Molecular biology and biochemistry of human recombinant follicle

Molecular Human Reproduction vol.2 no.5 pp. 371-382, 1996

Molecular biology and biochemistry of human recombinant folliclestimulating hormone (Puregon®)

Wiebe Olijve1, Willem de Boer, John W.M.Mulders and Peter M.G.F. van Wezenbeek

NV Organon, PO Box 20, 5340 BH Oss, The Netherlands

^o whom correspondence should be addressed

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein hormone produced in the anterior pituitarygland. The hormone is essential in the regulation of reproductive processes, such as follicular developmentand ovulation. It is clinically used for treatment of anovulation and in assisted reproduction technologiessuch as in-vrtro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Until recently, the only sourcefor human FSH has been the urine from post-menopausal women. Such a natural source implies limitedavailability and potential product variability. Thus, we have cloned the genes encoding the a- and {J-subunrtsof human FSH and transfected these into Chinese hamster ovary (CHO) cells. A CHO-clone was isolatedcapable of secreting intact glycosylated FSH with identical amino acid sequences to natural FSH. This cellline was grown in perfusion culture and enabled us to isolate highly pure FSH (>99%). The complexity of thecharge distribution of human recombinant FSH was demonstrated by isoelectric focusing. The observedmicroheterogeneity is caused by the large number of carbohydrate chain structures which are added to thefour potential glycosylation sites in the afJ-dimer. Furthermore, the carbohydrates show a variation in theirdegree of sialylation which reflects the different pi values of the individual isohormones. Despite thecomplexity of post-translational modification, the isoform distribution of recombinant FSH produced in aCHO-cell line and grown in perfusion culture is surprisingly similar to that observed with pituitary FSH andurinary FSH. In conclusion, we have shown that FSH-gene transfected CHO-cells are capable of stable serum-free production of recombinant FSH. A process has been developed which assures the consistent andreproducible production of highly-purified recombinant FSH.

Key words: FSH/heterologous gene expression/isohormones/peptide mapping/recombinant glycoproteinhormone

IntroductionFollicle stimulating hormone (FSH) is produced by the gonado-trophic cells of the anterior pituitary and released into thecirculation. FSH together with the luteinizing hormone (LH),which is produced by the same cells of the pituitary, controlsoocyte maturation in females and spermatogenesis in males.FSH and LH belong to a family of glycoproteins that areheterodimers, containing two non-covalently linked a- and{J-subunits. The subunits are encoded by separate genes. Theother members of the glycoprotein hormone family are thethyroid-stimulating hormone (TSH) and human chorionicgonadotrophin (HCG). Within an animal species the aminoacid sequence of the a-subunits is identical, whereas the p"-subunits differ and confer biological specificity on the indi-vidual gonadotrophins (Pierce and Parsons, 1981). Both thea- and P-subunits are glycosylated. As shown in Figure 1, thea- and (J-subunit of FSH each have two potential asparagine-linked glycosylation sites, characterized by the consensussequence Asn-X-Ser, on positions a52, cc78 and p"7, p24respectively. Serine (or 0-)-linked glycosylation is found onlyin the C-terminal peptide of HCG.

After initial glycosylation in the endoplasmic reticulum(ER), further processing involves trimming by glucosidases

© European Society for Human Reproduction and Embryology

and mannosidases, and remodelling of the carbohydrates in acomplex series of biochemical reactions. The final structureof the Asn-linked carbohydrates on glycoproteins is dependenton the protein itself and the tissue in which it is produced. Inparticular, the terminal residues on the carbohydrate antennamay differ: LH carbohydrates terminate with sulphate-4-N-acetyl-galactosamine, whereas FSH bears more highlybranched sialylated structures (Green and Baenzinger, 1988;Boime et al, 1992). As a result of the extensive biochemicalprocessing each of the glycan chains demonstrate considerablemicroheterogeneity resulting in numerous glycoforms, whichcan be resolved by isoelectric focusing. (Stockell Hartree andRenwick, 1992; Thotakura and Blithe, 1995; de Leeuw et al,19%). The carbohydrates on the gonadotrophins serve manyimportant functions. They are required for proper folding,assembly and secretion of the gonadotrophins. Furthermore,carbohydrates are also highly relevant for the biologicalactivity. It is well-established that glycosylation determinesthe half-life of the gonadotrophins (Kalyan and Bahl, 1981;Ulloa-Aguirre et al, 1988; Ullua-Aguirre et al, 1995). Theincreased in-vivo bioactivity and longer plasma half-life ofHCG compared to LH is attributed to the four additional0-linked oligosaccharides on the C-terminal peptide. Also,

371

Dow

nloaded from https://academ

ic.oup.com/m

olehr/article/2/5/371/985716 by guest on 02 January 2022

W.OIijve et al.

a- chain

HCG121

145 A A

Figure 1. Schematic representation of the primary structure of Gl-and P-subunits of the gonadotrophin family. Glycosylation sites ineach subunit are indicated.

recombinant TSH produced by Chinese hamster ovary (CHO)cells has a longer elimination half-life due to addition of sialicacid as terminal sugar moiety instead of sulphate-4-N-acetyl-galactosamine (Cole et al, 1993). Furthermore, alterations inthe carbohydrate structures may result in molecules withdecreased ability to stimulate adenylate cyclase and steroido-genesis, but with unaffected receptor affinity (Sairam, 1983;Boime et al, 1992). Thus, post-translational modification,such as glycosylation, is an absolute requirement for properexpression and full biological activity of the glycoproteinhormones.

Human FSH is used for treatment of women with low orabsent endogenous FSH production and in assisted reproduc-tion technologies, such as in-vitro fertilization (IVF) andintracytoplasmic sperm injection (ICSI). Partially purified FSHpreparations for clinical use have been isolated up to nowfrom human post-menopausal urine and therefore contain LHand other contaminating proteins from human origin. The useof such a natural source implies limited product availabilityand consistency. Production of a recombinant FSH would havethe principle advantage that it can be extensively purified andis not contaminated with other fertility hormones. At Organon,in the early 1980s, we decided to develop such an alternativeproduction route via DNA technology. A preliminary reporton this work has been published elsewhere (de Boer andMannaerts, 1990; Van Wezenbeek et al, 1990; Mannaertset al, 1991). In this article we describe in further detail the

strategy used for cloning of the genes encoding the FSH a-and pVsubunits, their expression in mammalian cells, theproduction of recombinant human FSH in cell culture andsome of its biochemical characteristics.

