RHGH Synthesis

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18

Recombinant DNA Synthesis of Human


Growth Hormone
Michael J. Ross, Kenneth C. Olson, Michael D. Geier,
John V. O'Connor, and Andrew J. S. Jones

1. Introduction

1.1. Cloning and Expression


Human growth hormone (hGH) is a polypeptide of approximately 22,000
daltons in size that is synthesized in the pituitary. Mature hGH has been
produced in Escherichia coli bacteria using recombinant DNA technology(l) using
the scheme outlined in Fig. 1. The process involved cloning of cDNA to the
pituitary hGH mRNA (2) and the subsequent adapting of the cloned gene for
expression in E. coli. hGH, as it is synthesized in the pituitary, is made as a
prehormone containing a hydrophobic leader peptide of some 20 amino acids
in length. This leader peptide is proteolytically removed by the pituitary during
the secretion of hGH. However, most E. coli do not have the biochemical
machinery to do the same processing efficiently (Fig. 2), thus, 84 base pairs of
double-stranded DNA were synthesized to tailor the hGH cDNA for direct
expression. The process is illustrated in Fig. 3 and involved ligating the
restriction fragment of the cDNA coding for amino acids 24 through 191 of
growth hormone to a synthetic sequence which coded for a start codon (Le., a
methionine) and the first 23 amino acids of GH. Thus, the molecule synthesized
in E. coli contained the full 191 amino acids of mature GH and one additional
amino acid, an amino terminal methionine, from the start codon.

Michael]. Ross, Kenneth C. Oison, Michael D. Geier,John V. O'Connor, and Andrew ].S.Jones •
Genentech, Inc., South San Francisco, California 94080.

241

S. Raiti et al. (eds.), Human Growth Hormone


© Plenum Publishing Corporation 1986
242 III • Biosynthesis of Human Growth Hormone

Human Pituitary
~
m RNA
Re verse I Trans crlptase
DNA • Polymerase
ds DNA
l
III I 1IIIII jill i III i II i III I III11111 i I
Haelll Re stric tion Nuclease
i II iii I I I I I iI I II II iii Ii II i i Ii i i
551 bp FRAGMENT
Terminal l Transferase
ccccc JillIIDiiCllllIIDillll!IJillJlllIIDilCllllIIDIiClIIIJ:lilJlllIIIJIiCII cccccc
TAILED FRAGMENT
DNA l Ligase

MIXTURE OF
LIGATED PLASM IDS

Figure 1 Cloning of the human growth hormone (hGH) message. Size fractionated messenger
RNA isolated from human pituitaries was copied using reverse transcriptase to form a heteroduplex
molecule. The RNA strand was then displaced with a second DNA strand with DNA polymerase I
Klenow fragment. The resulting mixtures of double-stranded DNA molecules were cut with
restriction endonuclease Hae III and fractionated on an agarose electrophoresis gel. The hGH-
specific cDNA of 551 base pairs (bp) in length was "tailed" with deoxycytosine using deoxynucleotide
terminal transferase and ligated into dG tailed cloning vectors using DNA ligase. The mixture of
ligated plasm ids was used to transform E. coli K-12. Clones containing the hGH cDNA fragment
were isolated and the amplified 551-bp fragment purified.

1.2. Product Characterization


1.2.1. Chemical Structure
Material isolated from these E. coli has been extensively characterized, both
at Genentech and at other institutions. The material synthesized in E. coli has
been shown by tryptic analysis and Edman degradation to be identical to the
sequence one would expect from the gene.(3,4) The methionyl hGH has been
shown to have the correct two pairs of disulfide bridges between the four
cysteines in the molecule. (3) The major isohormone found in pituitary hGH is
identical in isoelectric point to methionyl hGH, and the minor acidic isohor-

