5 Recombinant human growth hormone

5 Recombinant H u m a n Growth Hormone L. M. F R Y K L U N D J. R. B I E R I C H M. B. R A N K E

Methionyl human somatotropin, Somatonorm (Met-hGH), can be produced in the bacterium Escherichia coli K12 by the use of a recombinant plasmid. The modified plasmid phGH 407 was derived from the plasmid pBr 322 by the insertion of a gene for Met-hGH. The promotors chosen were for [3-galactosidase (lac) and tryptophan (trp); in fact double lac promotors were used (de Boer et al, 1980). E. coli K12 were transformed with the plasmid phGH 407. The recombinant bacteria can be fermented in a conventional way and the Met-hGH produced is secreted into the cytoplasm. Isolation of the hormone cannot be achieved without disrupting the cell wall and inner membrane. The resulting lysate is then subjected to purification procedures which do not differ in principle from methods employed to isolate naturally occurring human growth hormone, pituitary human somatotropin (hGH), from pituitary tissue. Once pure, the recombinant hormone was subjected to a battery of analytical assays designed to show the structural and functional properties of the hormone. Protein structure has four levels. Primary structure is the unique sequence of amino acids comprising it. Disulphide bridges link cysteine residues in the chain to each other. Secondary structure is the way the amino acid chain is arranged spatially. Tertiary structure describes the three-dimensional folding of the protein molecule to form an active structure. Quaternary structure describes whether the monomers combine to form aggregates, dimers, trimers and so on. Evidence of structure can also be obtained by comparing the behaviour of the hormone with antibodies and specific receptors to that of a defined preparation.

DETERMINATION OF THE PRIMARY STRUCTURE

The sequence of bases in the deoxyribonucleic acid (DNA) which comprise the gene for Met-hGH has been determined. The plasmid structure has Clinics in Endocrinology and Metabolism--Vol. 15, No. 3, August 1986

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L. M. F R Y K L U N D ET AL

also been shown to be stable over 60 generations, more than twice the normal fermentation number. Since the DNA structure is correct, by inference the primary sequence should also be. However, misreadings during transcription or translation can lead to the insertion of incorrect amino acids in the sequence and also lead to changes in the secondary, tertiary, and quaternary structure. Primary protein structure is determined by examining the amino acid composition and the sequence of the various peptides formed from the protein in question by enzymatic or chemical hydrolysis. The sequence of amino acids is determined by removing and identifying these one by one, by the chemical procedure known as Edman degradation. There is a limit, usually between 20 and 40 residues, to the number of amino acids which can be sequentially removed and identified, hence the need to form and identify smaller peptides. Different hydrolytic procedures are used to form peptides of differing length which overlap each other. Primary structure cannot usually be determined in one experiment, but is generally deduced from a sequential series of overlapping peptides.

Amino acid composition The amino acid composition for five consecutive batches of Somatonorm compared to hGH as Crescormon is shown in Table 1. The ratios between the amino acids are in agreement with the theoretical values and are comparable between the batches evaluated.

Amino terminal sequence The determination of the amino terminal sequence of Somatonorm was determined for 50 residues by Edman degradation. Quantitative yields of the phenylthiohydantoin amino acids have been determined by integration of the elution curve, using the extinction coefficient for the respective amino acid extrapolated with an internal standard (dansylphenylalanine). The sequence was identical with that of hGH with the exception of an additional amino-terminal methionine residue immediately preceding phenylalanine at residue 1. The additional methionine at the amino terminal is henceforth assigned the position 0.

Carboxyl terminal sequence The sequence of the C-terminal was determined by carboxypeptidase A digestion followed by quantitative amino acid analysis without prior hydrolysis. The C-terminal was correctly phenylalanine and, as expected, no further amino acids were removed, owing to the proximity of the disulphide bridge at residue 189 (Figure 1).

Tryptic peptide mapping The enzyme trypsin hydrolyses peptide bonds carboxyl to the basic amino acids arginine and lysine (Figure 1).

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Table 1. Complete amino acid composition of Somatonorm and Crescormon. Somatonorm

Crescormon

Batch number:

8 0 2 8 4 81651 81943 82412 82953

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Tryptophan Arginine

20.18 20.05 20.02 10.33 10.14 10.16 17.28 17.35 17.54 27.21 27.19 26.87 8.89 8.65 8.57 8.55 7.61 7.53 7.56 3.48 7.53 7.44 7.36 4.05 7.75 7.76 7.87 24.34 24.65 24.42 7.98 8.04 8.06 13.29 13.32 13.42 3.14 3.07 3.13 9.28 9.19 9.27 0.89 0.97 0.99 10.57 10.71 10.87

8.71 3.56 3.80

9.48 3.45 4.14

20.08 9.72 17.61 26.65 10.14 9.07 7.49 4.52 6.63 3.43 6.82 23.60 8.05 12.71 3.05 9.2 --$ 11.17

Theoretical pit-hGH 20 10 18 27 8 8 7 4 7 3t 8 26 8 13 3 9 1 11

?Three Met in pit-hGH, four in Met-hGH. :)Not analysed. The amino acid analyses were performed by the Central Amino Acid Analysis Laboratory, Institute of Biochemistry, Uppsala. The analyser was a Durrum D-500, Dioniex Corp. Hydrolysis was done in 6 M HC1 and amino acid analysis performed after 24 h and 72 h hydrolysis, respectively, all according to the standard procedure as described by the Durrum D-500 manufacturer. Oxidized samples were used for cysteine as cysteic acid, methionine as methionine sulphone and proline analysis as described by Moore (1963). Tryptophan analysis was performed as described by Penke et al (1974). Values are expressed as the average of 24 h and 72 h hydrolysis.

T r y p t i c p e p t i d e s were p r e p a r e d f r o m M e t - h G H a n d the e l u t i o n p a t t e r n after H P L C (high p e r f o r m a n c e liquid c h r o m a t o g r a p h y ) was identical to that f o u n d for h G H with the e x c e p t i o n of o n e peak. A m i n o acid analysis of p e a k 2 s h o w e d that this p e p t i d e c o r r e s p o n d e d to the a m i n o - t e r m i n a l p e p t i d e of M e t - h G H , which in the case of S o m a t o n o r m has a n a d d i t i o n a l m e t h i o n i n e residue. T h e extra r e s i d u e increases the h y d r o p h o b i c i t y of the p e p t i d e a n d t h e r e b y delays its e l u t i o n f r o m the c o l u m n . T h e m e t h o d of f o r m i n g tryptic p e p t i d e s f r o m the n a t i v e p r o t e i n , i.e. with d i s u l p h i d e residues intact, is r e p r o d u c i b l e a n d p r o v i d e s a simple m e a n s of showing i d e n t i t y of s t r u c t u r e in r o u t i n e batch specification.