Cloning strategy and resultsRecombinant DNA-derived Pharmaceuticals represent a newclass of therapeutic agents with properties similar or identicalto those of proteins occurring in the human body. The firstproducts in this respect (human growth hormone and pro-insulin) are relatively simple polypeptide hormones, which areproduced in Escherichia coli. Later, other micro-organismssuch as Saccharomyces cerevisiae (Kingsman et al, 1987)were used as host cells for products such as hepatitis B vaccine,because of the need for proper folding of the HBsAg antigen(Valenzuela et al, 1982). Glycoproteins presented anotherchallenge because of the need for proper post-translationalmodification of the protein backbone. Moreover, the gonado-trophins are composed of two subunits, which are transcribedfrom separate genes. Although it has been attempted torecombine subunits into the intact hormone in vitro, thisprocess is not very efficient. Another potential problem in thisrespect is that individually produced subunits may exhibitaltered glycosylation. Therefore, it was an obvious choice toco-express the a- and (J-subunit genes in a single mammaliancell, analogous to the co-expression of the heavy-and lightimmunoglobulin chains (Ochi et al, 1983).

Host cellsIn the early 1980s a number of cell lines (such as mousefibroblasts, Vero cells, Baby hamster kidney cells and CHOcells) were available which were considered as suitable hostsfor secreted glycoprotein production. Selection criteria were,among others, the possibility to transfect cells with hetero-logous DNA, the growth characteristics in cell culture, theglycosylation patterns observed with other recombinant glyco-proteins and last but not least the potential safety hazardsconnected with the use of mammalian cells. In this respect theCHO cell line proved to be most promising. The CHO cellline Kl used in our experiments originates from a parentalCHO cell line, initiated by Puck (1958) from a biopsy of anovary of an adult Chinese hamster, and is probably of epithelialorigin. The CHO-K1 cell line can be easily transfected, andtransformants can be grown on a large scale. Moreover, manystrong promoters, such as obtained from Simian Virus (SV40)to direct transcription of foreign genes are active in CHO cells(McKnight and Tjian, 1986). Assurance of proper glycoproteinexpression and safety of the cell line was given by promisingpreliminary reports on the cloning and expression in CHOcells of other glycoproteins such as human tissue plasminogenactivator (tPA) and erythropoietin (EPO). Thus, the CHO-K1cell line was the obvious choice as the host cell line for theco-expression of the human a- and (J-FSH subunit genes.

Gene cloning

Cloning of the a-subunit geneThe a-subunit gene is common to all four glycoproteinhormones and is encoded by a single gene. Cloned a-subunit

372

Dow


ic.oup.com/m


Human recombinant follicle stimulating hormone

H HI I

X1

PR X1

P X H R X H P P HI I I I II I B

Isl\

UAA poly A

> B j .

AFigure 2. Restriction endonuclease map of the a-subunit gene (taken from Fiddes and Goodman, 1981). The shaded regions in the uppersection correspond to the four exons. These are separated by the three intervening sequences A, B and C. The locations of the predicted capsite (CAP), the initiation codon (AUG), the termination codon (UAA) and the approximate position of the polyadenylation site (poly A) aremarked.

cDNAs and genes of several species, including man, havebeen isolated and the complete nucleotide sequence has beenpublished (Fiddes and Goodman, 1979, 1981; Godine et al,1982; Talmadge et al, 1984).

The complete human a-FSH subunit gene spans ~10 kb andcontains three introns (Figure 2).

An FSHa-cDNA containing the complete coding sequencewas obtained from Dr I. Boime, Washington University, Schoolof Medicine, St. Louis, USA. The cDNA was used as a probefor screening of a human genomic gene library in bacteriophage^.gtlO. This yielded a 2.4 kb DNA fragment containing partof the third exon and the complete fourth exon. The remainderof the coding sequence was obtained from the cDNA clone.The initiator consensus sequence was provided by syntheticDNA. The three pieces of DNA (synthetic initiating sequence,cDNA and genomic DNA) were ligated and formed a 2.6 kbgene with naturally occurring splice signals of the third intron.This gene (FSHOg) was used in the construction of the FSHexpression plasmid.

Cloning of the fi-subunit geneThe P-subunits of the glycoprotein family are encoded bydifferent genes and determine the biological activity of eachhormone (Pierce and Parsons, 1981). Cloned complementaryDNA (cDNAs) or genes of (3-LH (Talmadge et al, 1984),P-HCG (Fiddes and Goodman, 1980; Boorstein et al, 1982;Talmadge et al, 1984) and p*-TSH (Gurr et al, 1983;Hayashizaki et al, 1985) have been isolated and describedpreviously. At the time of the construction no reports wereavailable on the nucleotide sequence of the human P-FSHgene. On the other hand, the amino acid sequences of theP-FSH protein from human (Parlow and Shome, 1974; Saxenaand Rathman, 1976; Pierce and Parsons, 1981), equine (Pierceand Parsons, 1981), porcine (Pierce and Parsons, 1981) andovine (Sairam et al, 1981) sources had been published. Withthese data as a guide synthetic oligonucleotide probes havebeen used for screening of cDNA and genomic libraries.

Complementary DNA libraries were prepared by reversetranscription of polyA+ RNA isolated from human pituitaries(obtained from Dr. H.S. Jansz, Laboratory for PhysiologicalChemistry, State University Utrecht, The Netherlands). TheDNA fragments obtained were cloned in phage XgtlO and theresulting cDNA library was screened with very long syntheticDNA (200-400 bp) probes, covering portions of the reportedamino acid sequence of P-FSH. This yielded five p*-FSH-

specific clones. However, sequence analysis revealed that nonecontained the complete coding sequence. The largest clonemissed the 14 N-terminal amino acids.

cDNA probes were used to screen a genomic library. About7.5X1O5 phages were screened, which yielded five positiveclones. After sequencing of the cDNA the deduced maturep-FSH protein sequence was found to be 111 amino acidslong. In comparison with the reported protein sequence bySaxena and Rathman (1976) the deduced sequence is sevenamino acids shorter. In the remainder of the sequence threeother differences were found. The nucleotide sequence of thecDNA clone was identical to the corresponding region in thegene sequence. During the course of our work the nucleotidesequence of a human P-FSH gene was published and confirmed.

Construction of the expression vector and selectorplasmid

Cloning vectors should enable the efficient transfer of hetero-logous genes into recipient cells and ensure stable inheritanceof the genes, preferably in high copy number. Furthermore,expression vectors should contain all the regulatory elements,such as promoters and enhancers needed for transcription.Efficient expression vectors based on lytic DNA viruses suchas SV40 and polyoma had been designed for this purpose butwere not considered appropriate for large-scale production ofrecombinant FSH. As an alternative, replicative DNA expres-sion vectors, such as those derived from bovine papillomavirus (BPV) or Epstein-Barr virus have been considered, butrejected in view of the limited host cell range and potentialinstability problems. Thus, an alternative strategy was choseni.e. to transfect the subunit genes by physico-chemical methodsinto the recipient CHO cells and select for cells that havestable integrated these genes randomly into the host cell DNA.It had been observed that such an integrative process mightresult in the integration of multiple copies of the expressionvector. Usually a variable number of gene copies are integratedinto a single chromosomal site, probably in correspondence tochromosomal breaks. When multiple copies are integratedthey are usually in a tandem array, mostly in a head-to-tailorientation. The presence of a high copy number of the clonedgenes is advantageous for high expression levels.

