0013-7227/91/1281-0341$02.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 128, No. 1 Printed in U.S.A.

Biological Activity and Metabolic Clearance of a Recombinant Human Thyrotropin Produced in Chinese Hamster Ovary Cells N. RAO THOTAKURA, RAJESH K. DESAI, LISA G. BATES, EDWARD S. COLE, BRUCE M. PRATT, AND BRUCE D. WEINTRAUB Molecular, Cellular and Nutritional Endocrinology Branch (N.R.T., R.K.D., L.G.B., B.D. W.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and Recombinant Protein Development Department (E.S.C., B.M.P.), Genzyme Corporation, Framingham, Massachusetts 01701

ABSTRACT. The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carbohydrate structure of a protein reflects the glycosylation apparatus of the host cells in which the protein is expressed, we examined the biological activity and metabolic clearance of a preparation of purified recombinant human (rh) TSH derived from a stable transfectant of Chinese hamster ovary cells. Carbohydrate compositional analysis of this rTSH showed it to be more highly sialylated than a nonrecombinant, cadaver-derived pituitary hTSH. In addition, no Af-acetyl galactosamine was detectable in rhTSH, which implies the absence of terminal sulfate moieties, both of which are present in pituitary-derived TSH. The immunologic activity and porcine TSH receptor-binding activity of the preparation of rhTSH were 3- to 4-fold lower than those of a standard pituitary hTSH. The rhTSH showed a maximum stimulatory activity similar to that of pituitary hTSH in two different in vitro bioassays. However, rhTSH elicited about 3-fold and 5-fold

less cAMP than pituitary TSH after stimulation of adenylyl cyclase in bovine thyroid membranes and the rat FRTL-5 cell line, respectively. Removal of sialic acid did not alter the immunologic activity of rhTSH. However, the potencies of rhTSH in receptor-binding, adenylyl cyclase, and FRTL-5 assays were increased 2.4-, 2.6-, and 26.7-fold, respectively after sialic acid removal. These data suggest that the in vitro biological activity of rhTSH is influenced by its highly sialylated oligosaccharide chains. The rhTSH had a 2-fold lower metabolic clearance rate than pituitary TSH, resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. After sialic acid removal, the rhTSH was cleared faster (7.5-fold) than pituitary hTSH, showing that its longer plasma half-life was due to its higher sialylation. Biologically active rhTSH should be of clinical value in the diagnosis and treatment of patients with thyroid cancer and as a pure hTSH reference preparation. (Endocrinology 128: 341-348, 1991)

T

SH is a member of the pituitary and chorionic glycoprotein hormones and contains two noncovalently linked subunits, a and (8 (1). The a-subunit of TSH has two Asn-linked complex-type oligosaccharide chains and the /3-subunit has one. The structure of the oligosaccharide chains differs not only between species, but within the same molecule at different sites (2). Normal human pituitary TSH contains oligosaccharide chains that terminate in sialic acid linked to galactose, as well as sulfate linked to JV-acetyl galactosamine (GalNAc) (3), the latter unique to pituitary glycoprotein hormones. The oligosaccharide chains have been shown to play an important role in biosynthesis and subunit association (4, 5), protection from intracellular degradation (4, 5), secretion (6), plasma survival, and biological activity of the glycoprotein hormones (7-12). We have

previously demonstrated that the structures of the oligosaccharide chains in TSH are altered in a variety of physiological and pathological states (13), resulting in subtle alterations in the bioactivity of the hormone (14). However, the exact role of the terminal or individual sugar moieties of the oligosaccharide chains in TSH is not known. With the availability of cloned genes for TSH (15) and other glycoproteins, several recombinant proteins have been expressed in vitro for structure-function studies (10; Lash, R. W., R. K. Desai, M. R. Flack, T. Yoshida, F. E. Wondisford, and B. D. Weintraub, submitted for publication) as well as for commercial scale production. Since these proteins are expressed in nonhuman, established cell lines, the recombinant glycoproteins are likely to have different glycosylation patterns and quantitatively different biological properties from the nonrecombinant proteins. Earlier work from our laboratory (15) has shown that recombinant human TSH (rhTSH) produced

Received August 20,1990. Address all correspondence and requests for reprints to: Dr. N. Rao Thotakura, Building 10, Rm 8D14, NIH, Bethesda, Maryland 20892.