INTRACELLULAR DIRECT EXPRESSION Figure 2 Intracellular direct expression of human


growth hormone (hGH). GH is synthesized in se-
cretory cells in the pituitary. There it is synthesized
.
pre huma n growth hormone as a pre hormone containing a hydrophobic leader
sequence which is cleaved off by a protease as it is
processed through the cell membrane into secretory
granules. The hGH is expressed directly inside E.
coli cells by deleting the hydrophobic amino acids
Ecoli-derived human growth hormone
between the methionine start codon and the first
NH ,-met phe- -- -- - - COOH amino acid ofthe mature hormone, a phenylalanine.
18 • Recombinant DNA Synthesis of Human Growth Hormone 243

Chemical Synthesis of 84 Base Pairs of DNA


e---e---e e e e----
---e ---e e-~---e ---e---e
DNA Ligase
+
Cloning
!

-0
Eco RI Haelll Endonucleases

IIIIII1IIIII11111 III1IIIIIIII

DNA 1 Ligase

Figure 3 Expression of met-hGH. The Hae III-Eco Rl fragment of the hGH eDNA contains the
coding sequence for amino acids 23-191 of hGH: an 84-base fragment of DNA which coded for a
methionine start codon and amino acids 1-22 of hGH. These two fragments were then ligated
together into an expression plasmid derived from pBR322. The expression plasmid contained
promoters, selection markers, and ribosome binding site to allow efficient expression of hGH.

mones that are found in the methionyl hGH preparation are very similar in
their isoelectric pattern to the isohormones found in pituitary-derived hGH.(5)
The major difference observable using isoelectric focusing is that there are
fewer isohormones present in met-hGH.

1.2.2 . Folding
The three-dimensional structure of the molecule has been shown to be
indistinguishable from pituitary hGH by circular dichroism studies.(6) Immu-
nochemical studies comparing met-hGH and pit-hGH in sera from both
hyperimmunized rabbits and patients have as yet found no new determinants
in the methionyl compound, indicating that the amino terminus of the molecule
is probably not accessible on the molecule's surface.

1.2.3. Bioassay
Finally, bioassay data using rat weight gain and rat tibia length models(4.7)
as well as radioreceptor and receptor modulation assays show met-hGH to be
equally potent with pit-hGH.(8)

2. The Production Process

The first step in the process of manufacturing met-hGH for clinical use is
fermentation, a highly reproducible and easily scalable process which has been
244 III • Biosynthesis of Human Growth Hormone

11 J.£
2/10,000"
Single Bacterium

8 Flask
/
o
00
00
000
000

10..l to 1,000,000..l

Figure 4 Controlled fermentation processes can be carried out at scales ranging from 10 liters to
106 liters. Each batch is derived from the single colony of bacteria which is grown up to yield a
master culture. This master culture is exhaustively characterized and subdivided into identical vials
and stored frozen. For each new batch, a single vial of this master culture is inoculated into a flask
of nutrient broth where it is grown and used to inoculate a fermenter, which can then be used to
inoculate larger fermenters.

used as a key tool in pharmaceutical manufacturing for many years. The


process outlined in Fig. 4 shows production of shaking flask inoculum cultures
and, from those flasks, stirred batch fermentations from single colonies of E.
coli transformed with met-hGH production plasmid. After vegetative cell growth
is complete, the bacteria are harvested by centrifugation and broken open to
release the met-hGH. The purification steps are, however, the key to the ability
to make acceptable clinical product. Modern chromatographic methods allow
protein separation to be carried out at large scale with high resolution so that
the complex mixture of E. coli proteins present with the met-hGH in the starting
material can be removed. The successive removal of these proteins as the met-
hGH progresses through the purification can be seen in Fig. 5.
In 1980, previous experiences with production of pharmaceutical peptides
from bacteria such as streptokinase(9) and asparaginase(lO) were limited to acute
care indications, yet the first clinically tested products of rDNA technology
were human insulin(lO) and hGH. Our experience since 1980 with the devel-
opment of the met-hGH, which Genentech, Inc., has chosen to call Protropin,
has led us to the inescapable conclusion (not fully foreseen in 1980) that the
details of the production process can have profound effects on the clinical
performance of the drug. Increasing the expression levels of the product in
the bacteria, (II) using process validation studies and final specifications to design
and challenge the purification process,(12) and development of new analytical
tools such as HPLC,(3) the endogenous pyrogen test,(l3) and ECP assays(14,15)
have all turned out to be key factors in producing clinically acceptable material.
Genentech has developed three distinct manufacturing processes for manufac-
turing met-hGH over the past several years, and KabiVitrum has reported two
more distinct processes. The clinical results from each of these do in fact differ
18 • Recombinant DNA Synthesis of Human Growth Hormone 245