Alignment of the peptides M e t - h G H c o n t a i n s t h r e e m e t h i o n i n e residues which are dispersed irregularly t h r o u g h o u t the m o l e c u l e in a d d i t i o n to that at the a m i n o t e r m i n a l . P e p t i d e b o n d s carboxyl to the m e t h i o n i n e can be split with c y a n o g e n

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80

30

~0

55

165 180

~

17! J

~-COOH Figure 1. Primary structure of methionyl human growth hormone (Somatonorm). Numbering as for pituitary human growth hormone, methionine at the amino terminal is given the number 0. Trypsin cleaves at the carboxyl side of lysine (Lys) and arginine (Arg) residues. bromide resulting in four peptides. The theoretical peptides so formed are as follows: 1-14, 15-125, 126-170, and 171-191. The peptides can be separated by gel filtration and HPLC. Sequence determination has been performed on three of the cyanogen bromide fragments, namely peptides 15-125, 126-170, and 171-191. The results show unambiguously that the sequence is as expected and the isolated tryptic peptides are found in the correct position in the molecule. The sum of the linear sequence of amino acids can be used to deduce the primary structure if all amino acids can be accounted for. However, this assumes that the appropriate integral value has been chosen for each amino acid and this is in turn based on the molecular weight of the respective peptides.

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Molecular weight determination

Molecular weight can be determined by examining the electrophoretic mobility on polyacrylamide gels in ionic detergents; the most generally used is sodium dodecyl sulphate (SDS). The migration position of Met-hGH has been compared with hGH and again found to be identical, as shown in Figure 2. It is therefore correct to assume a molecular weight of 22 000 for the protein. Figure 2a and b also show a comparison of the different results achieved with Coomassie blue and silver stain, respectively. Electrophoretic mobility can also be used for determining the net charge of the protein, since this is the sum of positive and negative charges. Deamidation of asparagine and glutamine is known to occur in hGH. Figure 3 shows an electrophoretic comparison of Met-hGH with hGH in the absence of denaturing media (the detergent, SDS). Two charged components are observed. The components are designated b, for the main band, and c, which is presumed to correspond to a product deamidated at asparagine residue 152 and shows a concomitant increase in anodic mobility. The electrophoretic behaviour of Met-hGH is therefore comparable to that of hGH.

DETERMINATION OF THE SECONDARY STRUCTURE

The sequence of amino acids confers certain folding properties on the protein molecule. It is conceivable that the immediate environment near the translation site and the ribosomes affects the mode of folding and consequently determines which cysteine residues interact to form which disulphide bridges. The positioning of the disulphide bridges in Met-hGH can be determined by isolating, using HPLC, the tryptic peptides derived from enzyme digestion of the native protein. The amino acid compositions of the disulphide-containing tryptic peptides are as expected from the structure proposed for hGH. Therefore, it is correct to assume that the disulphide bridges are between residues 53 and 165, and 182 and 189.

DETERMINATION OF THE TERTIARY STRUCTURE

If a protein can be crystallized, then the x-ray diffraction pattern can be computed to show the three-dimensional spatial array of the amino acids, with c~-helix, [3-sheet and the way the chain folds back on itself. It is therefore very easy to compare the structure of two supposedly identical molecules directly. hGH is rather hydrophobic and no one has yet succeeded in making usable crystals. An indirect method must therefore be used, for example fluorescence and ultraviolet (UV) spectroscopy (Larhammar et al, 1985).

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C694

(a)

iilili ¸ i ¸

(b) Figure 2. (a, b) SDS-PAGE. Comparison of five consecutive batches of Somatonorm with Somatonorm and Crescormon references. (a) Stained with Coomassie blue R-250; (b) silver stain.

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RECOMBINANT HUMAN GROWTH HORMONE

No differences in the fluorescence and UV spectra for Met-hGH and hG H are observed. A further possibility for examining conformation is to use the technique of circular dichroism (CD) to study the solution behaviour of Met-hGH compared with the h G H at a variety of pH values. As shown in considerable detail by Larhammar et al, no difference was observed between Met-hGH and hGH. However, some change is seen, relating to the deamidation of the asparagine residue at position 152, between the major b and the c (isohormone) form, indicating that a change in the region of tryptophan 86 has occurred. Component c from Met-hGH has an identical CD spectrum to component c from hGH. No evidence of structural changes effected by the N-terminal methionine residue in Met-hGH is found.

D E T E R M I N A T I O N OF THE Q U A T E R N A R Y S T R U C T U R E

h G H and Met-hGH can dimerize or polymerize, for example during lyophilization; polymer and dimer are present in Met-hGH in very low amounts. Some of the structural data have been described earlier (Fryklund et al, 1982).

CONFORMATIONAL INFORMATION AND R E C E P T O R - B I N D I N G ACTIVITY

FROM

IMMUNOLOGICAL

Receptor binding and immunological properties are other sensitive ways of determining whether the conformation is correct or not.

Immunological activity The reactivity of a protein with one or several antibodies can be used both as a means of identifying it and also as a possible way of assessing its conformational properties. Polyclonal antibodies used for the quantitative evaluation by radioimmunoassay (RIA) of h G H could not distinguish between hG H and Met-hGH. Twelve different monoclonal antibodies to pituitary growth

Figure 2: Samples from the left MW markers (Pharmacia LMW lot 3071): Crescormon 80 609 80 438 Somatonorm 87 478 87 238 88 234 80 753 80 335 Reference Somatonorm 80 524 As lane 1 (MW markers)

MW markers in descending order: Phosphorylase Serum albumin Ovalbumin Carbonic anhydrase Trypsin inhibitor Lactalbumin

94 000 67 000 43 000 30 000 20 000 14 000

5]8

L . M . FRYKLUND ET AL

Figure 3. Comparison of five consecutive batches of Somatonorm by PAGE to Somatonorm and Crescormon references. PAGE is performed in gel T=10%, C=3%.

Samples from left to right Heat treated Crescormon (hGH b, c, d components) Crescormon batches 80609, 80438 Somatonorm 80335, 80753, 88234, 87238, 87478, 80524 (ref.) as lane 1 Densitometric analysis shows 5% c in the Somatonorm batches.

hormone were also used to assess Met-hGH (J6nsdottir et al, 1983). Although these antibodies were capable of distinguishing between hGH, human chorionic somatomammotropin and prolactin, and also the reduced and carboxymethylated derivative of h G H , they were unable to distinguish between the h G H and Met-hGH. Likewise, Retegui et al (1984) studied the cross-reaction of h G H and Met-hGH with five different monoclonal

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519

antibodies and were again unable to distinguish between the two molecules. All these immunological data therefore indicate that Met-hGH is recognized as being growth hormone.