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BIOACTIVITY AND MCR OF RECOMBINANT hTSH

by transient transfection of embryonal kidney cells was bioactive, but detailed structural and biological characterization was not possible due to low levels of expression. Subsequent studies have revealed that protein structure, and particularly the glycosylation pattern, can be influenced by both the specific subclone and culture conditions used to produce the recombinant hormone (Cole, E. S., T. Edmunds, K. Lee, K. Lauziere, P. Peterson, and B. Pratt, manuscript in preparation). In the present study, we have used a preparation of rhTSH produced in large quantity by a stably transfected Chinese hamster ovary (CHO) cell line. The carbohydrate composition, in vitro biological activities, and in vivo clearance of the rhTSH were examined. We demonstrate that rhTSH is sialylated with no apparent sulfate and that it is bioactive. We also show that this preparation of rhTSH has lower in vitro specific activity and slower metabolic clearance rate (MCR), due in part to its higher sialic acid content as compared to a standard preparation of pituitary hTSH. Materials and Methods hTSH (AFP-8770B) and anti-hTSH were supplied by the National Hormone and Pituitary Program (Baltimore, MD). This preparation is used throughout this study as the standard pituitary preparation. A complementary DNA for human choriogonadotropin a as well as an hTSH /? minigene (15) were cotransfected into CHO cells, and stable transfectants with high rates of rhTSH production were selected. The rhTSH used in this study was derived from one of several research cell lines currently under investigation. The expressed rhTSH was purified from culture supernatants by chromatography on BlueTrisacryl M (IBF Biotechnics, Savage, MD), Q-Sepharose Fast Flow, and S-Sepharose Fast Flow (both from Pharmacia, Piscataway, NJ). For this preparation, total purification yield was 32%, and final purity of the TSH was greater than 97%. The purification and structural characterization of rhTSH will be the subject of a separate publication (Cole, E. S., T. Edmunds, K. Lee, K. Lauziere, P. Peterson, and B. Pratt, manuscript in preparation). Sources of other materials are described elsewhere (7). Carbohydrate compositional analysis One- to two-microgram aliquots of the rhTSH and standard hTSH were hydrolyzed with trifluoroacetic acid (Sequanal grade from Pierce, Rockford, IL) and analyzed for carbohydrate composition by ion-exchange HPLC on a Dionex BioLC system using an AS6 Ionpak column (Dionex, Sunnyvale, CA) as described earlier (7). For sialic acid compositional analysis, the samples were hydrolyzed with 0.2 N HC1 at 80 C for 1 h and analyzed on the same column. Sialic acid was eluted isocratically with 100 mM sodium acetate in 100 mM NaOH and detected by a pulsed amperometric detector essentially as described for monosaccharides (7).

Endo • 1991 Voll28«Nol

Preparation of desialylated TSH One hundred micrograms each of rhTSH and pituitary hTSH were incubated with 10 mU Vibrio cholerae neuraminidase (Calbiochem, La Jolla, CA) in 50 mM sodium acetate, pH 5.0, 1 mM CaCl2 for 2 h at 37 C. The released sialic acid was separated by filtration through a Centricon microconcentrator (Amicon, Danvers, MA). Sialic acid release was monitored by HPLC (as described above) in the filtrate as well as after hydrolyzing the desialylated TSH, for the completeness of its removal. Control TSH samples were treated identically but in the absence of neuraminidase. The intactness of the remaining carbohydrate of desialylated TSH was ascertained further by monosaccharide compositional analysis. Sodium docecyl sulfate-polyacrylamide gel electrophoretic analysis of the asialo rhTSH preparation revealed no apparent proteolytic cleavage. Amino acid analysis Quantitation of the rhTSH and the standard pituitary hTSH was done by amino acid compositional analysis on a Beckman 6300 (Beckman Instruments, Fullerton, CA) analyzer with post-column ninhydrin reaction. The quantities of the hormones represented in all assays described below are based on amino acid analysis. RIA Immunologic activities of pituitary and rhTSH were compared in an RIA using a polyclonal antiserum to hTSH by methods described previously (17). The labeling of hTSH was done using Na125I by lactoperoxidase method as previously described (9). RRA Pituitary and rhTSH were assayed for receptor binding activities by their ability to displace [125I]bovine TSH from a solubilized porcine thyroid membrane receptor preparation (Kronus, Dana Point, CA), as previously described (18). Adenylyl cyclase activity The in vitro bioactivity of standard and rhTSH preparations was assayed by their ability to stimulate adenylyl cyclase activity of bovine thyroid membranes as described previously (7). cAMP production in FRTL-5 cells The TSH preparations were also compared for their activities in another in vitro bioassay, stimulation of cAMP production in FRTL-5 cells (a kind gift of Dr. L. D. Kohn, Interthyr Research Foundation, Baltimore, MD). These cells are cloned normal Fischer rat thyroid cells and possess all the characteristics of thyroid cells in culture, except the capacity to organify iodine (19). The cells were plated in 96-well culture plates and maintained as described (19). After growing the cells for 7-9 days in the absence of TSH, they were incubated with serial dilutions of rhTSH and pituitary hTSH in hypotonic medium, and the amount of cAMP released into the medium was assayed by an RIA as previously described (7).

