Gene expression in mammalian cells

Proteins such as human .beta.-interferon or human erythropoietin are prepared by culturing mammalian cells which harbour a nucleic acid sequence comprising: (i) a coding sequence which encodes the desired protein and which is operably linked to a promoter capable of directing expression of the coding sequence in a mammalian cell in the presence of a heavy metal ion; and (ii) a first selectable marker sequence comprises a metallothionein gene and which is operably linked to a promoter capable of directing expression of the metallothionein gene in a mammalian cell in the presence of a heavy metal ion; and optionally (iii) a second selectable marker sequence which comprises a neo gene and which is operably linked to a promoter capable of directing expression of the neo gene in a mammalian cell.

This invention relates to the expression of genes in mammalian cells,
 particularly genes responsible for proteins whose biological activity in
 vivo is affected by a diversity of factors including specific
 glycosylation. Examples of such genes are the human .beta.-interferon
 (IFN.beta.), human erythropoietin (EPO), human chorionic gonadotropin,
 various other cytokines and growth factors as well as specific viral
 antigens such as Dengue viral proteins whose structure may be relevant for
 the development of vaccines.
 Previously, genes have been extensively expressed in mammalian cell lines,
 particularly in mutant Chinese Hamster Ovary (CHO) cells deficient in the
 dihydrofolate reductase gene (dhfr) as devised by the method of Urlaub et
 al, PNAS U.S.A. 77, 4216-4220, 1980. A variety of expression systems have
 been used. Many vectors for the expression of genes in such cells are
 therefore available. Typically, the selection procedures used to isolate
 cells transformed with the expression vectors rely on using methotrexate
 to select for transformants in which both the dhfr and the target genes
 are coamplified.
 The dhfr gene, which enables cells to withstand methotrexate, is usually
 incorporated in the vector with the gene whose expression is desired.
 Selection of cells under increasing concentrations of methotrexate is then
 performed. This leads to amplification of the number of dhfr genes present
 in each cell of the population, as cells with higher copy numbers
 withstand greater concentrations of methotrexate. As the dhfr gene is
 amplified, the copy number of the gene of interest increases concomitantly
 with the copy number of the dhfr gene, so that increased expression of the
 gene of interest is achieved.
 Unfortunately, these amplified genes have been reported to be variably
 unstable in the absence of continued selection (Schimke, J. Biol. Chem.
 263, 5989-5992, 1988). This instability is inherent to the presently
 available expression systems of CHO dhfr.sup.- cells.
 For many years, several promoters have been used to drive the expression of
 the target genes such as the SV40 early promoter, the CMV early promoter
 and the SR.alpha. promoter. The CMV and SR.alpha. promoters are claimed to
 be the strongest (Wenger et al, Anal. Biochem. 221, 416-418, 1994).
 In one report, the .beta.-interferon promoter has also been used to drive
 the expression of the .beta.-interferon gene in the mutant CHO dhfr.sup.-
 cells (U.S. Pat. No. 5,376,567). In this system, however, the selected CHO
 dhfr.sup.- cells had to be superinduced by the method of Tan et al (Tan et
 al, PNAS U.S.A. 67, 464-471, 1970; Tan et al, U.S. Pat. No. 3,773,924) to
 effect a higher level of .beta.-interferon production. In this system a
 significant percentage of the superinduced .beta.-interferon produced by
 the CHO dhfr.sup.- cells was not glycosylated.
 The mouse metallothionein gene (mMT1) promoter has also been used for the
 expression of .beta.-interferon genes in CHO cells, BHK and LTK.sup.-
 mouse cells (Reiser et al 1987 Drug Res. 37, 4, 482-485). However, the
 expression of .beta.-interferon with this promoter was not as good as the
 SV40 early promoter in CHO cells. Further, .beta.-interferon expression
 from these cells mediated by the mMT1 promoter was inducible by heavy
 metals. Heavy metals are however extremely toxic to the cells and this
 system was therefore abandoned. Instead, Reiser et al used the CHO
 dhfr.sup.- expression system in conjunction with the SV40 early promoter
 (Reiser et al, Drug Res. 37,4, 482-485 (1987) and EP-A-0529300) to produce
 .beta.-interferon in CHO dhfr.sup.- cells as derived by the method of
 Urlaub et al (1980).
 We have now expressed .beta.-interferon in wild-type CHO cells. Wild-type
 CHO cells were transfected with a vector comprising a .beta.-interferon
 gene under the control of a mouse sarcoma viral enhancer and mouse
 metallothionein promoter (MSV-mMT1), a neo gene under the control of
 promoter capable of driving expression of the neo gene in both E. coli and
 mammalian cells and a human metallothionein gene having its own promoter.
 Transfected cells capable of expressing .beta.-interferon were selected by
 first exposing cells to geneticin (antiobiotic G418) and thus eliminating
 cells lacking the neo gene and then exposing the surviving cells to
 increasing concentrations of a heavy metal ion.
 The heavy metal ion enhanced the MSV-mMT1 promoter for the
 .beta.-interferon gene, thus increasing .beta.-interferon expression. The
 heavy metal ion also induced the human metallothionein gene promoter,
 causing expression of human metallothionein. The human metallothionein
 protected the cells against the toxic effect of the heavy metal ion. The
 presence of the heavy metal ion ensured that there was continual selection
 of cells which had the transfecting vector, or at least the
 .beta.-interferon gene and the human metallothionein gene and their
 respective promoters, integrated into their genome.
 The selected cells that had been successfully transfected expressed
 .beta.-interferon. Expression was surprisingly improved when the cells
 were cultured in the presence of Zn.sup.2+. The .beta.-interferon had
 improved properties, in particular a higher bioavailability, than prior
 .beta.-interferons.
 These findings have general applicability. Accordingly, the present
 invention provides:
 a nucleic acid vector comprising:

(i) a coding sequence which encodes a protein of
 interest and which is operably linked to a
 promoter capable of directing expression of the
 coding sequence in a mammalian cell in the
 presence of a heavy metal ion;
 (ii) a first selectable marker sequence which
 comprises a metallothionein gene and which is
 operably linked to a promoter capable of
 directing expression of the metallothionein gene
 in a mammalian cell in the presence of a heavy
 metal ion; and
 (iii) a second selectable marker sequence which
 comprises a neo gene and which is operably linked
 to a promoter capable of directing expression of
 the neo gene in a mammalian cell;
 mammalian cells which harbour a nucleic acid sequence comprising:

(i) a coding sequence which encodes a protein of
 interest and which is operably linked to a
 promoter capable of directing expression of
 the coding sequence in a mammalian cell in
 the presence of a heavy metal ion;
 (ii) a first selectable marker sequence which
 comprises a metallothionein gene and which
 is operably linked to a promoter capable of
 directing expression of the metallothionein
 gene in a mammalian cell in the presence of
 a heavy metal ion; and optionally
 (iii) a second selectable marker sequence which
 comprises a neo gene and which is operably
 linked to a promoter capable of directing
 expression of the neo gene in a mammalian
 cell;
 a process for producing such cells, which process comprises

(a) transfecting mammalian cells with a vector of the
 invention;
 (b) exposing the transfected cells to geneticin to
 eliminate thereby cells lacking the neo gene; and
 (c) exposing the cells that survive step (a) to
 progressively increasing concentrations of a
 heavy metal ion to select thereby the desired
 cells.
 use of a neo gene and a metallothionein gene as selectable marker genes in
 a single vector; and a process for the preparation of a protein of
 interest, which process comprising culturing mammalian cells of the
 invention under conditions allowing expression of the desired protein and
 recovering the desired protein thus expressed.