Receptor cross-reactivity Similarly, Retegui et al (1984) have studied the binding properties of Met-hGH compared to hGH on the pregnant rabbit liver membrane and human IM-9 lymphocytes. The displacement curves were totally congruent. Relevance of the amino-terminal methionine

Since antibodies and immunological methods can be very sensitive in revealing small variations in conformation, we devoted a lot of time and effort to studying whether our in-house RIA and electroimmunoassay, at least 19 monoclonal antibodies, and a method (nephelometry), which measures antibody-antigen complex, could discern any differences. Again we drew a blank. With the analytical methods available a few years ago, it looked as if Met-hGH was superior to pituitary hGH (pit-hGH) both with regards to purity (extraneous proteins) and hGH components (isohormones, dimer, polymer). We were therefore taken by surprise when patients treated seemed to be responding with anti-hGH antibody formation. This, in spite of the fact that the main culprits implicated in antibody formation with pituitary preparations, dimer and polymer, were very low. Of course it could always be argued that some immunosuppressive effect was at work with the pituitary hormone, but that was not really a solution to the problem. The obvious suspect was naturally the amino-terminal methionine, although our exhaustive characterization studies implied that any differences must be very subtle. We therefore addressed the question of whether Met-hGH had different antigenic determinants to pit-hGH, in two ways: (1) by studying the properties of antisera induced in rabbits, and (2) by doing the same studies using patient sera of the highest titre. In the first instance, we used specific immunosorption of antisera on hormone coupled to Sepharose, and studied the ability of the following substances in displacing labelled hormone in a simple binding assay from the immunoadsorbed, hopefully specific antisera from Met-hGH and hGH respectively: intact hormone and its analogue, dimer from Somatonorm, formed by harsh treatment, polymer, a 16-residue peptide (= aminoterminal sequence of Met-hGH), a 15-residue peptide (= amino-terminal sequence of pit-hGH), other tryptic peptides, E. coli proteins similar in properties to hGH. The inclusion of the last was prompted by the description of the presence of immunoreactive insulin activity in E. coli. We were able to show virtually nothing by this series of experiments: a very small amount (compared to total) of specific antibodies to pit-hGH was found (could be contaminating pituitary proteins other than hGH). In

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L.M. FRYKLUNDET AL

the case of Met-hGH, we found an infinitesimal amount of Met-hGHspecific antibody. The criticism that could be directed towards these results is that rabbits might respond differently since hGH is a foreign protein. The studies were therefore repeated, using patient sera. We had extremely small amounts available and had to use a method to amplify the response. Human IgG was coupled to Sepharose and used in a solid phase assay mixing Sepharose-immunosorbent patient sera and label as above. The idea was to study competition for the human antibody by the various substances added with tracer. Nothing was seen. Dimers and polymers were in fact less effective than the monomers; tryptic peptides were without effect. The amino-terminal peptides were very inefficient, and E. coli had no effect. This set of experiments corroborated our experimental and clinical findings that the patient antibodies were directed against the same antigenic sites in both Met-hGH and pit-hGH. Some other factor was presumably involved. Overloading of electrophoretic gels stained with silver showed that protein bands appeared which were not related to hGH, and, when the gels were analysed by Western blotting techniques using E. coli antisera and patient antisera, bands were picked up in both rabbit and patient sera. Analysis of fractions during purification showed that some of these components disappeared very late in the purification process. Could there be some sort of adjuvant effect involved? The most notorious adjuvant known from E. coli is LPS (lipopolysaccharide). We had, however, been able to demonstrate that LPS was not present and did not seem to adsorb to hGH by studying the behaviour of labelled LPS during purification. Glutathione and the possibility that mixed disulphides were formed could also be discounted. DNA was only present in picogram quantities (pg/mg hGH). We were able to develop a highly sensitive immunoradiometric assay (IRMA) method for measuring parts per million of E. coli proteins, and in consequence to improve and optimize the purification steps. The increased purity was closely paralleled by lowering of titres and frequency, and diminution of binding capacity to almost unmeasurable levels. Our working hypothesis would therefore seem to be correct, that E. coli protein in quantities less than one part per hundred thousand could turn the highly purified Met-hGH into a weak antigen in the human. These antibodies have not been sufficiently 'good' to attenuate growth; in fact the clinical efficacy of Met-hGH is excellent and the product Somatonorm efficacious and safe. IMPURITIES IN S O M A T O N O R M

Met-hGH (Somatonorm), unlike its pituitary counterpart, hGH (Crescormon), is not contaminated by other pituitary hormones. Common contaminants for both proteins are bacterial pyrogens and aggregates. Contaminants unique to Met-hGH are bacterial proteins, DNA and

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tetracycline from the culture medium. Tetracycline is a necessary constituent of the fermentation medium. The contaminants causing the greatest problem are E. coli proteins (ECP) for two reasons: firstly a suitable population of proteins must be prepared in order to identify the ECP, and secondly an analytical method with a sensitivity of parts per million is necessary. There is finally a possibility that ECP can cross-react with M e t - h G H and distort assay results. D e s c r i p t i o n o f the p u r i f i c a t i o n p r o c e s s

The process has been designed to remove, in as few steps as possible, contaminating substances from the M e t - h G H , while still maintaining the biological activity of the product. During the development of Somatonorm, we became aware that ECP were not being removed in an entirely satisfactory way. Employment of sensitive analysis techniques allowed us to reduce this, in two stages, down to less than 10 parts per million. The three products that have been used for clinical evaluation are designated SI, SII and SIII (Somatonorm), respectively, and the minor process differences are depicted in Table 2. Table 2. Isolation of Somatonorm.

Host fermentation strain Recovery Purification

SI

SII

SIII (Somatonorm)

E. coli K12 RV 308 Live cell harvest Ion exchange (twice) Gel filtration (once)

E. coli K~2 RV 308 Live cell harvest Ion exchange (thrice) Gel filtration (once) -Glycinephosphate <200

E. coli K12 RV 308 Live cell harvest Ion exchange (thrice) Gel filtration (once) precipitation Glycinephosphate <10

--

Excipient ECP (ppm/vial)

Glycine phosphate >200

Production of antisera towards E C P Antisera were produced in rabbits using an ECP preparation prepared from E. coli not containing the recombinant plasmid. The preparation was purified by ion exchange and gel filtration to give a pool of E C P similar in size and charge to M e t - h G H . Development of a specific immunoradiometric assay for E C P ( I R M A / E C P ) The antiserum developed towards ECP could then be used in an immunoradiometric assay to measure ECP content. Analysis of the ECP preparation by polyacrylamide gel electrophoresis ( P A G E ) with SDS as detergent showed a large number of bands. The gels

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were blotted by Western blotting using anti-ECP (immunosorbed on ECP-Sepharose), and good correlation to the number of bands was observed. The ECP lev,A could be reduced by minor modifications of the purification process using the IRMA to optimize removal of ECP during each isolation step. The preparations prepared in this way are defined as SI and SII, respectively (see Table 2). Somatonorm (SIII) describes material which has an ECP content of less than 10 ng/vial. Determination of the DNA content