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BIOACTIVITY AND MCR OF RECOMBINANT hTSH Metabolic clearance of TSH The in vivo clearance in rat for pituitary hTSH and rhTSH preparations was determined by injecting 0.5-1.0 tig of the sample and measuring serum TSH concentrations at several time points. The experimental protocol was according to the procedures described earlier (20). Statistical analyses A curve-fitting program, Flexifit (obtained from Dr. P. J. Munson, NICHD, Bethesda, MD) was used to fit the doseresponse data and calculate EC50 values in RRA and in vitro bioactivity assays. MCR parameters were determined by computer-fit nonlinear regression analysis of the data using MLAB (20). Comparison of means was made using the unpaired Student's t test, and P < 0.05 was considered significant.

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nM for pituitary hTSH). Desialylation of rhTSH and pituitary hTSH did not have any effect on the immunoactivity of the hormones, showing that the lower observed immunoreactivity of this preparation of rhTSH was not due to its sialylation. It should be noted, however, that other preparations of rhTSH in similar assay formats displayed immunoactivity equivalent to pituitary hTSH (data not shown). The variations in the immunological and biological activities of different preparations of rhTSH are apparently due to differences in their carbohydrate structure arising from the different production conditions (23). In all of the assays performed in this study, the mock incubated control hormones showed identical activities to those of untreated hormones (data not shown). Receptor binding activity

Results Carbohydrate analysis Monosaccharide compositional analysis of the preparation of rhTSH used in this study, as shown in Table 1, revealed the presence of mannose, galactose, GalNAc, fucose, and sialic acid residues, demonstrating that the oligosaccharide chains are of complex type terminating in sialic acid. The composition of pituitary hTSH was in general agreement with previously established structures (3). The sialic acid content of the rhTSH was 2.7 residues per oligosaccharide chain, compared to 0.3 residues in pituitary hTSH. The absence of GalNAc in rhTSH suggests the absence of GalNAc-transferase activity in CHO cells, confirming reports from other laboratories (21, 22). Immunologic activity In the RIA using polyclonal antibodies, the dose-response curves for this preparation of rhTSH were parallel to the pituitary standard (Fig. 1). The rhTSH showed immunoreactivity about 3-fold lower than that of standard pituitary hTSH (EC50 of 0.09 nM for rhTSH vs. 0.03

Figure 2 shows the RRA results comparing rhTSH with pituitary hTSH preparations. The rhTSH showed an EC50 4.4-fold higher than that of pituitary standard (22.6 nM for rhTSH vs. 5.1 nM for pituitary hTSH). Desialylated rhTSH had a lower EC50 (9.6 nM vs. 22.6 nM for control rhTSH) showing that the asialo rhTSH has higher affinity to the receptor than the untreated rhTSH. In vitro bioactivity The stimulation of adenylyl cyclase activity in bovine thyroid membranes by rhTSH and standard preparations of pituitary TSH is shown in Fig. 3. Recombinant hTSH stimulated the enzyme to the same maximum activity as pituitary TSH, but 3.4-fold higher concentrations of rhTSH (85 nM vs. 25 nM for pituitary hTSH) were required to attain half-maximal stimulation. Figure 3 also shows the effect of sialic acid removal on adenylyl cyclase stimulation by rhTSH. Desialylation did not affect the maximal stimulation of the enzyme by rhTSH, but after desialylation rhTSH showed an EC50 2.6-fold