 By using both a neo gene and a metallothionein gene as selectable markers
 in a single vector, it is possible to select for transformed mammalian
 cells, such as wild-type CHO cells, which have multiple copies of the
 expression vector stably integrated into their genomes. This selection
 system therefore facilitates the preparation and identification of stably
 transformed mammalian cells such as the wild-type CHO cells and avoids the
 need for dhfr.sup.- cells. The transformed cells enable the stable
 expression of genes such as the human .beta.-interferon gene because they
 have multiple copies, typically at least 20-100 copies or more, of these
 genes integrated into their genomes.
 Moreover, the use of relatively high concentrations of Cd.sup.2+ (up to 200
 .mu.M) in the selection procedure eliminates inadvertent microbial
 contaminants such as mycoplasma that may become associated with the
 transfected cells during tissue culture procedures. Thus, the present
 invention minimises the possibility of microbial contamination of
 transfected cells.
 Further, one particular promoter/enhancer system according to the invention
 surprisingly gave a significantly higher level of expression than the
 strong promoter systems that have been used in the past. This
 promoter/enhancer is the MSV-mMT1 system which comprises the promoter of
 the mouse metallothionein gene 1 (mMT1) flanked upstream with a mouse
 sarcoma virus (MSV) enhancer.
 A promoter of a metallothionein gene, particularly the combined MSV-mMT1
 promoter/enhancer system, can be operably linked to a gene of interest
 such as the human .beta.-interferon gene or the human erythropoietin gene.
 A vector comprising such an arrangement can give a high level of
 expression of the gene product in wild-type CHO cells. Therefore, the
 inventors have identified a new and unexpectedly powerful expression
 system suitable for use in mammalian cells, particularly wild type
 mammalian cells. Products, such as human .beta.-interferon and human
 erythropoietin, may be expressed with unexpected/novel biological
 properties such as higher bio-availability. Such properties may result in
 higher efficacy/additional utility for the product.
 It is therefore possible according to the invention to express genes such
 as a .beta.-interferon gene and others in large quantities in wild-type
 mammalian cells such as wild-type CHO cells and to do so in a stable
 manner, without the need for continuing selection and dependence on the
 CHO dhfr.sup.- -methotrexate selection system. The invention can be
 applied to a large variety of mammalian cells. In this way, it enables the
 expression of appropriate target genes with a glycosylation pattern and a
 cellular environment unique to the cell type used.
 A vector according to the invention is an expression vector. It comprises
 three sequences that are expressible in mammalian cells. Thus a vector of
 the invention comprises:

(i) a coding sequence comprising a gene of interest
 whose expression is desired, for example the
 human .beta.-interferon gene;
 (ii) a first selectable marker sequence comprising a
 metallothionein gene which confers resistance to
 heavy metal ions, such as cadmium, copper and
 zinc, on mammalian cells expressing the gene of
 interest; and
 (iii) a second selectable marker sequence comprising a
 neo gene which confers resistance to the
 antibiotic kanamycin upon transformed bacterial
 cells expressing the gene and resistance to the
 geneticin (antibiotic G418) upon mammalian cells
 expressing the gene.
 Each of these three sequences will typically be associated with other
 elements that control their expression. In relation to each sequence, the
 following elements are generally present, usually in a 5' to 3'
 arrangement: a promoter for directing expression of the sequence and
 optionally a regulator of the promoter, a translational start codon, the
 coding/marker sequence, a polyadenylation signal and a transcriptional
 terminator. Further, the coding sequence and/or either or both of the
 marker sequences may optionally be operably linked to an enhancer that
 increases the expression obtained under the control of the promoter.
 Suitable enhancers include the Rous Sarcoma Virus (RSV) enhancer and the
 Mouse Sarcoma Virus (MSV) enhancer.
 Further, a vector according to the invention will typically comprise one or
 more origins of replication, for example a bacterial origin of
 replication, such as the pBR322 origin, that allows replication in
 bacterial cells. Alternatively or additionally, one or more eukaryotic
 origins of replication may be included in the vector so that replication
 is possible in, for example yeast cells and/or mammalian cells.
 The vector may also comprise one or more introns or other non-coding
 sequences 3' or 5' to the coding sequence or to one or more of the marker
 sequences. Such non-coding sequences may be derived from any organism, or
 may be synthetic in nature. Thus, they may have any sequence. Such
 sequences may be included if they enhance or do not impair correct
 expression of the coding sequence or marker sequences.
 In vectors of the invention, the coding sequence and the marker sequences
 are each operably linked to a promoter capable of directing their
 expression in a mammalian cell. Optionally, one or more of these promoters
 may also be capable of directing expression in other cells, for example
 non-mammalian eukaryotic cells, such as yeast cells or insect cells and/or
 prokaryotic cells. "Operably linked" refers to a juxtaposition wherein the
 promoter and the coding/marker sequence are in a relationship permitting
 the coding/marker sequence to be expressed under the control of the
 promoter. Thus, there may be elements such as 5' non-coding sequence
 between the promoter and coding/marker sequence. Such sequences can be
 included in the construct if they enhance or do not impair the correct
 control of the coding/marker sequence by the promoter.
 Any promoter capable of enhancing expression in a mammalian cell in the
 presence of a heavy metal ion such as Cd.sup.2-, Cu.sup.2- and Zn.sup.2+
 may be operably linked to the coding sequence. A suitable promoter is a
 metallothionein gene promoter. The mouse metallothionein gene I (MMT1)
 promoter is preferred.
 Suitable promoter/enhancer combinations for the encoding sequence include
 the mTM1 promoter flanked upstream with MSV enhancer (MSV-mMT1) and the
 combination of the RSV enhancer and the MMTV promoter. MSV-mMT1 is
 preferred.
 Similarly, any promoter capable of enhancing expression in a mammalian cell
 in the presence of a heavy metal ion such as Cd.sup.2+, Cu.sup.2+ and
 Zn.sup.2+ may be operably linked to the metallothionein gene such as a
 human metallothionein gene. Preferably, the marker sequence gene is a
 human metallothionein gene, such as the human metallothionein gene IIA,
 which has its own promoter.
 The second selectable marker sequence is a neo gene. More than one type of
 this gene exists in nature: any specific neo gene can be used in a vector
 of the invention. One preferred neo gene is the E. coli neo gene.
 The promoter for the neo gene is capable of directing expression of the
 gene in a mammalian cell. Suitable promoters are the cytomegalovirus (CMV)
 early promoter, the SV40 promoter, the mouse mammary tumour virus
 promoter, the human elongation factor 1 .alpha.-P promoter
 (EF-1.alpha.-P), the SR.alpha. promoter and a metallothionein gene
 promoter such as mMT1. The promoter may also be capable of expressing the
 neo gene in bacteria such as E. coli in which a vector of the invention
 may be constructed.
 Whilst the protection against antibiotics conferred by the neo gene is
 qualitative in the sense that once expressed neo gene will confer
 antibiotic resistance on a cell, the protection against heavy metals
 conferred by the metallothionein gene is quantitative. The greater the
 level of expression of the metallothionein gene in a cell the greater the
 cell's resistance is to heavy metals. Thus, cells having a high copy
 number of metallothionein genes will be expected to have a high resistance
 to heavy metals.