Figure 5 The purification of met-


hGH. The met-hGH has E. coli pro-
teins removed from it at each stage
of the purification process. The fig-
ure shows a silver-stained SDS-
PAGE analysis of pools made during
the purification. Lane 1 is of crude
lysate and lane 5 is of bulk product.
Similar amounts of total protein were
loaded in each lane . It should be
noted that color differences in the
silver stain cause the intensity of the
hGH band to be far weaker than the
E. coli bands.
246 III • Biosynthesis of Human Growth Hormone

markedly; so one must precisely define the manufacturing process used to


produce hGH before evaluating the results of a clinical trial.

2.1. Process Design/Validation


2.1.1. Gll002: The Use ofSDS-PAGE
The first purification process that was developed at Genentech (Code
Gl1002) was designed using Coomassie stained SDS-PAGE purity estimates as
the primary tool. The process involved anion exchange chromatography and
gel filtration chromatography.(5) Figure 6 shows an SDS-PAGE analysis across
the fractions of a DEAE ion exchange column. The data from similar experi-
ments were used to optimize the parameters for this chromatography step.
Single protein contaminants of approximately 1% the amount of an overloaded
met-hGH band could be detected using this kind of SDS-PAGE analysis, and
thus final product of approximately 99% purity was achieved for Code G 11002.
Met-hGH, code Gl1002 was formulated in glycine-phosphate and lyoph-
ilized. The material produced by this process appears homogeneous by SDS-
PAGE, whereas in even the purest pituitary hGH preparation, one can detect
minor bands of either other hGH forms or impurities (Fig. 7). When tested in
adult human volunteers, the met-hGH behaved identically to pituitary hGH

Figure 6 An SDS-PAGE analysis (Coomassie blue stain) of column fractions from the initial ion
exchange column used to purify Protropin.
18 • Recombinant DNA Synthesis of Human Growth Hormone 247

except that some classical signs of endotoxin contamination appeared in spite


of the fact that the material passed both USP pyrogen and LAL tests (Hintz, et
al.).(16) Repeat LAL and pyrogen assays yielded no clue as to the cause of the
endotoxin reaction; so a material of considerably higher purity was prepared
(see Code G 11008 below) which eliminated the problem. Subsequent analysis
using a human monocyte activation assay(12) revealed that LAL and rabbit (but
not human) reactivity to E. coli endotoxin was irreversibly inhibited when
lyophilized in the presence of glycine.

2.1.2. Gll008: The Initial Preparation Used in Treating hGH Deficiency


A far more sensitive staining method (Silver versus Coomassie) for detecting
proteins on SDS_ PAGE(l7.18) was applied to the process design problem. Using
this technique, several new bands were detected in the GII002 preparation

Figure 7 Analysis of Process Code GII002 met-hGH by


SDS-PAGE. The original clinical preparation of met-hGH
was denatured in SDS/BME and separated by SDS-PAGE
and stained with Coomassie blue and compared with
clinical grade pit-hGH. Lanes I and 4 are 20 fLg of pit-
hGH. Lanes 2 and 3 are 20 fLg of met-hGH Proces Code
GII002. Though several bands other than 22K Monomer
hGH can be seen in the pituitary preparation . no bands
1 2 3 4 except 22K hGH are visible in the met-hGH.
248 III • Biosynthesis of Human Growth Hormone