TABLE 1. Carbohydrate compositional analysis of rhTSH and pituitary hTSH pmol sugar/^g protein

Fucose Galactosamine Glucosamine Galactose Mannose Sialic acid

Sugar residues/chain (Man = 3)

Pituitary hTSH

Recombinant hTSH

Pituitary hTSH

Recombinant hTSH

26.6 120.9 383.1 41.6 192.4 20.2

38.4 ND° 365.2 227.8 261.3 181.0

0.4 1.9 6.0 0.6 3.0 0.3

0.4 0.0 4.2 2.6 3.0 2.1

An aliquot (1-2 fig) each of rhTSH and pituitary hTSH was hydrolyzed and analyzed for monosaccharides on the Dionex BioLC system as described in Materials and Methods. The monosaccharide peaks were integrated, and the values represent the average of three determinations. Values are also represented taking mannose as three residues/chain. 0 ND, not detectable.

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BIO ACTIVITY AND MCR OF RECOMBINANT hTSH

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Endo• 1991 Vol 128* No 1

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1 •

60

40

A

Asialo pit hTSH

20 -2

10

.1 -3

-Ih

.01

TSH (nM)

.1 TSH (nM)

FlG. 1. Immunological activity of rhTSH. Increasing concentrations of control (filled symbols), pituitary hTSH (triangles), and rhTSH (circles), and those treated with neuraminidase (open symbols) are incubated with [12BI]hTSH and an antibody to hTSH as described in Materials and Methods. The quantities of both hormones are based on amino acid analysis. Binding in the absence of TSH is taken as 100%. Results are from a representative experiment repeated at least three times. Pit, pituitary.

lower than that of untreated hormone, and the EC50 of desialylated rhTSH was virtually identical to that of pituitary hTSH. As expected, the activity of pituitary hTSH was not altered by neuraminidase digestion, since it contains a minimal amount of sialic acid. Figure 4 shows the stimulation of cAMP production by TSH in FRTL-5 cells, a rat thyroid cell line. Again, the rhTSH stimulated cAMP production to similar maximal levels as pituitary TSH. RhTSH stimulated cAMP production half-maximally at a concentration of 1.6 nM, whereas pituitary hTSH did so at about 5-fold lower concentration (0.3 nM). However, desialylation of rhTSH increased its activity in this assay to about 26-fold higher than that of the unmodified rhTSH. Thus, desialylated rhTSH was more potent than pituitary hTSH. Neuraminidase treatment of pituitary hTSH caused minimal changes in its activity.

100

FIG. 2. Receptor binding activity of rhTSH. Pituitary hTSH (triangles) and rhTSH (circles) are assayed at the concentrations indicated in an RRA as described in Materials and Methods. Control preparations are represented by filled symbols and desialylated by open symbols. Values are represented as percentages of label bound in the absence of any hormone and are from a typical experiment repeated at least three times. Pit, pituitary.

Metabolic clearance rate

The MCR of injected rhTSH and standard pituitary hTSH before and after sialic acid removal are shown in Fig. 5. It is apparent that rhTSH is cleared significantly more slowly (about 2-fold) than pituitary hTSH resulting in a greater than 10-fold higher serum concentration of rhTSH at 3 h as compared to pituitary hTSH. This difference in MCR is most likely due to the highly sialylated oligosaccharide chains of the rhTSH. This contention is confirmed by the dramatic increase in the clearance of rhTSH from 0.5 ml/min to 7.5 ml/min after desialylation. However, the clearance of pituitary hTSH was not significantly affected by neuraminidase digestion, again consistent with the observation that its sialic acid content is very low. The individual metabolic clearance parameters of rhTSH and pituitary hTSH are shown in Fig. 6. The MCR (upper left panel) of rhTSH was significantly slower than both pituitary hTSH and desialylated rhTSH. The volume of distribution (upper

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BIOACTIVITY AND MCR OF RECOMBINANT hTSH



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Biological activity and metabolic clearance of a recombinant human thyrotropin produced in Chinese hamster ovary cells.

The presence and specific structures of the oligosaccharides on TSH have been shown to be important for its production and bioactivity. Since the carb...
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