 Therefore, cells including many copies of a vector of the invention have a
 higher resistance to heavy metals than cells comprising one or a few
 copies. Accordingly, it is possible to select for transfected cells having
 high copy numbers of a vector of the invention (and therefore high copy
 numbers of the coding sequence for a gene such as human .beta.-interferon)
 by progressively increasing the concentration of heavy metals to which the
 cells are exposed. Thus, cells having progressively higher copy numbers of
 the vector according to the invention are selected.
 Therefore, the combination of selectable markers found in the vectors of
 the invention allows a two stage selection process for transfected cells
 of interest. First, cells are exposed to geneticin (antiobiotic G418)
 which eliminates cells lacking the neo gene and therefore lacking the
 vector of the invention altogether. The neo gene serves no further
 function after this step.
 Second, selection is effected with progressively increasing levels of heavy
 metal ions, which selects cells having multiple copies of the vectors,
 especially cells having multiple copies integrated into their genomes. In
 this selection process, cells that survive high concentrations of heavy
 metal ions express metallothionein to a high degree, for example because
 they include a large number of vectors of the invention and/or because the
 vector or vectors that have integrated into their genome are in a
 chromosomal location that encourages strong expression.
 Any suitable heavy metal ions may be used. Thus, any heavy metal ion that
 is toxic to cells of the invention and to which an expressed
 metallothionein gene confers protection may be used. For example, zinc
 ions (Zn.sup.2-), copper ions (Cu.sup.2-) or preferably cadmium ions
 (Cd.sup.2+) may be used. Concentrations of a heavy metal ion of from 5 to
 100, indeed up to 200, .mu.M may be applied to effect selection. A
 concentration of from 130 to 170 .mu.M, preferably about 150 .mu.M
 Zn.sup.2-, is suitable.
 In order to effect selection using heavy metal ions, these ions may be
 provided as salts, in combination with any suitable counterion such as
 sulphate or chloride.
 Because selected cells are resistant to the toxicity of heavy metals which,
 as it happens, are inducers of the promoter for the coding sequence, the
 expression of the protein of interest can be maximised by heavy metal ions
 such as 130 to 170 .mu.M Zn.sup.2- which are inducers of the promoter.
 In addition to the neo and metallothionein genes, the vector may also
 contain one or more further selectable marker genes, for example an
 ampicillin resistance gene for the identification of bacterial
 transformants.
 In the vectors of the invention, the nucleic acid may be DNA or RNA,
 preferably DNA. The vectors may be expression vectors of any type. The
 vector must of course be compatible with the mammalian cell which it is
 going to transfect. The vector may be in linear or circular form. For
 example, the vector may be a plasmid vector, typically a DNA plasmid. A
 preferred plasmid vector is pMMTC (Example 2; FIG. 3).
 Those of skill in the art will be able to prepare suitable vectors starting
 with widely available vectors which will be modified by genetic
 engineering techniques such as those described by Sambrook et al
 (Molecular Cloning: A Laboratory Manual: 1999). So far as plasmid vectors
 are concerned, a suitable starting vector is the plasmid pRSN (Low et al
 (1991): JBC 266; 19710-19716), which is widely available. A further
 suitable plasmid starting vector is pBR322.
 Vectors of the invention may be able to effect integration of some or all
 of their nucleic acid sequence into a host cell genome or they may remain
 free in the host cell. Integrative vectors are preferred. This is because
 they give stable expression of coding sequences such as that of the human
 .beta.-interferon gene.
 The transfected mammalian cells may be BHK, COS, VERO, human fibroblastoid
 such as ClO, HeLa, or human lymphoblastoid cells or cells of a human
 tumour cell line. Preferably, however, the cells are CHO cells,
 particularly wild-type CHO cells.
 Desirably, transfected cells will have all or part of a vector of the
 invention integrated into their genomes. Such cells are preferred because
 they give stable expression of the coding sequence contained in the
 vector. Preferably, one or more copies of the entire vector will be
 integrated, with cells having multiple integrated copies of the vector,
 for example from 20 to 100 copies or more, being particularly preferred
 because these cells give a high stable level of expression of the coding
 sequence contained in the vector. However, cells having less than complete
 sections of vectors of the invention integrated into their genomes are
 also included within the invention if they are functionally equivalent to
 cells having the entire vector integrated into their genomes, in the sense
 that the integrated sections of the vector enable the cell to express the
 coding sequence and to be selected for by the use of heavy metals, as
 described above. Thus, cells exhibiting partial integration of vector of
 the invention are included in the invention if the integrated element or
 elements include the coding sequence operably linked to its associated
 promoter and the metallothionein marker sequence operably linked to its
 associated promoter.
 The cells may be transfected by any suitable method, such as the methods
 disclosed by Sambrook et al (Molecular cloning: A Laboratory Manual,
 1989). For example, vectors comprising nucleic acid sequences according to
 the invention may be packaged into infectious viral particles, such as
 retroviral particles. The vectors may also be introduced by
 electroporation, calcium phosphate precipitation or by contacting naked
 nucleic acid vectors with the cells in solution. Preferred methods of
 transfection include those described by Low et al (JBC 266; 19710-19716;
 1991).
 The invention also provides a process for producing proteins encoded by the
 coding sequence in a vector of the invention. Such processes comprise
 culturing cells transfected with a vector of the invention under
 conditions that allow expression of the coding sequence and recovering the
 thus produced protein. Preferred proteins that may be produced in this way
 include interferons, for example human interferons. .beta.-interferons are
 preferred and human .beta.-interferon is most preferred. Other proteins
 are interleukins (such as interleukin-12), human chorionic gonadotropin,
 growth factors, human growth hormone and human erythropoietin, cell
 membrane components, viral proteins and other proteins of biomedical
 relevance.
 The selected cells may be cultured under any suitable conditions known in
 the art and these conditions may vary depending on the cell type and the
 type of protein being produced. The promoter for the coding sequence can
 be a constitutive promoter so that the protein encoded by the coding
 sequence is expressed in the absence of a heavy metal ion. The cells may
 however be cultured in the presence of a heavy metal ion, particularly in
 an amount which is not toxic to the cells. That can lead to higher
 expression of the desired protein.
 The concentration of the heavy metal ion in the culture medium is typically
 from 100 to 200 .mu.M. Cells may therefore be cultured in the presence of
 from 100 to 200 .mu.M of a heavy metal ion selected from Cd.sup.2+,
 Cu.sup.2+ and Zn.sup.2+. for example from 130 to 170 .mu.M of the heavy
 metal ion. A useful concentration is about 150 .mu.M, particularly when
 the heavy metal ion is Zn.sup.2+. The use of Zn.sup.2+ has a beneficial
 effect on the yield of .beta.-interferon and erythropoietin production.
 Unexpectedly, it was observed that human .beta.-interferon production was
 increased two- to three-fold and human erythropoietin production was
 increased three- to five-fold.
 The protein that is produced may be recovered by any suitable means known
 in the art and the method of recovery may vary depending on the type of
 cells employed, the culture conditions and the type of protein being
 produced. Desirably, the protein produced will be purified after recovery.
 Substantially pure protein can thus be obtained.
 The present invention enables a novel human .beta.-interferon to be
 provided. This .beta.-interferon has a high degree of sialylation. Like
 natural human .beta.-interferon produced by primary diploid human
 fibroblasts, it is well glycosylated. However, it has a higher
 bioavailability than the natural .beta.-interferon or recombinant
 .beta.-interferon produced in E. coli (BETASERON).
 The higher bioavailability of the .beta.-interferon can be characterised.