Foreign DNA arising from the E. coli host could be present in Met-hGH and be introduced into the human patient. Since the purification process is aimed at isolating Met-hGH with a molecular weight (MW) of 22 000, the contaminating DNA would be in that range. The final gel filtration step should ensure removal of DNA except in that MW range. Anomalous behaviour is also to be expected from DNA since it is cylindrical in shape and would probably be excluded from the gel filtration matrix faster than its protein counterpart with the same MW. The purification process has however involved separation not only according to MW but also according to charge by the use of three ion exchange steps. These steps have been developed varying both pH and ionic strength which leads to an optimal removal of components differing in isoelectric point (pI), DNA having a net charge in the low acidic range would therefore be retained during ion exchange and be eluted long after Met-hGH which has a higher pI at around pH 5.0. The method used for measuring DNA is dot hybridization. Detection of non-specific E. coIi sequences can be accomplished at a level of sensitivity of 8 pg/IU. The principle of the method is as follows: sample DNA is extracted and concentrated by phenol/chloroform extraction, followed by an ethanol precipitation. The samples are serially diluted and applied to nitrocellulose filters which are dried and treated with excess denatured salmon sperm DNA to saturate any DNA sites remaining. DNA probe from E. coli with 32p-labelled phosphate is added and incubated at 42°C for 3-48 hours. The completed hybridization filters are then applied to x-ray film. Hybridized DNA exposes the film as a result of the 32p-labelled probe. The size and intensity of the exposed spots are proportional to the amount of hybridized probe present and therefore to the amount of E. coli DNA present in the sample or standard. Quantitation is accomplished by determining a visual standard end-point and comparing this directly to sample end-points. Dilution factors are applied to yield corrected values. Clinical data have shown how essential it is to have a high purity of the recombinant Met-hGH. Dimers and polymers have not been active as in pit-hGH in promoting antibody formation and have, in fact, from the start been consistently low.

RECOMBINANTHUMAN GROWTH HORMONE

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SUMMARY OF PHARMACOLOGICAL DATA Pharmaeodynamics

Somatotropin is an anabolic hormone exerting a broad spectrum of effects in vivo and in vitro (for review see Kostyo and Reagan, 1976): stimulation of somatic growth after birth; insulin-like effects in vitro in isolated tissues or cells, and in vivo; diabetogenic effects; lipolytic effects; lactogenic effects; refractoriness; feminizing activity. The binding of the hormone to its receptor and the subsequent stimulation of metabolic pathways are fundamentally dependent on the tertiary structure of the molecule. Small changes in the structure in the binding regions of the molecules could cause a marked impairment of the binding to receptors or antibodies. In all the different binding studies performed, using 125I-labelled human somatotropin as radioligand and either product for displacement, Somatonorm and Crescormon show equivalent potency (IM-9 lymphocytes: half-maximal binding at - 1 0 ng/ml; rat liver membranes: half-maximal binding at 20-25 ng/ml; human adipocytes: half-maximal binding at 20-25 ng GH/ml) (Retegui et al, 1984). Down-regulation of receptors on IM-9 lymphocytes, measured after a preincubation of the cells with somatotropin followed by a binding assay as above, was seen at 10 -l° mol/1 of either product (Retegui et al, 1984).

Primary effects The major effect of somatotropin is the stimulation of somatic growth. This can be measured in a number of different ways, the most common being the weight gain assay. Other methods are measurements of longitudinal bone growth and of uptake of radioactive sulphate into cartilage proteoglycans. Comparison between Met-hGH (Somatonorm) and hGH (Crescormon) has been performed in studies concerning somatic growth in GH-deficient animals (Skottner et al, 1981). Parameters such as total body weight gain, stimulation of longitudinal bone growth and indirect cartilage growth, measured as uptake of radioactive labelled sulphate, have been studied. However, the results from bioassays conclude that the two products are equal in growth-promoting effect (dose range 83-1200 mlU/kg).

Other pharmacodynamic effects The biological properties of hGH in animals and humans have been extensively documented in the literature since the hormone has been in clinical use for more than two decades. The binding to receptors of Somatonorm and Crescormon in a number of tissues of human or animal origin has been compared. A variety of different biological responses and postreceptor events have been measured (Tables 3 and 4).

524

L. M. FRYKLUND ET AL Table 3. Biological responses to hGH.

Test procedure

References

Weight gain, longitudinal bone growth and indirect cartilage growth in hypophysectomizedrats.

Skottner et al (1981)

Binding to specificcell surface receptors (IM-9 lymphocytes,rat liver membranes) and monoclonalantibodies.

Retegui et al (1984) Jonsdottir et al (1983)

Binding to adipocytes,insulin-like activityand refractorinesson adipose tissue of hypophysectomizedrats.

Unpublished data

Acute hypoglycaemicaction and effects on membrane transport in vivo

Unpublished data

Insulin-like activityand lipolyticactivityon adipose tissue of hypophysectomizedrats.

Goodman (1984)

Conclusion

In conclusion, an extensive pharmacological comparison of the biological effects of Somatonorm and Crescormon has been carried out in different in vivo and in vitro assays. These studies show identical biological effects of the two products (Table 4). No difference in the pharmacokinetic properties between the two products has been possible to detect. The time for peak serum levels of h G H after subcutaneous administration of both Somatonorm and Crescormon is similar. It is also shown that both products show equal patterns of elimination after intravenous injection. The only difference between the two products, namely the additional methionine at the N-terminal in Somatonorm, does not interfere in any of the assays referred to. G E N E R A L TOXICITY

The general toxicity of M e t - h G H and pituitary derived human growth hormone, Crescormon, has been compared in rats and dogs, and the mutagenic potential of both growth hormone preparations has been studied in Ames' bacterial test and also in vivo in bone marrow cells from hamster by analysis for chromosomal aberrations and by chromosomal analysis of sister chromatid exchanges. The results have been described elsewhere (Flodh et al, 1982). When interpreting and evaluating the animal data for the safety evaluation of biosynthetic M e t - h G H for human use, it is important to emphasize that the animal studies were designed according to generally accepted principles for toxicological studies, recommending daily dosing and the use Of dose levels high enough to induce observable biological effects. The dose levels were selected on the basis of the results from dose-range finding studies in rats and dogs. Although it was possible to carry out these studies successfully, it should be emphasized that the dose levels were extremely high compared to those clinically used in man. No mutagenic potential was demonstrated for either Crescormon or

525

RECOMBINANT HUMAN GROWTH HORMONE Table 4. Pharmacological studies performed on Somatonorm and Crescormon. Diabetogenic activity in obese mice and insulin-like activityin adipose tissue of hypophysectomizedrats

Unpublished data

Lactogeniceffects

Un ~ublished data

Feminization of prolactin-binding sites in rats

Un ~ublished data

Effect on the cardiovascularsystem in anaesthetized cats

Un ~ublished data

Half-life, intravenous

Un ~ublished data

Disappearance time; Cm,×,rats

Un ~ublished data

Disappearance time, C.... dogs

Un 9ublished data

biosynthetic M e t - h G H in a bacterial test designed to demonstrate induction of chromosomal damage in the intact animal (chromosomal aberrations and induction of sister chromatid exchanges in bone marrow cells of Chinese hamster). General conclusion

Results obtained in the toxicological studies summarized above provide sufficient safety for the use of M e t - h G H in therapeutic dose levels in man. Effects observed at high doses were similar to those observed with pit-hGH, and were related to the potent biological effect of the hormones.