1 2 4 3 4

Figure 8 Met hGH (Process Code GII002) was analyzed by SDS-PAGE using silver staining and
immunoblotting: 20 IJ.g, 10 IJ.g, 2 IJ.g, and 1 IJ.g of met-hGH (denatured in SDS/BME) were loaded
onto three identical sections of the same gel in lanes 1, 2, 3, and 4, respectively. The proteins in
the gel section in panel A were transferred to nitrocellulose and blotted with rabbit anti pituitary
hGH antibody. Antibodies bound to the blot were visualized by 125I-labeled protein A followed by
autoradiography. The major monomer band and the dimmer band can clearly be identified as
hGH as well as some minor higher- and lower-molecular-weight species. Panel B is a section of gel
stained with silverY7) A contaminant between hGH monomer and dimer can be easily seen as some
higher-molecular-weight species (larger than hGH dimer). Panel C is a section of gel which was
transferred to nitrocellulose and blotted with anti-E. coli protein antibody. The contaminants can
be clearly identified as being from E. coli origin.

(Fig. 8). Similar SDS gels were subjected to radioimmunological staining with
both anti-E. coli and anti-hGH antibodies to unambiguously assign the origin
of the newly detected bands. Two bands of E. coli origin were thus identified
and the remaining higher-molecular-weight species « 1% of the total protein
by Coomassie stain) were identified by anti-hGH antibody.
The purification process was then modified by monitoring SDS-PAGE
silver gels and by using data from experiments in which radio labeled E. coli
18 • Recombinant DNA Syntheris of Human Growth Hormone 249

Manufacturing Proce•• Validation

1. Grow Normal Cell. 1. Grow Labeled Cell. Without


with hGH Gene hGH Gene Under Same Condition.
I
2. Harve.t
I
2. Harve.t
I'~--------------~.I
Mix 1000:1
3. Purification 3. Purification
at 100% Scale At N1% Scale

I I
4. Final Product
4. Final Product

Purity Estimate no Better Purity Estimate Dependent


Than Method Precision on Specific Activity of Label

Figure 9 Radioactive spike process validation. Production organisms (E. coli missing the hGH
gene) are grown in media containing 14C_ or s5S-labeled amino acids. The labeled extract is added
to hGH containing cell paste from a normal production run. The purification is carried out as it
would be to produce Protropin except that the scale is reduced about 100X.

Process Validation of DEAE Ion Exchange Chromatography

14C Label
35 8 Label

--
Protein

-
4

--
(mg.ml-',
cpm.ml- 1 1. Protein
(x10·3 ,
Img.ml·',
1.

--
3
14 cpm.mr 1
(x10·4 ,
12
10 2


6
4

102030 40 50 60
Fraction Number Fraction Numbar

Figure 10 Monitoring of an ion exchange purification step using radioactive spikes. Radioactively
labeled E. coli were added during the Protropin T. purification process as described in Fig. 9. The
separation of contaminants (radioactivity) from met-hGH and the final step of the GIl 008 process
when the met-hGH was 99.7% pure coming into this step are illustrated. Two separate experiments
are shown; the experimental designs were similar, but in one experiment the E. coli were grown
with 35S04 and in the other, grown with l4C-Iabeled glucose.
250 III • Biosynthesis of Human Growth Hormone

0.2

300

35S 200
cpm/tube
100

Figure 11 HPLC analysis of 35S impurity in Protropin process validation. Size exclusion HPLC
on a TSK-3000SW column run in 100 mM sodium phosphate 0.1% SDS, pH 7.0.