 When 1.5.times.10.sup.6 International Units (I.U.) of the interferon is
 injected subcutaneously into the back of a rabbit of about 2 kg: (a)
 .gtoreq.128 I.U./ml of the interferon is detectable in the serum of the
 rabbit after 1 hour, and/or (b) .gtoreq.64 I.U./ml of the interferon is
 detectable in the serum of the rabbit after 5 hours.
 The maximum level of interferon is typically observed; after 1 hour.
 According to (a), therefore, 128 to 256 I.U./ml such as 140 to 190 I.U./ml
 of the interferon may be detectable in the rabbit serum after 1 hour.
 After 5 hours according to (b), .gtoreq.70 I.U./ml such as .gtoreq.80
 I.U./ml of the interferon may be detectable in the rabbit serum. Typically
 according to (b), an amount of interferon in the range of 64 to 128
 I.U./ml such as 80 to 110 I.U./ml can be detected.
 Additionally or alternatively, the interferon can be characterised by its
 specific activity. It can have a specific activity in the range of from
 4.8.times.10.sup.8 to 6.4.times.10.sup.8 I.U. per mg equivalent of bovine
 serum albumin protein. The specific activity may be from 5.times.10.sup.8
 to 6.times.10.sup.8, for example from 5.2.times.10.sup.8 to
 5.8.times.10.sup.8 such as from 5.3.times.10.sup.8 to 5.5.times.10.sup.8,
 I.U. per mg equivalent of bovine serum albumin protein.
 The specific activity can be referenced to a standard, in particular the
 Gb23-902-531 standard distributed by the Natl. Inst. Allergy and
 Infectious Disease, NIH, U.S.A. Specific activity is determined according
 to a modification of the method of Armstrong (1971) in which 0.2 .mu.g/ml
 of actinomycin D is included in the viral challenge and the viral-induced
 C.P.E. is read directly. The assay cells were MRC-5 fibroblasts.
 The .beta.-interferon may also be characterised by one or more of the
 following properties:
 1. The .beta.-interferon according to the present invention typically has
 an apparent molecular weight of 26,300 as determined by 15% sodium dodecyl
 sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
 2. When injected as a neat intravenous bolus into a rabbit, the half life
 of the interferon is typically in the range of from 12 to 15 min such as
 about 131/2 min. The bolus is injected into the rabbit ear vein and blood
 samples are withdrawn from the rabbit ear artery. Rabbit serum is assayed
 for the antiviral activity of the interferon according to the modification
 of the method of Armstrong (1971).
 3. The antiviral activity of the interferon in a human hepatoblastoma cell
 line (HepG2) is at least equal to and, typically, about 1.5 times the
 activity of natural .beta.-interferon from primary diploid human
 fibroblast cells. The interferon is also about 2.2 times more effective
 than betaseron in protecting Hep2 cells against a viral challenge.
 Antiviral activity is again determined according to the modified method of
 Armstrong (1971). Actinomycin D was omitted in the antiviral determination
 in HepG2 cells. The oligosaccharides associated with the .beta.-interferon
 of the invention may also characterise the .beta.-interferon. The
 .beta.-interferon carries oligosaccharides which can be characterised by
 one or more of the following features:
 1. Neutral (no acidic substituents): 5 to 15%, preferably about 10%.
 Acidic: 95 to 85%, preferably about 90%
 2. The total desialylated oligosaccharide pool is heterogeneous with at
 least six distinct structural components present in the pool.
 3. Matrix-Assisted Laser Desorption Ionisation--Time of Flight (MALDI-TOF)
 mass spectrometry and high resolution gel permeation chromatography data
 are summarised as follows:

Mass Compo- Calculated gu
 detected sition Mass equivalent
 1786.2 5Hex, 1782 11.1
 4HexNAc, 1
 2AB, Na
 1929.9 5Hex, 1dHex, 1928 12.2
 4HexNAc,
 1 2AB, Na
 2295.5 6Hex, 2293 14.5
 1dHex,
 5HexNAc
 1 2AB, Na
 2660.1 7Hex, 2658 17.6
 1dHex,
 6HexNAc
 1 2AB, Na
 3019.1 8Hex, 3023 20.7
 1dHex,
 7HexNAc,
 1 2AB, Na
 The carbohydrate moiety of the .beta.-interferon of the invention consists
 of bi-, tri- and tetra-antennary complex type N-linked oligosaccharides.
 These oligosaccharides contain repeating lactosamine(s). About 30 to 80%,
 for example 35 to 60% or 35 to 50%, of the oligosaccharides are
 bi-antennary oligosaccharides. About 15 to 65%, for example from 25 to 50%
 or 25 to 40%, of the oligosaccharides are tri-antennary oligosaccharides.
 About 5 to 55%, for example from IS to 45% or 20 to 40%, of the
 oligosaccharides are tetra-antennary oligosaccharides.
 The .beta.-interferon of the invention exhibits antiviral activity, cell
 growth regulatory activity and an ability to regulate the production of
 intracellular enzymes and other cell-produced substance. Accordingly, the
 .beta.-interferon may be used to treat various viral and oncologic
 diseases such as hepatitis B, hepatitis C, viral encephalitis, viral
 pneumonia, viral warts, AIDS/nasopharyngeal carcinoma, lung cancer,
 melanomas,CML renal cell carcinoma and brain tumours as well as diseases
 like multiple sclerosis, hemangiomas and cervical intraepithelial
 neoplasia.
 Pharmaceutical compositions that contain the .beta.-interferon of the
 invention as an active principal will normally be formulated with an
 appropriate pharmaceutically acceptable carrier or diluent depending upon
 the particular mode of administration being used. For instance, parenteral
 formulations are usually injectable fluids that use pharmaceutically and
 physiologically acceptable fluids such as physiological saline, balanced
 salt solutions, or the like as a vehicle. Oral formulations, on the other
 hand, may be solids, e.g. tablets or capsules, or liquid solutions or
 suspensions. The interferon of the invention will usually be formulated as
 a unit dosage form that contains from 10.sup.4 to 10.sup.9, more usually
 10.sup.6 to 10.sup.7, I.U. per dose.
 The interferon may be administered to humans in various manners such as
 orally, intravenously, intramuscularly, intraperitoneally, intranasally,
 intradermally, and subcutaneously. The particular mode of administration
 and dosage regimen will be selected by the attending physician taking into
 account the particulars of the patient, the disease and the disease state
 involved. For instance, viral infections are usually treated by daily or
 twice daily doses over a few days to a few weeks; whereas tumor or cancer
 treatment involves daily or multidaily doses over months or years.
 The interferon may be combined with other treatments and may be combined
 with or used in association with other chemotherapeutic or chemopreventive
 agents for providing therapy against viral infections, neoplasms, or other
 conditions against which it is effective. For instance, in the case of
 herpes virus keratitis treatment, therapy with interferon has been
 supplemented by thermocautery, debridement and trifluorothymidine therapy.
 The following Examples illustrate the invention.

EXAMPLE 1
 Identification of MSV-mMT1 as a powerful promoter for wild-type CHO cells
 The strengths of 5 promoter/enhancer systems were compared. These were:
 mouse metallothionein gene 1 promoter flanked upstream with mouse sarcoma
 virus enhancer (MSV-mMT1: mMT1 is described by Glanville et al (1981)
 Nature 292, 267-269; MSV is described by Dhar et al (1980) PNAS 77,
 3937-3941);
 the cytomegalovirus early promoter (CMV);
 RSV-SV40 (a fusion between the rous sarcoma virus [RSV] enhancer and SV40
 early promoter);
 RSV-mouse mammary tumour virus long terminal enhancer/promoter (RSV-MMTV);
 and
 SR-.alpha. promoter (Yutaka Takebe et al (1988) Mol. Cell. Biol. 8,
 466-472).