The construction of the final expression vector pKMS.FSHfXgPg (Figure 3) was complex. The a- and P-FSH geneswere cloned into a single vector in order to assure that the

373

Dow


ic.oup.com/m


W.OIijve of a/.

S V « E VTIIA

FSH eg

pBR322

FSHpg

PBRJ22

PBR..27

Figure 3. Schematic representations of the follicle stimulating hormone (FSH) expression plasmid pKMS.FSHotgPg and the selector plasmidpAG60/MT2. The direction of transcription is indicated by the arrows. CDS A 1,2 and Bl,2 indicate the location of the coding sequences(exons) of the a- or pVsubunit genes respectively.

copy number of the a- and p-FSH genes in the transfectedcell is the same. Furthermore, the vector contains a numberof DNA elements that are needed for further vector assemblyand efficient expression of the FSH subunit genes. Since allthe DNA construction work is done in E. coli, the vector isbasically an E. coli plasmid (pBR327), containing an originof replication and an ampicillin resistance gene for selectionof transformants. Transcription of the oc-gene is under thecontrol of the SV40 early promoter and is terminated at thepoly(A) signal of the a-gene. The human metallothionein-IIA

promoter (MT-IIA), drives transcription of the p^FSH gene.Furthermore, enhancer sequences from SV40 and murineleukaemia virus (MuLV) have been added to enhance thetranscription rate of the a- and (J-FSH genes.

The correct assembly of all the DNA elements inpKMS.FSHctgPg was confirmed by digestion of the vector withrestriction endonucleases and determination of the size ofthe fragments obtained by agarose gel electrophoresis. Theexperimentally-obtained values matched perfectly with thetheoretically-deduced fragment lengths.

Several methods have been developed to amplify clonedgenes by linkage to an amplifiable marker. The most widelyemployed amplification strategy involves the use of dihydro-folate reductase (DHFR) expression vectors. DHFR transfect-ants are subjected to increasing amounts of methotrexate,which is an inhibitor of DHFR. Methotrexate-resistant cellsshow increased levels of the DHFR as a consequence ofamplification of copies of the DHFR genes. The gene ofinterest is co-amplified because of its close physical proximity.Though valuable for research purposes, this technology isless suited for production processes because of the potentialinstability of these amplified genes in cells grown withoutmethotrexate. Weidle etal. (1988) showed consistent instabilityin the absence of selective pressure in methotrexate/DHFRamplified tPA-cell lines containing 300-1100 integrated copiesof the expression construct This genetic instability was accom-panied by a decrease in the production of tPA. Instability wasnot observed in the presence of methotrexate, thus necessitatingthe presence of such a compound during the productionprocess. In contrast to a gene amplification strategy we

374

have directly selected for high copy number clones by co-transfection of the FSH-expression plasmid with the selectorplasmid pAG60/MTlla (Figure 3). This plasmid contains aneomycin resistance gene (neo1) and the human metallothioneingene (MT-IIA). Co-transfection of the FSH expression vectortogether with a neomycin resistance gene allows the selectionof transfectants in media containing the antibiotic geneticin(G418). The human MT-HA gene confers resistance to thetoxic effects of heavy metal ions such as Cd2+. Since co-transfected plasmids often integrate at the same position intothe chromosome, and thus are located in the same expressionenvironment, a high expression of MT-IIA is likely to beassociated with a high expression of FSH.

Transfection and selection of FSH-producing CHOcells

Transfection of CHO-K1 cells with the FSH expression plasmidpKMS.FSHotgPg and the selector plasmid pAGoWMnr1 (ratioof 10:1) was carried out essentially following a calciumphosphate-DNA co-precipitation protocol (Wigleref a/., 1979).Geneticin resistant cells were subsequently grown in thepresence of different concentrations of cadmium chloride (upto 10 uM). A number of pools of Cd2+ resistant cellsindeed produced significant amounts of recombinant FSH, asdetermined by an FSH-specific enzyme-linked immunosorbentassay (ELISA). These pools have been subjected to single cellcloning procedures. Finally, a CHO clone coded CHO.FSH.30was selected for further characterization of genetic stability,cell growth and FSH productivity.

Genetic stability

Genetic stability is an important requirement for cell clonesto be used in a production process for a therapeutic protein.Thus, the CHO.FSH.30 line was subjected to detailed geneticanalysis. Since the vectors used do not contain an origin ofreplication, they must integrate into the chromosome of theCHO cell in order to be stably passed to the daughtercells during growth. Proof of integration was obtained byfluorescence in-situ hybridization (FISH). Metaphasic chromo-somes and interphase nuclei of CHO.FSH.30 were hybridized

Dow


ic.oup.com/m


Human recombinant follide stimulating hormone

Figure 4. Fluorescence in-situ hybridization (FISH) of follicle stimulating hormone (FSH) expression vector in the Chinese hamster ovary(CHO) cell genome.

with a biotin-labelled probe containing the bacterial DNAsequences present in the original expression plasmid. Hybrid-ization of probe and metaphasic chromosome was visualizedby incubation with fluorescein-coupled avidine (Figure 4).This strongly indicates the integration of the vector DNA at asingle position of the CHO cell genome.

The presence of a high gene copy number was confirmedby Southern blotting. Genomic DNA was isolated fromCHO.FSH.30, digested with restriction endonucleases andsubjected to agarose gel electrophoresis. Gels were blottedonto nitrocellulose paper and hybridized with a vector-specificradioactive probe. The size and intensity of the bands revealedthe presence of 150-450 gene copies, most likely in a head-to-tail arrangement Since this CHO cell proved to be stable, wedecided to develop a production process for recombinant FSH.

Production and purification of recombinant FSH(Puregon®)The culturing of mammalian cells on a production scale istechnically much more difficult compared with microbialfermentation. This is caused by the low growth rate (usuallywith population doubling times in the order of 16-24 h) andthe fragility of mammalian cells. Furthermore, they usuallyrequire growth factors, such as those present in fetal calfserum. Because of the complexity of sera and the potentialpresence of adventitious agents the use of such a componentin production media should be avoided. Therefore, cells mustbe adapted to growth on serum-free media. As an alternative,growth of cells and production may be uncoupled whereas thelatter is carried out in a serum-free medium.

The CHO cell line is an anchorage-dependent cell line,which implies that a proper surface must be provided forgrowth of the cells. In order to obtain a favourable surface/volume ratio cells are grown on small beads with a diameterof ~0.2 mm. The use of microcarriers in cell culture alsoprovides an opportunity for easy physical separation of the cells

"5o

(D

tomaE

co

o

ICO

105

10*4

0 5 10 15 20 25 30 35

culture time (days)

Figure 5. Growth ( • ) of CHO.FSH.30 cells and folliclestimulating hormone (FSH) production (O) in perfusion culture.The bar at the bottom represents the perfusion phase, indicating thestart of culture perfusion (open box), while cross hatching indicatesthe harvesting period.

from the culture supernatant. This enabled the development ofa perfusion-type continuous culture of CHO cells. Comparedwith batch cultures, perfusion cultures have the advantages ofa high cell concentration, due to retention of the cells, easyseparation of supernatant and a short residence time preventingproduct degradation by proteases. A typical example of a FSH-production process is provided in Figure 5.