CLINICAL STUDIES In 1981, KabiVitrum A B , Stockholm, asked us (JRB and MR) in the auxological group in Tiibingen whether we would be prepared to carry out a comprehensive collaborative study on the M e t - h G H preparation. The reason for this request was that we had successfully introduced pit-hGH, Crescormon, produced by KabiVitrum 10 years previously, and had published our experiences two years previously (Ranke et al, 1979). The multicentre study comprised a total of 13 university clinics in West G e r m a n y and Austria (Table 5). One report was presented in Baltimore in N o v e m b e r 1983 and one in Stockholm (Bierich, 1983). Since then the whole study has been finalized and we can report on the definitive results. Table 5. Somatonorm studies I to III: participants,

JR Bierich & MB Ranke, Tiibingen P Beyer, Berlin P Bittner, Giessen M Borkenstein, Graz J Br~imswig, Mfinster O Butenandt, Munich U Schweikert, Bonn

D Sch6nberg & U Heinrich, Heidelberg W Sippell, Kiel N Stahnke, Hamburg H Stolecke, Essen P Stubbe, G6ttingen R Ziegler, Heidelberg

526

L. M. FRYKLUNDET AL

All available preclinical, chemical, pharmacological and toxicological evidence indicated that the preparation was safe. Short term administration to human volunteers showed good compatibility. Permission was therefore granted by the German authorities to start the study. In order to have as homogeneous a group as possible, we restricted the inclusion criteria to children with pituitary dwarfism, in particular to patients with the idiopathic form. Altogether, we have performed three studies which differed with regards to which Met-hGH preparation was used. In study I, preparations were used which contained between 1400 and 190 ng ECP per ampoule (SI); in study II, ampoules contained 170-30 ng (SII), and in study III (Somatonorm, SIII) 8-2 ng ECP per ampoule (Table 2). In studies I and II, a total of 40 children with idiopathic pituitary dwarfism took part, as well as one boy with septo-optic dysplasia, two girls who had been operated on for brain tumours and one boy with a growth disorder which already existed in utero, most probably belonging to a type A1 dwarfism according to Illig (1970). In the Somatonorm study III, there were 11 children, nine with idiopathic pituitary dwarfism or with pituitary lesions due to head injuries, one patient with intrauterine growth retardation and one, in addition, with hypoplastic genitalia and undescended testicles, suggesting genetically determined pituitary dwarfism. The idiopathic and other cases will be discussed separately. The protocol which we put together provided the following inclusion criteria for admitting a patient to our study: Height lower than 2.5 standard deviations (SD) or more. Growth velocity decreased by 2 or more SD, i.e. SDScA (chronological age), or was less than 4 cm/year. Bone age <12 years in boys and <11 years in girls--in order to avoid interference from the pubertal growth spurt. GH deficiency was always assessed by two provocation tests, one of them invariably an intravenous insulin tolerance test. Maximal GH increments <5 ng/ml were considered to be total GH deficiency, increments between 5 and 10 ng/ml incomplete or partial deficiency. Furthermore, the usual endocrinological tests for evaluation of the function of the anterior pituitary were done, and in addition further routine laboratory investigations such as serum electrolytes, creatinine, alkaline phosphatase, somatomedin-C, somatomedin activity in porcine cartilage, liver enzymes and haematological parameters. Importance was placed on the immunological investigations. Antibodies (Ab) were determined in three ways: (1) by means of intradermal prick tests which were performed only prior to treatment; (2) antibody against the preparation proper, the Met-hGH; (3) antibody against the contaminating ECP, so-called anti-ECP Ab (as described above).

Therapeutic protocol The therapeutic protocol corresponded to the regimen applied in 1979 and was identical to the scheme used in the Children's Hospital in Ziirich as

527

RECOMBINANT HUMAN GROWTH HORMONE published in 1982 (Sorgo M e t - h G H i.m. per week. treatment, hypothyroidism in addition. Follow-up investigations

et al, 1982). All patients received 3 x 4 I U If either initially, or during the course of was manifest, thyroxine r e p l a c e m e n t was given were p e r f o r m e d every three months.

Case histories and exclusion criteria

W e will m a k e only a few short c o m m e n t s on the individual case histories of the children. In general, great significance is placed on the o r d e r of birth of the children. T w e n t y - t h r e e of the 49 patients with so-called idiopathic h y p o p i t u i t a r i s m were firstborns. Complications during p r e g n a n c y , e.g. vaginal bleeding and toxaemia, were r e c o r d e d 14 times, complicated deliveries totalled 15. A s we have d e m o n s t r a t e d previously, b r e e c h deliveries play an i m p o r t a n t role. T h e y were r e c o r d e d in 11 cases (22%) c o m p a r e d with 4% in the n o r m a l population. Tables 6 and 7 show the height o f o u r idiopathic cases in studies I and II, calculated in relation to b o t h chronological age ( C A ) and b o n e age ( B A ) . Table 6. Somatonorm (SIII) German multicentric study. IGHD--auxology at diagnosis.

Study

I

N Age Bone age (TW2-RUS) Height Height velocity

28 9.1 5.5 -3.2 -2.7

II

III

12 9.9 6.5 -3.3 -2.0

10 8.2 5.4 -3.0 -2.2

(yrs) (yrs) (SDScA) (SDSca)

G r o w t h velocity a m o u n t e d to - 2 . 5 S D in the first and second groups c o m b i n e d . In the third group, the m e a s u r e m e n t s r e p o r t e d by the parents of several children were inaccurate, so exact m e a n values c a n n o t be given. C o n c e r n i n g the endocrinological tests, 41 patients had a c o m p l e t e lack of G H and eight a partial deficiency. Fifteen children had s e c o n d a r y h y p o t h y r o i d i s m and received thyroxine r e p l a c e m e n t , eight children h a d a s e c o n d a r y adrenal deficiency which r e m a i n e d untreated. Table 7. Height in relation to bone (BA) and chronological age (CA).

N

Study I

N

Study II

N

StudiesI+II

CA D Rx

28 26

-3.27 --+ 0.92 -3.4 - 0.96

12 11

-3.32 -+ 0.84 -3.29 +-+-0.72

40 37

-3.29 + 0.89 -3.37 _+ 0.88

BA D Rx

22 26

0.88 _+ 1.36 0.45 + 1.74

10 11

-0.58 _+ 1.51 0.02 + 1.26

32 37

0.43 _+ 1.54 0.32 _+ 1.61

'D' means the time when diagnosis was established, 'Rx' the time when treatment was started. In relation to CA, an average height deficit of 3.3 SD was measured. In relation to BA, no height deficit existed, which means that height and bone age were equally retarded. In study III nearly identical results were registered.