 For this comparison, an EcoRI-XhoI cDNA encoding full-length dipeptidyl
 peptidase IV (DPPIV) (Hong and Doyle (1988) J. Biol. Chem. 263,
 16892-16898) was cloned into the respective expression vectors so that
 DPPIV expression was under the control of MSV-mMT1, CMV, RSV-SV40,
 RSV-MMTV, or SR-.alpha., respectively.
 For MSV-mMT1, the DPPIV fragment was inserted into XhoI-NotI sites of pMMTN
 vector (FIG. 2); for CMV, the fragment was inserted into the EcoRI and
 XhoI sites of pXJ41neo vector (Zheng and Pallen (1992); Nature,
 359,336-339); for RSV-SV40, see Low et al (1991) J. Biol. Chem. 266,
 19710-19716); for RSV-MATV, the fragment was inserted into the NhoI-XhoI
 sites of pMAMneo vector (from Clontech: catalogue number 6104-1; described
 by Lee et al (1981): Nature 294, 228 and by Sardet et al (1989): J. cell
 56, 271); for SR-.alpha., the fragment was inserted into the XhoI-BamHI
 sites of pSRalpha/neo vector (Yutaka Takebe et al (1988) MCB 8, 466-472;
 Nilsson et al, J. Cell Biol. 120, 5-13, 1993).
 Each expression vector was transfected into CHO cells and stably
 transfected cells were pooled for each vector. The strength of each
 expression vector was then measured by the protein levels of DPPIV using
 immunoblot analysis, with the amount of DPPIV detected giving an
 indication of the strength of each expression system.
 Immunoblot analysis to detect DPPIV in these transfected cells was
 performed as described by Hong et al (1989) (Biochemistry 28, 8474-8479).
 Briefly, cells were washed with Tris-buffered saline (TBS) (20 mM Tris,
 pH7.2, 150 mM NaCl), and then extracted with 1% TRITON X-100 in TBS with 1
 mM PMSF. The extracts were cleared of cell debris by centrifugation. The
 protein concentration of the extracts was determined using a BSA kit
 (Pierce Chemical Co.).
 About 100 .mu.g of proteins extracted from respective transfected cells
 were resolved by SDS-PAGE and then analysed by immunoblot as previously
 (Hong et al (1989) Biochemistry 28, 8474-8479). The results shown in FIG.
 1 show that the MSV-mMT1 expression system (lane 2) is much stronger than
 the remaining widely used ones.
 EXAMPLE 2
 Description of plasmid pMMTC
 Based on the above, a powerful expression vector pMMTC was constructed
 using MSV-mMT1 to drive the expression of foreign genes in conjunction
 with two selection markers (the neo gene for transfected cells and the
 human metallothionein gene IIA for cells that have integrated multiple
 copies of the vector into their genomes).
 pMMTC (FIG. 3) is a mammalian cell expression vector. The gene to be
 expressed is cloned into XhoI and/or NotI sites so that the expression of
 the gene is driven by a control region (MSV-mMT1) that comprises the mouse
 sarcoma virus enhancer (MSV) and the mouse metallothionein gene 1
 promoter. The SV40 splicing region and the polyadenylation site serve to
 terminate the transcription and to ensure proper control of
 post-transcriptional events.
 The bacterial neo gene is flanked upstream with the SV40 origin of
 replication and the SV40 early promoter and downstream by the SV40
 splicing region and the polyadenylation site. This neo expression unit
 confers transfected mammalian cells resistance to geneticin (G418) and
 also confers upon transformed E. coli. resistance to kanamycin.
 The pBR322 origin of replication (Ori) serves as the origin for autonomous
 replication of the plasmid DNA in E. coli. The human metallothionein
 structural gene IIA, which confers resistance to heavy metal ions such as
 Cd.sup.2+ on mammalian cells, was used to select for mammalian
 transfectants that have integrated multiple copies of the plasmid.
 Construction of mammalian expression vector pMMTC
 Plasmid pRSN (Low et al., JBC 266;19710-19716, 1991) was cut with the
 restriction enzyme BamHI and resolved by agarose gel electrophoresis. A
 DNA fragment of about 2685 bp was gel-purified. This BamHI fragment
 contains the expression unit for the E. coli neo gene in both mammalian
 cells and E. coli.
 Plasmid pBPV (Pharmacia: product number 27-4390; see FIG. 4 and below for
 full description) was cut with restriction enzyme BamHI and then treated
 with calf intestinal alkaline phosphatase (CIAP). After being resolved by
 agarose gel electrophoresis, a BamHI fragment of about 4570 bp was gel
 purified. This 4570 bp fragment contains the pBR322 origin of replication
 in E. coli, the Ampicillin (Amp) resistance gene and an expression
 cassette composed of the mouse sarcoma virus enhancer and the mouse
 metallothionein gene 1 promoter followed by a multiple cloning site and
 the SV40 splicing junction and polyadenylation signal.
 This 4570 bp fragment was ligated to the above 2685 bp BamHI fragment. The
 resulting plasmid was named pMMTN (FIG. 2). A plasmid phMT (Karin et al
 (1982): Nature 299, 797-802) was cut with HindIII and blunt-ended with
 Klenow fragment of DNA polymerase I. A 3100 bp fragment, containing the
 human metallothionein gene II A, was gel-purified. Plasmid pMMTN was cut
 with ScaI (which is within the Ampicillin resistance gene), treated with
 CIAP, and then ligated with the 3100 bp fragment obtained from phMT.
 The product of this ligation was transformed into E. coli. Selection was
 performed for kanamycin resistance (conferred by the neo gene) and Amp
 sensitivity (due to the insertion of the 3100 bp fragment into the
 structural gene for Amp resistance). The final plasmid construct was
 confirmed by restriction enzyme digestion and was named pMMTC. pMMTC is
 about 10350 bp in length.
 GENEOLOGY: pBPV (12516 bp)
 (Nucleotide numbers refer to numbering in the reference)
 MSV enhancer (388 bp)
 Dhar. R. et al, Proc, Natl. Acad Sci. U.S.A. 77, 3937 (1980). Nuc 529-142
 BamHI/BglII linker CCGGATCTG
 5'-end of metallothionein promoter (295 bp) Nuc 1-295
 3'-end of metallothionein promoter (368 bp) Glanville, N. et al Nature 292
 267 (1981). Nuc 300-68
 Multiple cloning site and additional nucleotides from construction
 CTCGAGCCGCGGCCGCTTCGAGG (SEQ ID NO:1)
 SV40 small T-antigen splice (612 Patent Bulletin No.) and polyadenylation
 (235 Patent Bulletin No.) signals
 Buchman, A. R. et al DNA Tumor Viruses, Cold Spring Harbor Laboratory, pg
 799 (1980), Nuc 4713-4102 and 2772-2538
 BPV genome (7945 bp)
 Chen E. Y. et al Nature 299,529 (1982). Nuc 4451-7945 and 1-4450
 pML2: a derivative of pBR322 with a deletion between bases 1,095 and 2,485
 (2,632) bp)
 (1) Balbas. P., et al Gene 50.3 (1986).
