6-phosphate, probably derived from contaminating endogenous CHO enzyme. This contrasts with the presence of mannose 6-phosphate on naturally occurring alpha-N-acetylglucosaminidase secreted by diploid human fibroblasts and on recombinant human alpha-l-iduronidase secreted by the same CHO cells. Thus contrary to current belief, overexpressing CHO cells do not necessarily secrete recombinant lysosomal enzyme with the mannose 6-phosphate-targeting signal; this finding has implications for the preparation of such enzymes for therapeutic purposes.     

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.     

Carbohydrate structures of recombinant soluble human CD4 expressed in Chinese hamster ovary cells

Infection of T-lymphocytes and macrophages by human immunodeficiency virus (HIV) is mediated by the binding of the HIV envelope glycoprotein to the cell-surface receptor glycoprotein CD4. A soluble, recombinant CD4 molecule (rCD4), produced by expression of a truncated CD4 gene in Chinese hamster ovary (CHO) cells [Smith et al. (1987) Science 238, 1704-1707], is in clinical trials as a potential therapeutic agent in the treatment of acquired immunodeficiency syndrome (AIDS). In the present study, the structures of the Asn-linked oligosaccharides of soluble rCD4 have been elucidated. The rCD4 molecule has two potential sites for N-glycosylation, Asn-271 and Asn-300. Tryptic glycopeptides containing either of the sites were purified by reversed-phase HPLC, and their oligosaccharides were released enzymatically. The structures of the oligosaccharides were determined by methylation analysis, high-pH anion-exchange chromatography, fast-atom bombardment mass spectrometry, and 1H NMR spectroscopy at 500 MHz. Asn-271 was found to carry diantennary N-acetyllactosamine-type ("complex") oligosaccharides, of which 8% were asialo, 55% were monosialyl, and 37% were disialyl. Approximately 18% of these structures contained fucose alpha(1-->6) linked to the reducing GlcNAc residue. Two different hybrid structures were found to account for 34% of the oligosaccharides attached to Asn-300. The remainder of the oligosaccharides attached to Asn-300 were diantennary N-acetyllactosamine-type, of which 10% were asialo, 61% were monosialyl, and 29% were disialyl. Approximately 9% of the hybrid structures and 40% of the N-acetyllactosamine structures at Asn-300 were found to contain fucose alpha(1-->6) linked to the innermost GlcNAc residue.     

Asn-linked sugar chain structures of recombinant human thrombopoietin produced in Chinese hamster ovary cells

Human thrombopoietin (TPO) that regulates the numbers of megakaryocytes and platelets is a heavily N- and O-glycosylated glycoprotein hormone with partial homology to human erythropoietin (EPO). We prepared recombinant human TPO produced in Chinese hamster ovary (CHO) cells and analyzed the sugar chain structures quantitatively using 2-aminobenzamide labeling, sequential glycosidase digestion and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS).We found bi-, tri- and tetraantennary complex-type sugar chains with one or two N-acetyllactosamine repeats, which are common to recombinant human EPO produced in CHO cells. On the other hand, there were triantennary sugar chains with one or two N-acetyllactosamine repeats that were specific to the recombinant human TPO, and their distributions of branch structures were also different. These results suggested that proximal protein structure should determine the branch structure of Asn-linked sugar chains in addition to the glycosyltransferases subset.     

Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1, recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and 0.32 μM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying the biological effects of exogenous rhAng1 in the future.     

Purification and characterization of recombinant human interleukin 5 expressed in Chinese hamster ovary cells

Recombinant human interleukin 5 (rhIL-5) expressed in Chinese hamster ovary cells was purified and characterized. Molecular heterogeneity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two major components of Mr around 40,000 were detected under non-reducing conditions. However, under reducing conditions, the Mr of rhIL-5 was determined to be 22,000 and 20,000. After treatment with endoglycosidase F, a band with an apparent Mr of 18,000 was observed. Treatment of rhIL-5 with 2-mercaptoethanol followed by N-ethylmaleimide resulted in its dissociation into a monomeric form. This alkylated rhIL-5 was biologically less active than intact rhIL-5. These results suggest that rhIL-5 exists as a dimer, and that the heterogeneity of rhIL-5 is mainly due to the difference in the content of carbohydrate. Moreover, the formation of disulfide bond(s) might be important for the biological activity of rhIL-5.