528

L. M. FRYKLUND ET AL

Therapy with Somatonorm

Growth The therapeutic success with S o m a t o n o r m is best demonstrated if compared with the results of the two previous studies from Ttibingen (Ranke et al, 1979) and Ziirich (Sorgo et al, 1982) because exactly the same dosages were used. As is apparent from all observations, growth velocity is always maximal during the first half-year, the so-called catch-up, and decreases in the second and even more in the third half-year. The dynamics are clearly shown in Table 8. Table 8. Growth velocity measured as cm/year, at half-yearlyintervals. Study I ~

Study II

Half-year

N

SD

Prior to treatment I II III

25 3.46 1.76 26 10.16 3.30 26 8.99 2.91 18 7.98 2.15

N 8 8 1

~

Study I+II SD

2.94 1.12 8.49 1.95 5.80

N

~

SD

33 34 27 18

3.34 9.76 8.87 7.98

1.63 3.09 2.92 2.15

It is a consequence of this situation that one can compare only identical half-yearly periods of treatment. In Table 9, the results of the first S o m a t o n o r m study during the first year of treatment are compared to those obtained previously in Tiibingen and Ztirich: with SI growth was 9.5 cmJyear; with Crescormon in Tiibingen 7.7 crn/year, which is a statistically significant difference; and in Ziirich 8.4 crrdyear. In the lower part of the table these measurements are transformed into SDS values. S o m a t o n o r m appears superior, with 3.6 SD v. 2.3 and 2.73 SD, respectively, using this calculation.

Table 9. Growth velocity in the first year of treatment; Somatonorm (SI) compared with pituitary hGH. cm/year SDScA

Somatonorm I

Ttibingen 1979

Ztirich 1982

9.5 _+ 2.7 3.6 -+ 2.7

7.7 _+2.4 2.3 ± 2.5

8.4 ± 2.8 2.73 ± 2.8

The same was true for the results during treatment II as emerges from Table 8, in which the various half-yearly results are compared. The 12-month period with SII cannot be described since only a minority of the children have been treated for a full 12 months. This does not apply for our third study. All children who received Somatonorm have been treated for a full year or more. The results are again similar (Table 10). The results are a bit better than those with Crescormon; the difference compared to the Ztirich results is, however, not statistically significant.

RECOMBINANT HUMAN GROWTH HORMONE

529

Table 10. Growth velocity in the first year of treatment; Somatonorm (SIII) compared with pituitary hGH. cm/year

SDScA

Somatonorm III

Ttibingen 1979

Ziirich 1982

8.8 + 2.2 3.3 -+ 2.6

7.7 + 2.4 2.3 + 2.5

8.4 + 2.8 2.4 _+ 2.7

T h e b e s t m e a s u r e for t h e e v a l u a t i o n o f t h e t h e r a p e u t i c success is t h e s u m o f t h e g r o w t h v e l o c i t y in S D a t t a i n e d during t r e a t m e n t plus t h a t prior to t r e a t m e n t w h i c h in S D is a l w a y s a n e g a t i v e figure. I n T a b l e 11, this has b e e n c a l c u l a t e d for t h e SI p a t i e n t s , a g a i n in c o m p a r i s o n to t h e p r e v i o u s results f r o m T t i b i n g e n a n d Zfirich. F o r SII a n d S o m a t o n o r m , this k i n d of c a l c u l a t i o n was i m p o s s i b l e b e c a u s e t h e d a t a for g r o w t h v e l o c i t y p r i o r to t r e a t m e n t w e r e t o o inexact. F r o m this t a b l e it is e v e n m o r e e v i d e n t t h a t t h e a d m i n i s t r a t i o n o f S o m a t o n o r m gave b e t t e r results t h a n with t h e p i t - h G H p r e p a r a t i o n . T a b l e 12 c o m p a r e s S D S c a at h a l f - y e a r l y i n t e r v a l s for studies I, I I a n d III. Table 11. Growth velocity per year (SDScA) during treatment. Prior to treatment First year of treatment Total increase

Somatonorm I

Ttibingen 1979

Ziirich 1982

-2.67 _+ 2.16

-2.45 _+ 0.84

-2.38 + 1.87

+3.55 _+ 2.66 +6.22

+2.29 _+ 2.49 +4.47

+2.4 _+ 2.7 4.78

N o t all 49 p a t i e n t s w h o w e r e in fact t r e a t e d a p p e a r in t h e t a b l e s s h o w n . T h e r e a s o n is t h a t in s e v e r a l c h i l d r e n t r e a t m e n t h a d to b e a l t e r e d o r discontinued. One of the patients whose parents were Greek immigrants r e t u r n e d h o m e with his p a r e n t s . T w o p a t i e n t s - - b o t h of t h e m a t o p i c - d e v e l o p e d A r t h u s r e a c t i o n s in t h e s e c o n d w e e k of t r e a t m e n t so t h e a d m i n i s t r a t i o n o f S o m a t o n o r m h a d to b e s t o p p e d . A f t e r a s h o r t while w e c o n t i n u e d with p i t - h G H , with r a t h e r g o o d success. A f o u r t h p a t i e n t , w h o was also an a t o p i c i n d i v i d u a l , d e v e l o p e d v e r y high a n t i b o d y titres w i t h o u t such skin r e a c t i o n s using SI t r e a t m e n t . H i s g r o w t h c u r v e s h o w e d i n c r e a s i n g a t t e n u a t i o n a n d w e d e c i d e d also to c h a n g e t h e t h e r a p y to p i t u i t a r y h G H . T h e n c e f o r t h his g r o w t h b e c a m e n o r m a l .

Table 12. Growth velocity (SDScA) for half-yearly intervals. Study I

Study II

Half-year

N

~

SD

N

Prior totreatment I II III

22 23 23 16

-2.67 3.88 3.51 2.14

2.16 3.44 2.99 1.76

8 8 1

~

Studies I+II SD

N

~?

SD

-1.98 2.13 4.73 3.73 -0.30 - -

30 31 24 16

-2.49 -4.10 3.35 2.14

2.13 3.48 3.03 1.76

530

L. M. FRYKLUND ET AL

Four of the six non-idiopathic pituitary dwarfs grew just as quickly as the idiopathic cases. These were the two children with septo-optic dysplasia and the two girls who had been operated on for brain tumours. The other two patients suffered most probably from genetically determined G H deficiency and displayed very conspicuous responses during therapy. The first child received preparation II and grew well initially. After six months his growth velocity declined and after nine months growth came to a full stop. At the same time, urticarial reactions occurred at the injection sites, erythemas and itching infiltrations. The administration of Somatonorm was discontinued and two pit-hGH preparations were given successively; nevertheless the same reactions appeared. Already, long before these observations, the immunological investigations had demonstrated a very high antibody titre against Met-hGH with a high binding capacity. The second patient who displayed the same clinical symptomatology received Somatonorm. Also this child developed rather high antibodies against growth hormone. However, up to now, the growth curve remains indistinguishable from that expected.