 (2) Sarvor. N. et al Proc. Natl Acad. Sci U.S.A. 79,7147 (1982). Nuc
 376-1095, 2485-4363 and 1-33
 BglII/BamHI linker GAGATCCGG
 EXAMPLE 3
 Insertion of human .beta.-interferon expression DNA into pMMTC
 The .beta.-interferon coding sequence was retrieved from human genomic DNA
 by PCR with two oligonucleotides. The 5' oligo
 (GGGGTACCATGACCAACAAGTGTCTCCTC, SEQ ID NO:2) was modified in such a way
 that the sequence (CCACCATG) around the initiation ATG codon favours
 efficient translational initiation (Kozak (1984): NAR 12,857-872). The
 sequence of the 3' oligo was GGAATTCTTCAGTTTCGGAGGTAACCTGT (SEQ ID NO:3).
 This modified expression sequence for .beta.-interferon was inserted into
 the XhoI and NotI sites of pMMTC. The insertion and correct orientation
 was confirmed by restriction mapping, PCR or sequencing. The resulting
 plasmid was named pMMTC/IFN.beta..
 EXAMPLE 4
 Establishment of CHO cell clones that constitutively secrete high levels of
 functional human .beta.-interferon
 CHO cells were transfected with pMMTC/IFN.beta. as described (Low et al.,
 J. Biol. Chem. 266;19710-19716, 1991). Cells were selected in G418 (800
 .mu.g/ml) for 7-10 days to allow growth of stably transfected cells. The
 cells were then incubated in medium with 50-100 .mu.M Zn.sup.2- ions for
 24 to 48 hr to induce the expression of human metallothionein and then
 incubated in a medium with step-wise increasing concentration of Cd.sup.2+
 (final concentration 200 .mu.M). Individual colonies was cloned and
 expanded. The culture medium from the cloned cells accumulated
 .beta.-interferon to a concentration of 10.sup.6 I.U./ml or more and
 10.sup.6 I.U. or more of .beta.-interferon was secreted by 10.sup.6 cells
 in at most 24 hr.
 EXAMPLE 5
 Production of human .beta.-interferon in CHO cells
 Wild type Chinese hamster ovary cells CHO-K1 line (ATCC CCL-61) were
 propagated in Dulbecco's Minimum Essential Medium (DMEM) containing 10%
 fetal calf serum. Cells were grown at 37.degree. C. in an atmosphere of 5%
 carbon dioxide. These cells were transfected with the pMMTC/IFN.beta.
 plasmid to secrete constitutively high levels of functional human
 .beta.-interferon. Cells were selected as described in Example 4.
 During the selection with Cd.sup.2-, clones of transfected cells were
 measured for the antiviral activity of .beta.-interferon to show that they
 were constitutively secreting high levels of functional human
 .beta.-interferon. Antiviral activity was measured according to the method
 of Armstrong (Armstrong, Applied Microbiology 21, 723, 1971) modified by
 including 0.2 .mu.g ml of actinomycin D in the viral challenge and
 reacting the viral-induced C.P.E. directly. From these measurements,
 individual colonies were isolated and expanded. Indeed several lines were
 found to produce 10.sup.6 I.U./ml to 10.sup.7 I.U./ml of .beta.-interferon
 when grown in plastic roller bottles.
 One of these cell lines, GS38, was maintained in culture over 12 months to
 test its ability to maintain a consistent high level of .beta.-interferon
 production. The GS38 cell line was maintained in plastic culture flasks
 (80 cm.sup.2) in DMEM containing lot fetal calf serum, 100 .mu.g/ml
 penicillin, 100 .mu.g/ml streptomycin, 2.5 .mu.g/ml amphotericin and 150
 .mu.M zinc sulphate ("regular medium"). The seeding of a roller bottle
 (1700 cm.sup.2) was done by adding a culture flask (80 cm.sup.2) of GS38
 cells into one 1700 cm.sup.2 roller bottle and the cells were maintained
 in 200 ml of regular medium.
 The medium from the roller bottle was discarded on day 2 and day 4 and
 replenished with 200 ml of fresh regular medium each time. On day 6, the
 regular medium was discarded and the roller bottle was replenished with
 300 ml of serum-free DMEM medium which contained 2.5 mg/ml of human serum
 albumin containing the list of additional ingredients listed in Table 1
 ("serum-free medium").
 TABLE 1
 Component Conc.
 Penicillin 100 .mu.g/ml
 Streptomycin 100 .mu.g/ml
 Amphotericin B 2.5 .mu.g/ml
 ZnSO.sub.4 150 .mu.M
 EX-CYTE (trade mark)* 1:1000
 Transferrin 2.5-5.0 .mu.g/ml
 Insulin 5 .mu.g/ml
 *EX-CYTE is an aqueous liquid supplement from human serum sold by Bayer,
 Illinois, U.S.A..
 On day 7, the serum-free medium was discarded and replenished with another
 300 ml of serum-free medium. On day 8, the serum-free medium was again
 discarded and replenished with another 300 ml of serum-free medium. On day
 9, the serum-free medium (300 ml) was harvested and replenished by another
 300 ml of serum-free medium. This harvesting procedure was repeated daily
 for another 14 days.
 From each roller bottle, a total of about 4.2 liters of GS38-produced
 .beta.-interferon (or GS38-IFN.beta.) was harvested. From
 2.4.times.10.sup.6 to 3.6.times.10.sup.6 I.U. of .beta.-interferon was
 obtained per ml of crude harvest from GS38 cells. This is equivalent to
 1.35 mg to 2 mg of GS38-IFN.beta. per day from one roller bottle of GS38
 cells from about 5 mg to 6.7 mg per liter of GS38-IFN.beta. from 1 liter
 of crude harvest per day. The crude GS38-IFN.beta., when purified to
 homogeneity, had a specific activity of 5.37.times.10.sup.8 I.U./mg of
 protein (bovine serum albumin), standardized to the Gb23-902-531 standard
 (an NIH reference standard distributed by the Natl. Inst. Allergy and
 Infectious Diseases, NIH, U.S.A.).
 The harvest of crude GS38-IFN.beta. was pooled and subjected to
 purification by a combination of affinity and ion exchange column
 chromatography purification (Tan et al, J. Biol. Chem. 254, 8067-8073,
 1979; Edy et al, J. Biol. Chem. 252, 5934-5935, 1977; Knight et al PNAS
 U.S.A. 73, 520-523, 1976). Pure GS38-IFN.beta. was obtained with about
 70-80% recovery. The pure GS38-IFN.beta. when analysed was found to be
 homogenous according to the following criteria of homogeneity:
 A single molecular mass of an apparent molecular weight of 26,300 was
 observed on SDS-PAGE (15%) (FIG. 5a). This is similar to the molecular
 weight of a natural .beta.-interferon produced by primary human diploid
 foreskin fibroblasts after the superinduction procedure of Tan et al (1970
 and 1973) (see FIG. 5b). Note that the broad range molecular weight
 markers obtained from BIO-RAD were slightly different from the ones used
 as previously reported by ourselves and others. The identity of these
 g-interferons (GS38-IFN.beta. and human fibroblast-produced
 .beta.-interferon) were verified by Western Blot (FIG. 5b) to belong to a
 single average molecular mass of 26,300.
 When subjected to hplc (Hewlett Packard 1090) C18 column chromatography,
 the protein peak of the material corresponded directly with the antiviral
 activity of interferon.
 When subjected to amino acid sequencing, the material had the sequence of
 .beta.-interferon.
 The amount of GS38-IFN.beta. produced by GS38 cells over 12 months was
 found not to change much. The cells produced from 2.35 to
 3.6.times.10.sup.6 I.U./ml of GS38-IFN.beta. throughout that period.