contaminants were added to lysate containing met-hGH. A schematic of the


latter experimental protocol appears in Fig. 9. Both 14C_ and 35S-labeled E. coli
extracts were used in these experiments. The results of an optimization of a
DEAE ion exchange column are shown in Fig. 10. The 14C and 35S spikes have
almost superimposable profiles at each chromatography step indicating that
molecules containing both sulfur and carbon (proteins) are the major contam-
inants being detected. Significant differences between the 35S and 14C profiles
might indicate contamination by lipids, carbohydrates, or nucleic acids which
contain little or no sulfur. This conclusion is supported by analysis of the
product for the presence of E. coli DNA using hybridization techniques.(19) We
were unable to detect any DNA in the product to the sensitivity of the assay
(10 pg/mg of hGH) after the first ion exchange step in the process.
Though the spike experiments provided valuable guidance in designing
the Gl1008 process, they do have their limitations: 14C-Iabeled molecules have
a very low specific activity owing to the long half-life of the isotope; so the
number of counts left after a thorough purification is too small to characterize.
3H-Iabeled molecules are so prone to label exchange with solvent that their use
is precluded for this type of experiment. Even 35S labels have been complicated
by label migration, as can be seen with met-hGH.
We have characterized the 35S counts remaining after the Gl1008 purifi-
cation process was carried out to completion; these counts represented of the
order of 0.2% contamination. However, we found to our surprise that the
counts behaved like hGH when analyzed by HPLC (Fig. 11). Further charac-
terization by a monoclonal antibody affinity column showed that about half of
the label behaved identically to hGH while the other half of the labeled
molecules were not recognized by this single antibody. Our postulate is that
18 • Recombinant DNA Synthesis of Human Growth Hormone 251

free radicals generated by the f3 particles and chlorine atoms which are the
result of the sulfur decay cause low-molecular weight 35S E. coli contaminants
such as glutathione to become covalently attached to met-hGH. The damaged
hGH could co purify with met-hGH and could have antigenic sites damaged.
Since this process would be an artifact of the use of radioisotopes in the
experiment, the similar met-hGH adducts are unlikely to exist in the clinical
product. Therefore, absolute quantitation of impurities is probably not accurate
if they represent less than -0.1 % of the major protein.
Phase II clinical trials were initiated in children with hGH deficiency. The
detailed results of this 18-month trial are presented by S. Kaplan et al. (this
volume, Chapter 20), but we will summarize the results very briefly: The met-
hGH proved highly efficacious in promoting linear growth and no drug-related
side effects were noted. However, a majority of the patients developed low-
titer antibodies to hGH. In an effort to eliminate this immunological side effect
of therapy further, process improvements were undertaken.

2.1.3. Gl1015: The Use of an anti-E. coli. Assay


. A new tool was needed to improve the GIl 008 process since by the criteria
of SDS-PAGEISilver stain, no contaminants could be seen (Fig. 12). Immunol-
ogical techniques have historically been used to quantitate small amounts of
compounds and are particularly well suited to the relatively high-molecular-
weight substances that are present. Baker et al. (15) and Ross et al. (14) have
reported the development of an anti-E. coli protein (ECP) assay which was used
in the assays for purity of human insulin produced in E. coli. We attempted to
develop a similar tool for use with our hGH process but used different reagents
since such assays have proven to be highly process specific (data not shown).
A solid-phase sandwich immunoradiometric assay for 'the ECP antigens
likely to be present in the final steps of this met-hGH process was developed
as described below. The principle of the assay was to coat a microtiter plate
with anti-ECP antibody, adsorb the sample to the antibody, and quantitate the
e
antigen using other anti-ECP antibody 25 I-labeled). In order for such an assay
to be linear and accurate, there must be an excess of both coat and tracer
antibody to everyone of the antigens in the ECP mixture, or the assay would
vary in sensitivity for different antigens.
E. coli cells that did not contain the hGH gene were grown, harvested,
lysed, and carried through the first several steps of the met-hGH process to
select a crude mixture of ECP's. This was done to reduce the complexity of the
antigen mixture somewhat and therefore to improve the sensitivity of the assay.
A crude E. coli lysate was used to hyperimmunize rabbits, and the antiserum
was immunopurified on an ECP affinity column. A large excess of antiserum
was passed over the column before wash and elution to adjust the population
of antibodies used in the assay to better reflect the quantitites of the various
antigens in the ECP preparation (Fig. 13). An assay was developed that measured
ECP's accurately down to 10 ng ECP/mg hGH. The upper range of the assay
was limited to 100 ng/mg to ensure conditions of antibody excess.
252 III • Biosynthesis of Human Growth Hormone