Somatomedin and alkaline phosphatase In order to further evaluate the effectiveness of the G H preparations, somatomedin (SM) was measured in two different ways: (1) somatomedin activity, according to the method of Van den Brande and de Caju (1974), using rib cartilage of young pigs; (2) somatomedin-C (SM-C) by RIA. The results for the first two studies are depicted in Figure 4. With a mean level of 0.26 U/ml the entry values of the patients were markedly diminished. Already, after two days' treatment with Met-hGH, the SM activity had risen to more than double that level. After six months, a maximum of 1.61 U/ml was attained. Later on, slowly decreasing levels were recorded, but even after a year and a half the mean level exceeded that of normal age matched controls. Also the SM-C levels, determined by RIA, were initially low and rose significantly during treatment, but not nearly as high as the SM activity. Hence it follows again that the biological test certainly measures more than SM-C. Basal levels of alkaline phosphatase were low before treatment and rose significantly during Met-hGH treatment.

Undesired side-effects As mentioned in the beginning, numerous additional laboratory parameters were checked in blood and urine. Summing up all the results of these investigations, no hint of metabolic disturbance or lack of compatibility was found.

Allergic and immunological reactions As with all preparations that are biosynthetically produced, i.e. by bacteria, the formation of antibodies against bacterial substances plays a

RECOMBINANT HUMAN GROWTH HORMONE

531

D/m~ 1.5-

1.3-

1.1

- 40C

0.9 - 350 0.7-

.300 0.5 25O

0.3

200

O.t

_4/ Itbetore 1:5

3

6 9 months of treatment

12

15

//_z I'8

Figure 4. Somatomedin-C and alkaline phosphatase values during the SI trial. SM-C RIA

(&), SM activity (porcine cartilage) (O), alkaline phosphatase (®).

decisive role in the compatibility of the products. Therefore, the search for antibodies was intensive from the beginning. As already mentioned, prior to treatment, intradermal prick tests were performed, which were negative in all patients. In the two cases mentioned earlier, who at the start of our investigations in 1981 received relatively impure preparations, the prick tests became positive in the second week. Somatonorm was discontinued and exchanged for pit-hGH which was well tolerated and successful. In all children, antibodies against M e t - h G H were radioimmunologically measured. If the test was qualitatively positive (twice blank), titration of the antibodies followed. The results were expressed as logarithms of the final dilution. Moreover, the binding capacity was determined since this gives the best measure of the antibody characteristics. Titres became positive after six weeks in all patients and reached their maxima after six months. In six cases the titre exceeded 4 (log final dilution) (Figure 5). The antibody titres from the patients in the second study were lower. The difference from the study ! results was statistically significant after six weeks and six months. The purification of the preparations which had been undertaken in the meantime had obviously b e e n successful, although limited. Figure 5 demonstrates the differences in the curves of the mean values for SI and SII. Also the binding capacity of the antibodies was considerably lower in study II than in study I. In the first series we obtained after six months a

532

L.M. FRYKLUND ET AL 1000 ~CP,ng/vial~

STUDY I

I00

lO

GH'AS ( T i l e r ) ~/~

~'/STUDY[II

A = GH-AB (Binding capacity) O = Anti-ECP-AB (Z-Score)

1

0 0

1 0.1

0.2

2 0.3

0.4

3

0.5

0.6

0.7

:I-T] 0.8: ~ C ~

Figure 5. Somatonorm multicentre study antibody properties as a function of ECP content. GH antibodies, titre (logarithm of final dilution) (O), GH antibodies, binding capacity (mg/litre) (A), ECP antibodies Z score (see text for explanation) (O). The values are expressed as the mean. m e a n v a l u e of 0.6 mg/1, after 12 m o n t h s 0.66 mg/1. I n the s e c o n d series the v a l u e after six m o n t h s was o n l y 0.24 mg/1; the difference is highly significant. ( F i g u r e 5). T h e next t a b l e ( T a b l e 13) shows the a n t i b o d y against h G H o b s e r v e d d u r i n g S o m a t o n o r m ( S i l l ) t r e a t m e n t . T h i r t y of the 36 m e a s u r e m e n t s were n e g a t i v e ; the r e m a i n i n g six s h o w e d r a t h e r low titres. I n a g r e e m e n t h e r e w i t h , the b i n d i n g capacities were e x t r a o r d i n a r i l y low. T h e highest b i n d i n g capacity m e a s u r e d was o n l y 0.01 rag/1. F o r the d e t e r m i n a t i o n of the circulating a n t i b o d i e s against Escherichia coli p e p t i d e s ( E C P ) a n E L I S A ( e n z y m e - l i n k e d i m m u n o s o r b e n t assay) was used ( E r i k s s o n et al, u n p u b l i s h e d data). T h e test is a d o u b l e - a n t i b o d y p r o c e d u r e . T h e s e c o n d a n t i b o d y is labelled with p h o s p h a t a s e . T h e E C P

Table 13. Anti-hGH antibodies during Somatonorm (SIII) treatment. Titre = log final dilution. No.

0

6W

3M

6M

9M

12M

905/805 905/806 907/803 907/804 909/806 914/802 914/803 917/806 921/801

NEG NEG NEG NEG NEG NEG NEG NEG NEG

NEG -NEG NEG NEG -NEG NEG NEG

NEG 2.0 NEG NEG NEG NEG NEG NEG NEG

NEG 2.3 NEG -NEG NEG NEG -1.4

NEG 2.6 1.4 -NEG NEG NEG NEG NEG

--1.1 -NEG NEG NEG -NEG

905/804 905•807

NEG NEG

NEG NEG

3.5 NEG

3.2 NEG

2.9 NEG

2.3 --

533

RECOMBINANT HUMAN GROWTH HORMONE

standard is a mixture of polypeptides extracted from E. coli K20, as described earlier. The result is expressed as quotient Z which is the UV absorption of the patient's serum divided by the UV absorption of standard serum. All children in study I developed markedly increased anti-ECP Ab in the course of treatment. As with the GH Ab, maximal peaks were attained after six months, quotient Z being 0.73 on the average compared with 0.05 as entrance values prior to treatment. In study II the maximal values attained were markedly lower, only 0.2 after six and nine months. This difference between the two studies is highly significant (Figure 5). As mentioned earlier, comparison between study I and II with regard to all parameters pointed to a much better purification of the preparations used in study II than in study I. This was considerably more so when the first two studies were compared to the third one. It emerges from Table 14 that 25 out of 36 measurements of anti-ECP Ab were entirely negative. Only in two of the remaining 11 were slightly increased values measured. These were the same cases in which anti-hGH Ab with low titres had also been measurable. The purification steps carried out in Stockholm had caused an enormous reduction of ECP in the preparations. As it turns out, this improvement was followed by a decrease of the antibody, against GH itself as well as against ECP. A remarkable and scientifically most interesting exception was case 905/804 in study III, the second child with congenital pituitary dwarfism who probably belonged to type A of Illig and Prader. As in the first case in study I, we found high titre anti-GH Ab with a binding capacity of 0.4 mg/1, whereas the Z-scores against ECP remained zero in all samples. For both of the patients who were already growth retarded in utero, the human GH which they themselves could not produce was a foreign protein, i.e. an antigen. This led in both instances to high antibody titres against GH, but not against ECP. A possible correlation between the parameters height, antibody titres and growth velocity of the patients, was calculated--always six-month

Table 14. A n t i E C P antibodies during S o m a t o n o r m (SIII) t r e a t m e n t (Z-score). No.