Clone CHO.FSH.30 was grown to high cell density in acontinuously-stirred bioreactor. The bioreactor was designedfor aseptical operation and maintenance of optimal growthconditions. The perfusion of the culture was started at the endof the exponential growth phase with serum-containing mediumat a dilution rate of 0.5 volume/day. After increasing theperfusion rate to 1.0 volume/day a maximum cell concentrationof 107 cells/ml was obtained. At this stage the supply medium

375

Dow


ic.oup.com/m


W.OIijve et al.

1.20

£ 0.80

* 0.40

0.00

rFSH

excipients

10 15 20 25

Tims (min)

Figure 6. High performance size exclusion chromatography (HP-SEC) of recombinant human follicle stimulating hormone (rhFSH).rhFSH (-20 ug) was injected on a HP-SEC column (Zorbax GF250) equilibrated in 0.2 M sodium phosphate (pH 7.0) at a flowrate of 1 ml/min. Absorbance was monitored at 210 nm.

was changed to a serum-free formulation. After a number ofperfusions for removal of serum components the culturesupernatant was collected and used as source for recombinantFSH. Under conditions of serum-free production the culturescould be maintained for at least 3 months, without significantloss of productivity. Recombinant FSH was isolated frompooled culture supernatant by a series of chromatograpnicsteps including anion and cation exchange chromatography,hydrophobic interaction chromatography and size exclusionchromatography. The overall recovery was ~50%. The finalproduct was stored as a lyophilized powder.

Characterization: purity and identityComplex biotechnological products like recombinant FSH canonly be identified and characterized properly by the combinedresults of several physico-chemical methods such as gelelectrophoresis, high-performance liquid chromatography(HPLC), amino acid analysis, Edman degradation analysis,peptide mapping, mass spectrometry (MS) and carbohydrateanalysis (Garnick et al, 1988; Geisow, 1991; Federici, 1994).Some of these methods also allow demonstration of productpurity (e.g. peptide mapping, HPLC and electrophoresis).Residual DNA and CHO cell-derived proteins were analysedby hybridization and enzyme immunoassay, respectively.

Purity of rhFSH

The purity of rhFSH was determined by several complementarymethods including sodium dodecyl sulphate-polyacrylamidegel electrophoresis (SDS-PAGE), Western blotting, high per-formance size exclusion chromatography (HP-SEC), ELISAand DNA hybridization. In Figure 6 the HP-SEC profile ofrhFSH is shown.

The major peak represents the rhFSH ap*-dimer whereas thesecond peak contains excipients used for stabilization of theprotein during freeze-drying and storage. Apart from the rhFSHheterodimer no other peaks are detectable thus indicating theabsence of FSH aggregates (oligomers). The purity of the FSHpreparation was further established by SDS-PAGE (Laemmli,1970). When solubilized at room temperature in SDS-samplebuffer which does not contain reducing agent, rhFSH migrates

376

9666

45

31

21

14

f

M 1Figure 7. Sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) of recombinant human folliclestimulating hormone (rhFSH). 10 (ig rhFSH was dissolved at roomtemperature (lane 1) or by boiling (lane 2). Lane M: molecularmass marker proteins (Biorad) from top to bottom: phosphorylase B(96 kDa), serum albumin (66 kDa), ovalbumin (45 kDa), carbonicanhydrase (31 kDa) and trypsin inhibitor (21 kDa) and lysozym (14kDa). Proteins were visualized using Coomassie Brilliant BlueG250.

as intact ap"-dimer. As can be seen in Figure 7 lane 1,rhFSH (10 (ig protein) does not contain detectable amounts offree subunits.

When the gels were overloaded, trace amounts of freesubunits were visible (<]%, data not shown). In controlexperiments, complete dissociation of rhFSH into subunits isobserved (Figure 7, lane 2). The occurrence of CHO cell-derived proteins and culture medium components was assessedby specific immuno assays including a multi-analyte assay forthe detection of residual CHO cell-derived proteins. The limitof detection for each of these assays is between 1 and 20 ppm.No concentrations exceeding the low ppm range have beenfound in different batches of rhFSH. Similarly, Western blottingusing polyclonal antisera against CHO cell-derived proteinsdid not reveal CHO proteins in purified rhFSH (data not shown).The concentration of contaminating DNA was determined bya slot blot hybridization procedure using 32P-labelled genomicDNA extracted from CHO cells. Purified rhFSH contained<10pgDNA per 500 IU.

Identity of rhFSH

Molecular mass and sizeThe molecular mass of the rhFSH-dimer and the a- andP-subunits was determined by SDS-PAGE using non-dissociat-ing/dissociating solubilization conditions. Both the intact

Dow


ic.oup.com/m



protein and the subunits appear as a diffuse band due to theheterogeneity introduced by the attached oligosaccharides. Themolecular mass of the dimer was found to be 40-45 kDa whilethe subunits migrate at a molecular mass of ~25-30 kDa(Figure 7). These masses correspond well with the calculatedmasses of the protein plus oligosaccharides. The molecularsize was determined by size exclusion chromatography bycomparing the retention time of rhFSH with the retention times

0.80

0.60

CDCMCM

0.40

0.20

0.0010 20 30 40

Time (min)

50

Figure 8. C4 reverse phase high performance liquidchromatography (RP-HPLC) separation of rhFSH into its a- andP-subunits. Peaks labelled 1 and 2 represent [}- and a-subunitsrespectively. The small peak labelled with the asterisk (*) alsocontains a-subunit as determined by Western blotting (data notshown).

of a number of calibration proteins with known size; rhFSHelutes with an apparent molecular size of ~45 kDa (Figure 6).

Reversed phase HPLCThe two-subunit structure of rhFSH was further demonstratedusing hydrophobic interaction chromatography on a C4reversed phase HPLC column (Figure 8). Two peaks labelled1 and 2 represent the (J- and a-subunit respectively, asdetermined by amino- and carboxy-terminal sequence analysis,amino acid analysis, Western blotting and peptide mapping(for details see below).

Amino acid analysisAmino acid analysis involves the complete hydrolysis of aprotein to its constituent amino acids. This hydrolysate isseparated by ion exchange chromatography followed by reac-tion with ninhydrin and detection at 440 and 570 nm (TableI, intact rhFSH). Alternatively, the amino acid composition ofthe two subunits was determined by pre-column derivatizationof the amino acid hydrolysates of the isolated subunits witho-phthalaldehyde followed by separation of the modified aminoacids by RP-HPLC and UV detection (Table I, subunits). Thedata show that the experimental composition of rhFSH and itssubunits is in agreement with the theoretical composition.