 Five biological activities of GS38-IFN.beta. were assayed. The
 .beta.-interferon from primary human fibroblasts referred to below was
 produced from early to mid-passage primary human foreskin fibroblasts
 according to the superinduction method of Tan et al (1970) with additional
 priming of cells by 100 I.U. of .beta.-interferon about 16 hours before
 superinduction. The resulting .beta.-interferon was purified by affinity
 chromatography. The five activities which were assayed are:
 1. Antiviral activity of .beta.-interferon was assayed on either human MRC5
 fibroblasts or human hepatoblastoma cell line (HepG2) after the modified
 method of Armstrong (1971). Accordingly, the specific activity of
 GS38-IFN.beta. was 5.37.times.10.sup.8 I.U./mg protein as assayed in MRC5
 human fibroblasts and referenced to the NIH .beta.-interferon standard.
 The antiviral activity of GS38-IFN.beta. in HepG2 cells was at least equal
 to or 1.5 times more effective than natural .beta.-interferon from human
 fibroblast cells. GS38-IFN.beta. is also about 2.2 times more effective
 than BETASERON (recombinant human .beta.-interferon produced in E. coli)
 in protecting HepG2 cells against a viral VSV challenge.
 2. Cell growth inhibition assay of .beta.-interferon (Tan, Nature 260,
 141-143, 1976) on the human hepatoblastoma cells as described for primary
 human cells but applied to HepG2 cells was performed. However, the assay
 was performed in 2 cm.sup.2 wells containing 1 ml of regular medium and an
 initial HepG2 cell count of 3 to 5.times.10.sup.4 cells/well. Accordingly,
 GS38-IFN.beta. was as effective as natural primary human diploid
 fibroblast .beta.-interferon in inhibiting HepG2 cell growth as measured
 by this in vitro assay.
 3. Pharmacokinetics of subcutaneously injected .beta.-interferon was
 performed in rabbits. Purified .beta.-interferon from either GS38 or
 primary human fibroblasts, or BETASERON from E. coli, was separately
 reconstituted in 4 mg of human serum albumin in 1 ml of phosphate buffered
 saline (0.15M NaCl) pH 7.0 containing 20 mg trehalose. 0.7.times.10.sup.6
 or 1.5.times.10.sup.6 I.U. of an interferon was separately injected
 subcutaneously into the back of albino rabbits of a weight of about 1.5 kg
 and about 2 kg respectively.
 Whole blood (500 .mu.l) was withdrawn from the rabbits at 15 min., 30 min.,
 1 h, 2 h, 3 h, 4 h and 5 h. Serum from the drawn blood was then assayed
 for the antiviral activity of .beta.-interferon according to the modified
 method of Armstrong (1971). The results are presented in FIGS. 10 (a) and
 (b) showing that GS38-IFN.beta. has a higher bio-availability than
 .beta.-interferon produced from primary human fibroblasts and BETASERON.
 The maximum level of GS38-IFN.beta. (128-256 I.U./ml) occurred after 1
 hour, and significant levels of GS38-IFN.beta. (64-128 I.U./ml) were found
 for at least 5 hours in the serum of rabbits injected with
 1.5.times.10.sup.6 I.U. of GS38-IFN.beta.. This was unexpected. It is
 generally known that subcutaneous or intramuscular injection of human
 .beta.-interferon results in no or low serum levels of circulating human
 .beta.-interferon.
 4. Pharmacokinetics of a neat intravenous bolus of .beta.-interferon in
 rabbits was also performed. 1 ml of each kind of .beta.-interferon
 (GS38-IFN.beta., natural interferon produced from primary human
 fibroblasts and E. coli BETASERON) containing approximately equal amounts
 of .beta.-interferon (0.7.times.10.sup.6 I.U.) was injected into the
 rabbit ear vein. Blood (500 .mu.l) was withdrawn at 5 min, 10 min, 15 min,
 30 min, and 90 min. The serum was assayed for the antiviral activity of
 .beta.-interferon according to the modified method of Armstrong (1971).
 The result is shown in FIG. 11 where the half-life (t1/2) of
 GS38-IFN.beta. is 13.6 min, compared to primary human fibroblast-produced
 .beta.-interferon (t1/2=4.4 min) or BETASERON (t1/2=3.8). According to
 standard methodology, the total amount of .beta.-interferon injected was
 divided by the blood volume to estimate the starting concentration of
 .beta.-interferon at time zero. The blood volume was assumed to be 5% of
 the body weight of the rabbit. The time to decay to 50% of this starting
 concentration was t1/2.
 5. The dosage effect of injecting increasing amounts of GS38-IFN.beta. was
 investigated. Rabbits of about 2 kg were injected subcutaneously with
 increasing amounts of GS38-IFN.beta., in particular with
 1.2.times.10.sup.6 I.U., 2.5.times.10.sup.6 I.U. and 10.1.times.10.sup.6
 I.U. of GS38-IFN.beta.. The results in FIG. 12 show that increasing doses
 of GS38-IFN.beta. injected subcutaneously proportionally increase the
 measurable level of GS38-IFN.beta. in the serum of the injected rabbits.
 EXAMPLE 6
 Analysis of oligosaccharides associated with GS38-IFN.beta.
 GS38-IFN.beta. is a glycoprotein. The oligosaccharides associated with
 GS38-IFN.beta. were quantitatively released and recovered. The N and O
 linked glycans were released by treatment with anhydrous hydrazine. In
 this procedure, the protein backbone is converted into amino acid
 hydrazones. Intact reducing glycans are separated, recovered and labelled
 fluorimetrically with 2-aminobenzamide.
 More specifically, a sample of GS38-IFN.beta. (1-2 mg) was subjected to
 vigorous sample preparation, involving lyophilisation (&lt;50 mill Torr, &gt;24
 hours), introduced to a GLYCO PREP 1000 (an automated system for release
 and recovery of glycans from glycoproteins, (Oxford Glyco Systems, GB) and
 the oligosaccharides were released and recovered using the "N+O" program.
 The sample was fluorescently labelled by reductive amination with
 2-aminobenzamide.
 The sample was then applied to Whatman 3MM chromatography paper and
 subjected to ascending paper chromatography using 1-butanol/ethanol/water
 (4:1:1). The labelled sample remaining at the origin was subsequently
 eluted with water. This procedure leads to the quantitative (and
 non-selective) recovery of the total pool of oligosaccharides associated
 with the GS38-IFN.beta. sample as 2-aminobenzamide labelled
 oligosaccharide.
 The pool of labelled oligosaccharides was fractionated and analysed as
 follows:
 The labelled oligosaccharides were analysed for their charge distribution
 by hplc anion exchange chromatography. Accordingly, an aliquot of the
 total pool of 2-aminobenzamide-labelled oligosaccharides was subjected to
 hplc anion exchange chromatography on a GLYCO SEP C column (Oxford
 GlycoSystems, GB) using acetonitrile and ammonium acetate as eluent. The
 labelled glycans eluted from the column were detected using the
 fluorometer at .lambda.ex=356 mm .lambda.em=450mm. The resultant
 chromatogram is shown in FIG. 6.
 It is shown from FIG. 6 that the oligosaccharides associated with
 GS38-IFN.beta. consist of both neutral and acidic components. To determine
 the nature of the acidic substituents, an aliquot of the total pool of
 fluorescently labelled oligosaccharides was incubated with neuraminidase
 (derived from Arthrobacter ureafaciens). An aliquot was again subjected to
 GLYCO SEP C chromatography. The resultant chromatogram is shown in FIG. 7.