0

6W

3M

6M

9M

12M

905/805 905/806 907/803 907/804 909/806 914/802 914/803 917/806 921/801

NEG 0.2 0.2 NEG NEG NEG NEG NEG NEG

NEG -0.2 NEG NEG -NEG NEG NEG

NEG 0.3 0.2 NEG NEG NEG NEG NEG NEG

0.2 0.2 NEG -NEG NEG NEG NEG NEG

NEG 0.2 0.2 -NEG NEG NEG NEG NEG

--0.2 -NEG NEG --0.2

905/804 905/807

NEG NEG

NEG NEG

NEG NEG

NEG NEG

NEG NEG

NEG --

534

L. M. FRYKLUNDET AL

periods of growth v. titres and binding capacities. Statistically, no correlations were found. Growth was apparently inhibited solely in the one boy already discussed (case 901/806) who developed the highest titre of hGH Ab ever observed and simultaneously a rather high binding capacity, but at the same time only negligible antibody against ECP. This child was also an atopic patient with extraordinarily high IgE who suffered hay fever.

SUMMARY 1. All batches of Met-hGH examined stimulated statural growth to approximately the same extent. The growth rates measured partly exceeded the results obtained in previous studies with pituitary preparations in the same dosage. 2. Under treatment with SI, i.e. the preparation with the highest amount of ECP, high antibody titres with high binding capacity against GH and ECP were found. With SII all antibody determinations showed much lower titres. With Somatonorm (SIII), in the large majority of cases no antibodies were detectable. The titres registered in a few children were low and the binding capacities were negligible. 3. The biologically determined somatomedin activity was initially pathologically low. During treatment it rose to supraphysiological levels. Also the radioimmunologically assayed somatomedin and the alkaline phosphatase increased significantly. 4. At the start of the first series, two patients showed allergic skin reactions which turned out to be caused by the insufficiently purified preparations. Therapy with extractive preparations was free of such side-effects and fully successful. Both of the patients were atopic. A third child who was also allergic developed after 6-9 months the highest antibody titres seen, combined with a high binding capacity. Also, with this boy, treatment was switched over to pit-hGH, with very good results. 5. Two children with pituitary dwarfism already developed in utero high antibody titres against Met-hGH but not against ECP. For this response, neither the Somatonorm nor its impurities can be implicated. Rather, it is the reaction to GH generally, which the organism recognizes as a foreign protein and thus as an antigen. One of the patients stopped growing after nine months. Likewise, pituitary GH did not lead to any further improvement.

Acknowledgements We would like to thank all our colleagueswho participated in the multicentrestudy and research staffinvolvedfrom KabiVitrum.

RECOMBINANT HUMAN GROWTH HORMONE

535

REFERENCES Bierich JR, Ranke MB, Flodh H & St6ver B (1983) Growth promotion by biosynthetic human growth hormone in pituitary dwarfism. XIV Acta Endocrinologica Congress, Stockholm, Sweden, June 27-30, 1983. Aeta Endocrinologica 103: (Supplement 256). De Boer HA, Comstock LJ, Yansura DG & Heyneker HL (1980) Construction of a tandem trp-lac promoter and a hybrid trp-lac promoter for efficient and controlled expression of the human growth hormone gene in Escherichia coli. In Rodriguez RL & Chamberlin MJ (eds) Promoters Structure and Function, pp 462-479. New York: Praeger Scientific. Flodh H, Johansson H-E, JOnsson M & Ekvfirn S (1982) General toxicity in rats of biosynthetic and pituitary human growth hormones. Proceedings of the FDA-USP Workshop on Drug and Reference Standards for Insulins, Somatropins, and Thyroid-axis Hormones, Bethesda, Maryland, May 19-21, 1982, pp 327-335. Fryklund L, Brandt J, Eketorp G e t al (1982) A comparison of human biosynthetic and pituitary somatotropins: purity and potency. Proceedings of the FDA-USP Workshop on Drug and Reference Standards for Insulins, Somatropins, and Thyroid-axis Hormones, Bethesda, Maryland, May 19-21, 1982, pp 319-327. Goodman HM (1984) Biological activity of bacterial derived human growth hormone in adipose tissue of hypophysectomized rats. Endocrinology 114: 131-135. IIlig RJ (1970) Growth hormone antibodies in patients treated with different preparations of human growth hormone (hGH). Journal of Clinical Endocrinology and Metabolism 31" 679. J6nsdottir I, Ekre HP & Perlman P (1983) Identity of pituitary and bacterial human growth hormone as determined by monoclonal antibodies. (Comparative study of pituitary and bacteria derived human growth hormone by monoclonal antibodies.) Molecular Immunology 20 (8): 871-876. Kostyo JL & Reagan CR (1976) The biology of growth hormone. Pharmacology and Therapeutics (B) 2: 591-604. Larhammar H (1985) Comparative conformational analysis of human somatotropin and biosynthetic methionyl human somatotropin by spectroscopic fitrations. International Journal of Pharmaceutics 23: 13-23. Moore S (1963) On the determination of cystine as cysteic acid. Journal of Biological Chemistry 238: 235. Penke B, Ferenczi R & Kovacs K (1974) A new acid hydrolysis method for determining tryptophan in peptides and proteins. Analytical Biochemistry 60" 45. Ranke M, Weber J & Biericb JR (1979) Long-term response to human growth hormone in 36 children with idiopathic growth hormone deficiency. European Journal of Pediatrics 132: 221-238. Retegui LA, Keefer LM, Fryklund L & Demeyts P (1984) Monoclonal antibodies and specific cell surface receptors do not discriminate between human growth hormone prepared by DNA recombinant techniques and the native hormone. Acta Physiologica et Pharmacologica Latinoamericana 34: 193-197. Skottner A, Forsman A, Fh61enhag K et al (1981) Human growth hormone produced by E. coli. A preliminary study of effects on hypophysectomized rats. In Gueriguian J (ed.) Insulins, Growth Hormone and Recombinant DNA Technology, pp 109-115. New York: Raven Press. Sorgo W, Zachman M, Tassinari D, Ferndandez F & Prader A (1982) Longitudinal anthropometric measurements in patients with growth hormone deficiency, effect of human growth hormone treatment. European Journal of Pediatrics 18: 38-45. van den Brande JL & de Caju MVL (1974) An improved technique for measuring somatomedin activity in vitro. Acta Endocrinologica 75: 233-242.