Amino- and carboxy-terminal sequence analysisQuality control sequencing of rhFSH was performed usingEdman degradation to elucidate the first 13 residues of theamino- (N-) termini of the 2 subunits (Table H). This methodis used to confirm the cDNA-predicted amino acid sequenceand to detect potential proteolytic clips. Edman degradation isbased on the coupling reaction of the N-terminal amino acidof a protein with phenyl thiocyanate. The derivatized aminoacid is cleaved from the protein in a subsequent reaction,exposing the next amino acid for further cycles of couplingand cleavage. HPLC is used to separate and identify thereleased amino acids. As can be seen in Table n, significantheterogeneity of the N-terminus of the P-subunit is observed:

Table I. Amino

Ammo acid

AsxThrSerGlxProGlyAlaCys2ValMetHeLeuTyrPheLysHisArg

acid composition of recombinant

rhFSH

experimental

15.0*18.5 ± 0.613.1 ± 1.219.1 ± 0.512.3 ± 0.410.2 ± 0.411.3 ± 0.67.8 ± 1.0

13.8 ± 1.03.3 ± 0.66.6 ± 0.39.0 ± 0.47.2 ± 0.97.6 ± 0.8

13.5 ± 0.86.1 ± 0.37.4 x 0.5

human follicle

theoretical

152015191210111113479

117

1368

stimulating hormone (rhFSH)

a-subunit

experimental

6.3 ± 0.68 .0 :7.4 :9.7 :7.3 :3.8 i5.2 25.7 i7.0*3.0 i1.6 i4.4 j4.2 24.3 24.5 23.4 2

t 0.6t 1.2t 0.2t 0.5t 0.5t 0 . 3t I.I

t 0.1t 0.1t 0.1t 0.3t 0.1t 0.7t0 .2

3.3 T 0.1

and its subunits1.

theoretical

68897455731444633

Data represent the mean

p^-subunit

experimental

8.6 ± 0.410.7 ± 0.65.9 ± 0.5

10.8 ± 0.75.7 ± 0.35.6 ± 0.66.2 ± 0.36.1 ± 2.16.0*1.2 ± 0.16.1 ± 0.25.6 ± 0.36.9 ± 0.43.3 ± 0.15.1 ± 0.73 3 ± 0.25.1 ± 0.2

± SD of six batches

theoretical

9127

105666616573735

"Normalization of amino acid ratios was performed by assuming Asx = 15 (rhFSH) or Val=7 (a-subunit) or 6 ((J-subunit). Asx = Asn + Asp; Glx = Gin +Glu; Cys2 = Cystine. Tip is not recovered as it is destroyed during acid hydrolysis.

377

Dow


ic.oup.com/m


W.OIijve et al.

Table IL Amino termina] sequence analysis' of recombinant human folliclestimulating hormone (rhFSH) by automated Edman degradation

Cycle a-subunit ^-subunit

123456789

10111213

aminoacid

AlaProAspValGinAsp(Cys)ProGlu(Cys)ThrLeuGin

yield(pmol)

517293129443333179-16965

-12712184

aminoacid

AsnSer(Cys)GluLeuThr(Asn)HeThrHeAlaHeGlu

(Cys)GluLeuThr(Asn)HeThrlieAlalieGluLysGlu

yield(pmol)

141174-194167170-320216276175134109

_

112219194-189119320216276

73134109

'Data are shown for the first 13 cycles. Amino acids in parentheses (Cysand Asn) were not recovered. Amino terminal heterogeneity of theP-subunit (start at position 1, left column) or 3 (right column) has also beendescribed for pituitary FSH (Shome et al., 1988). Routinely low amounts(«S5%) a-subunit were found to start at residue 3 (data not shown).

EcoCM

L7

batsL5

L6

L5-6 L1-4

alfa

L5

L1

20 30 40 50 60

Time (mln)

70

Figure 9. Peptide maps of a- (lower trace) and p"-folliclestimulating hormone (FSH) (upper trace). Recombinant FSHsubunits, prepared by C4 reverse phase high performance liquidchromatography (RP-HPLC), were reduced, pyridylethylated anddigested with LysC. Peptides were separated by C18 RP-HPLCusing a linear gradient of acetonitril. Peptide L2 of the a-subunit(Lys45) was not recovered; its presence in rhFSH, however, wasconfirmed by electro-spray mass spectrometry (ES-MS) of partialdigests (Table IV).

~50% of the protein starts at the expected amino acid residue1 (asparagine) whereas the other half of the sequence starts atamino acid residue 3 (cysteine). The amino terminal sequenceheterogeneity results in the typical doublet appearance ofrhFSH isohormones as seen in isoelectric focusing experiments(Figure 10; for further details see below). Similar aminoterminal heterogeneity is observed for the a-subunit, althoughthe amount of truncated a-subunit is much less: ~5% starts atresidue 3 (aspartic acid) and 95% starts at the expected residue1 (alanine). The amino terminal heterogeneity observed forthe P-subunit has also been described for pituitary FSH (Shomeet aL, 1988) and is thought to originate from the processing

Table III. C-terminal sequence analysis of recombinant human folliclestimulating hormone (rhFSH) a- and p-subunit as determined bycarboxypeptidase Y. The order shown is the order in which specified aminoacids are released by CPaseY. Numbers refer to the theoretical positionwithin the sequence

Order of release of amino acidresidues

123

Amino acid released

a-subunit

Ser 92Lys 91His 90

p-subunit

Glu 111Lys 110Met 109

(cleavage of the leader sequence) of the protein. Neither thea- nor the P-subunit contain proteolytic clips.

Up till now no reliable and staightforward routine analysismethod for the sequencing of the carboxy- (C-) terminus ofproteins (such as the Edman degradation for the TV-terminus) hasbeen available (Inglis, 1991). We have used carboxypeptidaseY (CPaseY) digestion of rhFSH subunits to determine theC-termini. The enzyme CPaseY releases the C-terminal aminoacid from a protein. Thus by following the sequential releaseof amino acids over time, the C-terminal sequence can bedetermined. In Table HI the C-terminal sequences of the twosubunits of rhFSH are shown. In each case these are infull agreement with the expected sequences. No C-terminalheterogeneity was detected. Similar results were obtained fromamino acid composition and electro-spray mass spectrometry(ES-MS) analysis of the C-terminal peptides which wereobtained by peptide mapping (see below).