 No acidic oligosaccharides were detectable after incubation with the
 neuraminidase. Hence, the oligosaccharides that carry an acidic
 substituent do so only because they possess a covalently linked
 non-reducing terminal outer-arm sialic acid residue. The relative molar
 content of neutral and acidic oligosaccharides in the total pool was
 determined by integration of the chromatographic peaks (FIG. 6). The
 results are as follows:

Neutral 10% .+-. 0.8% (to 1 s.d.)
 Acidic 90% .+-. 0.6% (to 1 s.d.)
 s.d. = standard deviation
 2. Size-distribution of the total pool of deacidified oligosaccharides
 released from GS38-IFN.beta.
 An aliquot of the total pool of deacidified 2-aminobenzamide labelled
 oligosaccharides was subjected to high resolution gel permeation
 chromatography using the RAAM 2000 (Oxford Glyco Systems, GB). The
 resulting gel permeation chromatogram is shown in FIG. 8. As a note of
 explanation, the fluorescently labelled deacidified oligosaccharides were
 suspended in an aqueous solution of a partial acid hydrolysate of dextran,
 and applied to a RAAM 2000 (eluent of water, maintained at 55.degree. C.,
 constant flow 80 .mu.l/min over 10.6 hours). Detection was by an in-line
 fluorescence flow detector (to detect fluorescently labelled sample), and
 an in-line differential refractometer (to detect individual glucose
 oligomers).
 Numerical superscripts in FIG. 8 represent the elution position, of the
 non-fluorescent, co-applied, glucose oligomers in glucose units (gu), as
 detected simultaneously by refractive index. The hydrodynamic volume of
 individual 2-amino-benzamide labelled oligosaccharides is measured in
 terms of glucose units, as calculated by cubic spline interpolation
 between the two glucose oligomers immediately adjacent to the
 fluorescently labelled oligosaccharide.
 It is clear that at least 6 discrete oligosaccharide are identifiable
 within the dextran calibration range, and their effective hydrodynamic
 volumes are as follows:

20.7 gu 14.5%
 17.6 gu 23.4%
 14.5 gu 29.8%
 12.2 gu 26.4%
 11.1 gu 2.1%
 1.0 gu 3.8%
 Annotation of hydrodynamic volume is accurate to .+-.0.1 gu for all volumes
 .ltoreq.20 gu. The conjugation of the glycans with 2-aminobenzamide (2-AB)
 decreases the hydrodynamic volume of the glycans by a constant value. The
 hydrodynamic volume of the 2-AB labelled glycans (.lambda.f) is calculated
 from hydrodynamic volume of the unreduced glycans (.lambda.) using the
 following equation:
EQU .lambda.f=1.2.lambda.-1.96
 3. Molecular weight distribution of the de-acidified glycans released from
 GS38-IFN.beta.
 Since peaks were detected outside the dextran calibration range (FIG. 8)
 and particularly in the void volume, it was necessary to obtain a
 molecular weight distribution in order to establish what carbohydrate
 species were present. An aliquot of the de-acidified glycan pool was
 prepared on a matrix of 3.5-dihydroxybenzene. A Matrix-Assisted Laser
 Desorption Ionisation-Time of Flight (MALDI-TOF) mass spectrum was
 obtained in positive ion mode (i.e. molecular ion plus sodium). The
 following ions could be assigned to carbohydrates (FIG. 9).

Molecular Ion Na
 1929.9
 2292.5
 2660.1
 3019.1
 4. SUMMARY
 The Glycoprotein GS38-IFN.beta. carries oligosaccharides with the following
 structural characteristics:

(i) Neutral (no acidic substituents): 10% .+-. 0.8%
 Acidic: 90% .+-. 0.6%
 (ii) The total desialylated oligosaccharide pool is
 heterogeneous, with at least 6 distinct
 structural components present in the total pool.
 (iii) The MALDI-TOF mass spectrometry data and the RAAM
 2000 data can be summarised as follows:
 Mass Composi- Calculated gu
 detected tion Mass equivalent
 1786.2 5Hex, 1782 11.1
 4HexNAc 1
 2AB, Na
 1929.9 5Hex, 1dHex, 1928 12.2
 4HexNAc,
 1 2AB, Na
 2295.5 6Hex, 2293 14.5
 1dHex,
 5HexNAc
 1 2AB, Na
 2660.1 7Hex, 2658 17.6
 1dHex,
 6HexNAc
 1 2AB, Na
 3019.1 8Hex, 3023 20.7
 1dHex,
 7HexNAc,
 1 2AB, Na
 Hex = Hexose, dHex = deoxyHexose, HexNAc = N-Acetylhexosamine, 2AB =
 2-aminobenzamide, Na = sodium ion.
 NB. The peak which elutes at 1.0 gu will be included in the matrix in
 MALDI-TOF mass spectrum and is therefore not detected.
 EXAMPLE 7
 Expression of human erythropoietin in CHO cells using pMMTC
 cDNA encoding human erythropoietin (EPO) was derived by PCR with pfu
 polymerase using human kidney mRNA that had been reverse transcribed. The
 nucleotide sequence of the 5' and 3' PCR primers are as follows:
 5'PCR Primer:
 5'GTGGATCCGCCGCCACC/ATG/GGG/GTG/CAC/GAA/TGT/CCT/GCC/TG-3' (SEQ ID NO:4, the
 CCGCCGCCACC sequence (SEQ ID NO:5) before the ATG initiation Met codon was
 designed for optimal translation of the resulting mRNA); and
 3'PCR Primer:
 5'-GATCTAGACAGTTCTTGTCAATGAGGTTGAAG-3' (SEQ ID NO:6)
 The PCR product was gel-purified, cut with restriction enzymes BamHI and
 XbaI, and then ligated into pGEM-11Z plasmid that has been cut with BamHI
 and XbaI. After confirming the nucleotide sequence of the EPO coding
 region, the cDNA was retrieved from pGEM-11Z plasmid by cutting with XhoI
 and NotI. The EPO cDNA was gel-purified, and inserted into the XhoI-NotI
 sites of pMMTC, giving rise to a plasmid referred to pMMTC/EPO.
 Wild type CHO cells (CHO-K1) were transfected with pMMTC/EPO and initially
 selected with G-418 for 7 days and then with gradually increased
 concentrations of Cadmium (4, 8, 16, 32, 64 and 92 .mu.M). About a few
 thousand colonies were obtained after the initial G418 selection. When the
 Cadmium concentration was 64 .mu.M, about 100 colonies remained viable.
 Among these 100 colonies, 60 were individually isolated and expanded, and
 assayed for the levels of EPO in their culture media by Western blot,
 resulting in identification of 8 high expressing colonies (referred to as
 E15/1, E15/3, E15/8, E15/10, E15/13. E15/18, E15/26 and E15/30,
 respectively).
 The remaining colonies were further selected in media with 92 .mu.M Cadmium
 and several colonies remained viable after this selection, from which 6
 colonies (termed C5, C10, C11, C12, C14 and C15, respectively) were
 individually isolated and assayed for the levels of EPO expression. The
 levels of EPO secretion by the 8 high expression colonies after 64 .mu.M
 Cadmium selection and the 6 colonies after 92 .mu.M Cadmium selection were
 further compared by Western blot.
 Cells of the selected colonies were seeded onto 35 mm (in diameter) culture
 dishes. Upon confluency, 1 ml of culture medium was added to each of them
 and cultured for 24 hrs. The media were then harvested and 10 .mu.l of
 each, together with a control 50 ng of EPO (lane 15) were resolved by SDS
 PAGE and analyzed by Western blot using the Amersham ECL detection system.
 The Western blot B shown in FIG. 13.