Peptide mappingPeptide mapping is routinely used to compare the proteinstructure of a product to that of a reference material or tothose of previous lots to confirm the primary structure and toensure lot-to-lot consistency of protein structure. It also sup-ports the genetic stability of FSH genes in CHO cells duringculture (Dougherty et al, 1990; Lu et al, 1993; O'Connor,1993; Rohde and Rush, 1994). Peptide mapping of rhFSH isaccomplished by reducing and modifying all disulphide bondsin each of the two subunits. This is followed by cleaving thesubunits into a number of small fragments by digestion withthe endoproteinase LysC, which specifically cleaves the proteinat specific lysine residues (Table TV). The resultant peptidesare then separated by RP-HPLC with on-line UV-monitoring(Figure 9) and/or ES-MS (Table IV). Thus a map or 'fingerprint'is obtained which allows for differentiation between proteinsof similar, but not identical, primary structure. The selectivityof the peptide mapping procedure was determined by analysingthe effects of mutation (by purposeful creation of mutants ofrhFSH using site-directed mutagenesis) as well as by studyingthe effects of oxidation (by exposing rhFSH to an H2O2~containing buffer). Both types of modified forms of rhFSHwere easily discriminated from normal (wild-type) rhFSH bychanges in the retention times of the mutated or oxidizedpeptides (data not shown). Peptide mapping in combinationwith amino acid analysis, Edman degradation and ES-MS hasbeen used to verify the entire primary structure of rhFSHwhich was shown to be in full agreement with the cDNA-

378

Dow


ic.oup.com/m



Table IV. Identification of LysC peptides* by electro-spray massspectrometry (ES-MS)

Peptides Residues Expected mass Observed mass

Panel A

aL4aL6aL3aL5aLl-2aLl

Panel B

pL5PL7p"L6PL5-6p

52-6376-9246-5164-75

1^51-44

50-5487-11155-8650-86

1-49

multiplemultiple718.4

1357.75500.65372.5

703.33002.73863.84549.7glycopeptide

glycopeptideglycopeptide

718135855035374

703300338644550multiple

'Peptides are listed in order of appearance during C18 reverse phase highperformance liquid chromatography (RP-HPLC) analysis. Peptide aLl-2was only detectable in incomplete digests, whereas peptide (5L1-4 was notaccessible to further digestion. Panel A: LysC peptides of the oc-subunit,panel B: LysC peptides of the pVsubuniL

A B

pi 6.5-

pl 3.5-

- p l 5.4

< 1<2

J -pi 4.0

Figure 10. Isoelectric focusing (IEF) of recombinant human folliclestimulating hormone (rhFSH). Panel A: rhFSH (10 ug/lane) wassubjected to IEF (pH 3.5-6.5) under non-denaturing (lanes 1 and 2)or denaturing conditions (lanes 3 and 4). Following focusing,proteins were transferred electrophoretically onto PVDF andvisualized by a-subunit specific (lanes 2 and 4) or p%subunitspecific (lanes 1 and 3) monoclonal antibodies. Panel B: IEF ofrhFSH in a pH 4.0-5.4 gradient. Recombinant FSH was solubilizedunder denaturing conditions. Following focusing and Westernblotting, protein was visualized by staining with CoomassieBrilliant Blue. Bands labelled 1 and 2 were subjected to automatedEdman degradation (Table V).

Table V. Amino terminal sequence analysis of bands 1 andFigure 10*

Band excised

12

Major amino acid sequence

Asn-Ser-Xxx-Glu-Leu-ThrXxx-Glu-Leu-Thr-Xxx-Ile

Identity

PIP3

2 from

Yield (pmol)

2020

"The identification was done on the basis of the known amino acid sequenceof the pVsubunit. Xxx represent gaps in the sequence caused by Cys (residue3) or glycosylated Asn (position 7). The amount of sequencable protein(initial yield in pmol) is reported.

5.3 5.5

Figure 11. Isohormone distribution of recombinant human folliclestimulating hormone (rhFSH) as determined by immobilized pHgradient gel electrophoresis followed by Coomassie Brilliant Bluestaining and densitometry. Data represent the mean ± SD fromthree independent experiments.

6

CO

c

2. 4

oECO

<D

jo

cr

JQ

rFSH

LJJ urFSH

Fuc Man GlcNAc Gal NeuNAcmonosaccharide

Figure 12. Monosaccharide composition of recombinant humanfollicle stimulating hormone (rhFSH) in comparison with urinaryFSH. Data represent the mean ± SD of 13 batches.

derived sequence. In addition, it has provided additionalinformation on the positions of the glycosylation sites, i.e. inthe a-subunit at positions 52 and 78, and in the pVsubunit atpositions 7 and 24 'gaps' were sequenced where asparaginewas expected. This is indicative of the attachment of carbo-hydrate side chains. Also, the glycopeptides showed in ES-MS analysis multiple masses characteristic of heterogeneityintroduced by oligosaccharides. Consequently, this techniqueis particularly important in the quality control of rhFSH.

Isohormone analysisOligosaccharides of FSH have been implicated importantdeterminants of the circulatory half-life (and in-vivo bio-activity) as well as in the process of signal transduction(Kalyan and Bahl, 1981; Sairam, 1983; Ulloa-Aguirre et al.,1995; de Leeuw et al., 19%). Typically, microheterogeneitycauses charge heterogeneity due to differences in the amountof terminal sialic acid. Therefore, isoelectric focusing andchromatofocusing are widely employed to visualize micro-heterogeneity (Sairam, 1983; Blum and Gupta, 1985; de Leeuwet al., 1996). In Figure 10 a typical example of rhFSHheterogeneity is shown. rhFSH was separated in a pH 3.5-6.5immobilized pH gradient (IPG), transferred to a polyvinylidene

379

Dow


ic.oup.com/m


W.OIijve et al.

Figure 13. Carbohydrate profile analysis of recombinant human follicle stimulating hormone (rhFSH). Oligosaccharides released from200 |ig rhFSH were analysed by high performance anion exchange chromatography (HPAEC-PAD). Windows for neutral, mono-, di-, tri-and tetra-sialo-carbohydrate peaks run from 19-22, 32^2, 44-56, 56-66 and 66-80 min respectively.

fluoride membrane (Millipore, Etten-Leur, The Netherlands)and probed with monoclonal antibodies specific for the a-subunit (lanes 2 and 4) or P-subunit (lanes 1 and 3). Intact,heterodimeric FSH yields the same staining pattern typicallyshowing ~7-9 bands at pi 3.9-5.5, irrespective of the antibodyused. Each band resolves as a doublet. In order to study furtherthe origin of this doublet appearance, rhFSH was loaded onan IPG gel covering pH 4.0-5.4, blotted onto PVDF andstained with Coomassie Brilliant Blue (Figure 10, panel B).Bands labelled 1 and 2 were subjected to automated Edmandegradation for six cycles (Table V). As can be seen the majorsequence of band 1 is the p*-subunit which starts at Asn-1,whereas the major sequence of band 2 starts at Cys-3. Apartfrom this amino terminal protein heterogeneity the majorcharge heterogeneity is, however, caused by differences inglycosylation in general and more particularly due to variationin the sialic acid content (for details see also de Leeuw et al,1996). When rhFSH was dissociated into subunits by includingurea during the focusing experiment, the doublet nature of thepVsubunit is retained (lane 3) while the a-subunit appearedless heterogeneous (~3—4 bands are observed, lane 4) andless acidic. The observed differences may relate to spatialconstraints imposed on terminal glycosylation in the Golgisystem which occurs after the assembly of the two subunits.Indeed, it has been shown for HCG and free a-subunitthat the a-subunit in HCG contains more mono-antennaryoligosaccharides whereas the latter contains more tri- andtetra-antennary oligosaccharides (Hard et al, 1990; Blithe,1994). The isohormone distribution was determined by densito-metry of gels similar to the one shown in Figure 10, exceptthat protein staining was done by Coomassie Brilliant Blue.Figure 11 shows a typical example. As can be seen the majorityof rhFSH focuses between pH 4.3-5.3. This is slightly lessacidic than FSH from urinary origin (Metrodin®) (Lambertet al, 1995; de Leeuw et al. 1996).

Oligosaccharide analysisThe carbohydrate moiety was investigated by monosaccharidecompositional analysis, oligosaccharide profiling and structure

380

50

~ 40

o 30

20

10

Charge

Figure 14. Distribution of oligosaccharides from recombinanthuman follicle stimulating hormone (rhFSH) over neutral (0),mono- (1-), di- (2-), tri- (3-) and tetra-sialo-oligosaccharides (4-).Data represent the mean ± SD of 13 batches. The relative amountof each charge window was calculated as the ratio of the peak area/window and the total peak area (five windows, see Figure 13).

determination. In Figure 12 the overall composition of rhFSHis compared with that of purified urinary FSH (see Hardetal, 1990).

The monosaccharides found represent those typically foundin TV-linked, complex type oligosaccharides; ~50% of theoligosaccharides of rhFSH are fucosylated. Monosaccharideanalysis of the isolated subunits revealed that fucose is exclus-ively found in the p*-subunit and not in the a-subunit (data notshown). The content of N-acetyl glucosamine and galactoseindicates that an 'average' oligosaccharide contains 2.5antennas, 80% of which is sialylated. Based on the content ofgalactose and W-acetyl glucosamine of the two subunits itappears that the a- and p^-subunit contain 'on average' di- andtri-antennary oligosaccharides, respectively (data not shown).The oligosaccharide moiety of rhFSH was further studied bycarbohydrate profiling. Oligosaccharides were released byhydrazinolysis and separated by high performance anionexchange chromatography (HPAEC) on a CarboPac PA-1column (Dionex) using a linear gradient of NaOAc. Oligo-saccharides are detected by pulsed amperometric detection. In

Dow


ic.oup.com/m



Figure 13 a typical profile of rhFSH oligosaccharides isshown illustrating again the heterogeneity of the sugar moietyof rhFSH.

Based on the retention time of reference oligosaccharideswindows can be denned during which neutral, monosialo-,disialo-, trisialo- and tetrasialo-oligosaccharides elute. Thepeak areas of each of these windows are depicted in Figure14. As can be seen >70% of the oligosaccharides is mono-or di-sialylated, whereas only a minor portion is neutral ortetra-sialylated.

Separate from the composition and profiling studies reportedhere, oligosaccharide sequencing of rhFSH has also beenperformed (Hard et al., 1990). This study showed only minordifferences in the structure of carbohydrate antennas betweenrecombinant and pituitary FSH, most notably the absence ofintersecting N-acetyl glucosamine moieties in rhFSH. More-over, N-acetyl neuraminic acid is linked in an a2-3 conforma-tion while in natural FSH both a2-3 and a l - 6 occur (Greenand Baenzinger, 1988; Hard et al, 1990). The impact of theglycosylation on the biological properties of rhFSH will bediscussed elsewhere (de Leeuw et al., 1996).

AcknowledgementIn the course of the project many people contributed their excellenttechnical skills towards the cloning and biochemical characterizationof recombinant FSH. In particular, the authors would like to thankDr. Jan Damm of the Department for Analytical Research for theanalysis of carbohydrate composition and structure.

ReferencesBlithe, D.L. (1994) Structure and function of the gonadotrophin free alpha

molecule of pregnancy. In Lustbader, J.W., Puett, D. and Ruddon, R.W.(eds), Glycoprotein Hormones. Springer Verlag, New York, pp. 156-166.

Blum, W.F.P. and Gupta, D. (1985) Heterogeneity of rat FSH bychromatofocusing: studies on serum FSH, hormone released in vitro andmetabolic clearance rates of its various forms. J. Endocrinol., 105, 29-37.

de Boer, W. and Mannaerts, B. (1990) Recombinant follicle stimulatinghormone. II. Biochemical and biological characteristics. In KrommelinDJ.A. and Schellekens H. (eds). From Clone to Clinics, Developments inBiotherapy. Vol. 1. Kluwer Academic Publishers, Dordrecht, pp. 253-259.

Boime, I., Keene, J., Galway A.B., Fares, F.M. et al. (1992) Expression ofrecombinant human FSH, LH, and CG in mammalian cells: a structure-function model for therapeuUc drug design. Sem. Reprod. Endocrinol., 10,45-50.

Boorstein, W.R., Vamvakopoulos, N.C. and Hddes, J.C. (1982) Humanchorionic gonadotrophin beta-subunit is encoded by al least eight genesarranged in tandem and inverted pairs. Nature, 300, 419-422.

Cole E.S., Lee, K., Lauziere, K. et al. (1993) Recombinant human thyroidstimulating hormone: development of a biotechnology product for detectionof metastatic lesions of thyroid carcinoma. Biotechnology, 11, 1014-1024.

Dougherty, JJ., Snyder, L.M., Sinclair, R.L. and Robins, R.H. (1990) High-performance tryptic mapping of recombinant bovine somatotropin. Anal.Biochem., 190, 7-20.

Federici, M.M. (1994) The quality control of biotechnology products.Biological*, 22, 151-159.

Fiddes, J.C. and Goodman, H.M. (1979) Isolation, cloning and sequenceanalysis of the cDNA for the alpha-subunit of human chorionicgonadotrophin. Nature, 281, 351-356.

Fiddes, J.C. Goodman, H.M. (1980) The cDNA for the beta-subunit of humanchorionic gonadotrophin suggests evolution of a gene by readthrough intothe 3'-untranslated region. Nature, 286, 684-687.

Fiddes, J.C. and Goodman, H.M. J. (1981) The gene encoding the commonalpha subunit of the four human glycoprotein hormones. /. Mol. Appl.Genet., 1,3-18.

Gamick, R.L., Solli. NJ. and Papa, P.A. (1988) The role of quality control inbiotechnology: an analytical perspective. Anal. Chem., 60, 2546-2557.

Geisow. MJ. (1991) Characterizing recombinant proteins. Biotechnology, 9,921-924.

Godine, J.E, Chin, W.W. and Habener, J.F. (1982) Alpha subunit of rat pituitaryglycoprotein hormones. Primary structure of the precursor determined fromthe nucleotide sequence of cloned cDNA's. J. Biol. Chem., 257, 8368-8371.

Green, E.D. and Baenzinger, J.U. (1988) Asparagine-linked oligosaccharideson lutropin, follitropin and thyrotropin. J. Biol. Chem., 263, 25-35.

Gurr, J.A. Catterall, J.F. and Kouides, I.A. (1983) Cloning of cDNA encodingthe pre-beta subunit of mouse thyrotropin. Proc. Nail. Acad Sci. USA, 80,2122-2126.

Hard, K. Mekking, A., Damm, J.B.L. et at. (1990) Isolation and structuredetermination of the intact sialylated AMinked carbohydrate chains ofrecombinant human follitropin expressed in Chinese hamster ovary cells.Eur. J. Biochem., 193, 263-271.

Hayashizaki, Y., Miyai, K., Kato, K. and Matsubara, K. (1985) Molecularcloning of the human thyrotropin-beta subunit gene. FEBS Lett., 188,394-400.

Inglis, A.S. (1991) Chemical procedures for C-terminal sequencing of peptidesand proteins. Anal. Biochem., 195, 183-1%.

Kalyan, N.K. and Bahl, O.P. (1981) Role of carbohydrate in human chorionicgonadotrophin. Effect of deglycosylation on the subunit interaction and onits in vitro and in vivo biological properties. J. Biol. Chem., 258, 67-74.

Kingsman, S.M., Kingsman, AJ. and Mellor, J. (1987) The production ofmammalian proteins in Saccharomyces cerevisiae. Trends Biolechnoi, 5,53-57.

Laemmli, U.K., (1970) Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature, 227, 680-685.

Lambert, A., Rodgers, M., Mitchell, R. et al. (1995) In-vitro biopotency andglycoform distribution of recombinant human follicle stimulating hormone(Org 32489), Metrodin and Metrodin-HP. Hum. Reprod., 10, 1928-1935.

de Leeuw R., Mulders, J., Voortman, G. et al. (1996) Structure-functionrelationship of recombinant FSH (Org 32489). Mol. Hum. Reprod., 2,361-369.

Lu, H.S., Fausset, PR., Sotos, L.S. et al. (1993) IsolaUon and characterizationof three recombinant human granulocyte colony stimulating factor His —*Gin isoforms produced in Escherichia coli. Protein Expression Purific., 4,465-472.

Mannaerts, B., de Leeuw, R., Geelen, J. et al. (1991) Comparative in vitroand in vivo studies on the biological characteristics of recombinant humanfollicle stimulating hormone. Endocrinology, 129, 2623-2630.

McKnight, S. and Tjian, R. (1986) Transcriptional selectivity of viral genesin mammalian cells. Cell. 46, 795-805.

Ochi, A., Hawley, R.G., Hawley, T. et al. (1983) Functional immunoglobulinM production after transfection of cloned immunoglobulin heavy and lightchain genes into lymphoid cells. Proc. Nail. Acad. Sci. USA, 80, 6352-6355.

O'Connor (1993) The use of peptide mapping for the detection of heterogeneityin recombinant DNA-derived products. Biologicals, 21, 111-117.

Parlow, A.F. and Shome, BJ. (1974) Human follicle stimulating hormone(hFSH). First proposal for the amino acid sequence of the alpha-subunit(hFSH-alpha) and first demonstration of its identity with the alpha-subunitof human luteinizing hormone (hLH.alpha). Clin. Endocrinol. Metab., 39,199-202.

Pierce, J.G. and Parsons, T.F. (1981) Glycoprotein hormones: structure andfunction. Ann. Rev. Biochem., 50, 465-495.

Puck T.T. (1958) Genetics of somatic mammalian cells. III. Long termcultivation of euploid cells from human and animal subjects. J. Exp. Med.,108, 945.

Rohde, M.F., Lu, H.S. and Rush, R.S. (1994) Peptide mapping of recombinantproteins. In Brown, F. and Lubiniecki, A.S. (eds), Genetic Stability andRecombinant Product Consistency. Karger, Basel, pp. 121—127.

Sairam, M.R. (1983) Gonadotropic hormones: relationship between structureand function with emphasis on antagonists. Hormonal Proteins and PeptidesXI, Academic Press, New York, pp. 1-79.

Sairam, M.R., Seidah, N.G. and Chretien, M. (1981) Primary structure of theovine pituitary follitropin beta-subunit. Biochem. J., 197, 541-552.

Saxena B.B. and Rathman, P. J. (1976) Amino acid sequence of the beta-subunit of follicle-stimulating hormone from human pituitary glands. Biol.Chem., 251, 993-1005.

Shome, B., Parlow, A.F, Liu, W.-K. et al. (1988) A re-evaluation of theamino acid sequence of human follitropin beta-subunit. J. Prof. Chem., 7,325-339.

381

Dow


ic.oup.com/m


W.OIijve of al.

Stockell Hartree, A. and Renwick, A.G.C. (1992) Molecular structures ofglycoprotein hormones and functions of their carbohydrate components.Biochem. J., 287, 665-679.

Talmadge, K., Vamvakopoulos N.C. and Hddes, J.C. (1984) Evolution of thegenes for the beta subunits of human chorionic gonadotrophin and luteinizinghormone. Nature, 307, 37^0 .

Thotakura, N.R. and Blithe, D.L. (1995) Glycoprotein hormones: glycobiologyof gonadotrophins, thyrotrophin and free a subunil. Glycobiology, 5, 3—10.

Ulloa-Aguirre, A., Espinoza, R., Damian-Matsumura, P. and Chappcl S.C.(1988) Immunological and biological potencies of the different molecularspecies of gonadotrophins. Hum. Reprod., 3, 491-501.

Ullua-Aguirre, A., Midgley, A.R., Beitins, I.Z. and Padmanabhan, V. (1995)Follicle-stimulating isohormones: characterization and physiologicalrelevance. Endocrine Rev., 16, 765-787.

Valenzuela, P., Medina, A., Rutter, WJ. et al. (1982) Synthesis and assemblyof hepatitis B virus surface antigen particles in yeast. Nature, 298, 347-350.

Van Wezenbeek, P., Draaijer, J., van Meel, F. and Olijve, W. (1990)Recombinant Follicle Stimulating Hormone I. Construction, selection andcharacterization of a cell line. In Crommelin, DJ.A. and Schellekens, H.(eds), From Clone to Clinics, Developments in Biotherapy. Vol. 1. KluwerAcademic Publishers, Dordrecht, pp. 245-251.

Weidle, U.H., Buckel, P. and Wienberg, J. (1988) Amplified expressionconstructs for human tissue-type plasminogen activator in Chinese hamsterovary cells: instability in the absence of selective pressure. Gene, 66,193-203.

Wigler, M., Pellicer, A., Silverstein, S. et al. (1979) DNA-mediated transferof the adenine phosphoribosyltransferase locus into mammalian cells. Proc.Natl. Acad. Sci. USA, 76, 1373-1376.

Received on January 4, 1996; accepted on March 4, 1996

382

Dow


ic.oup.com/m


Documents

Molecular biology and biochemistry of human recombinant follicle