A c

1 2 3

Figure 12 Analysis of Process Code GII008 met-hGH by SDS-PAGE. This figure is analogous to
Fig. 8 in that 20 ILg. 10 ILg. 2 ILg. and 1 ILg of met-hGH (denatured in SDS/BME) were loaded on
three identical sections of the same gel in lanes 1.2.3. and 4. respectively. The proteins in the gel
section in panel A were transferred to nitrocellulose and blotted with anti-hGH antibody. Only
met-hGH monomer. a small amount of dimer. and a hint of trimer can be seen in the resulting
autoradiogram. Panel B is stained with silver. All of the visualized bands except a trace of a species
with a molecular weight lower than hGH monomer in the overloaded 20 ILg lane are identified in
Panel A as being of hGH origin. In panel C the proteins on the gel were transferred to nitrocellulose
and then blotted with anti-E. coli protein antibody. No bands can be seen on this autoradiogram.

The assay, however, was very process dependent. Only those antigens
which could contaminate the hGH prepared by this manufacturing process
(Code GllOl5) were selected during the preparation of those ECP's. The assay
is therefore specific for these contaminants. A further complication is that the
conditions (e.g., pH, buffer) for the extraction and handling of the ECP's have
the potential for changing the tertiary structure of the antigen molecules and,
hence, their antigenic determinants. This process with different first steps could
give falsely low values in this ECP assay. In fact, Process Code Gl1002 met-
hGH, which is prepared by a different process than GII015, gives an ECP
assay value of about 100-200 ppm although in fact we know the preparation
contains at least 10,000 ppm ECP contaminants.
This ECP assay was used to characterize the Gl1015 process for met-hGH
which products contain less than 30 ppm ECP's. There are five major separation
18 • Recombinant DNA Synthesis of Human Growth Hormone 253

Figure 13 SDS-PAGE analysis of antigens and antibodies


used in the ECP assay. ECP's were prepared as described
in the text and run on a 12.5% acrylamide SDS gel. In one
lane the proteins were strained with silver (panel A).(18)
From a similar lane the proteins were transferred to a
nitrocellulose by filter electroblouing.(21) The nitrocellulose
was then incubated with the antibodies used in the ECP
assay. Bound antibodies were visualized using 125I-labeled
protein A followed by autoradiography. This figure (panel
A B) represents a 60-hr exposure at - 80°C using an image-
B enhancing screen.
254 III • Biosynthesis of Human Growth Hormone

999

Fold 99
Purification
Per 99.9
Step

99

2 3 4 5
Process Steps

Figure 14 Prototropin purification. The five major purification steps used in the Gl1015 are
analyzed to determine their efficiency in removing E. coli contaminants. The fold purifications
(bars) are calculated by taking the ECP values/mg of hGH before process steps and dividing by the
ECP values after those steps. The cumulative effect of the steps can be seen by plotting the overall
purity at the completion of the steps. The material after step 5 is purified bulk ready for formulation.

steps in the process, and each contributes significantly to the overall purification,
as can be seen in Fig. 14. The met-hGH produced by process GII015 has been
tested in GS-deficient children for 6 months and has shown itself to be as
efficacious as, but significantly less immunogenic than, material produced by
process code GII008 .

99.999

99.99
..
I
Ph ••• II

• •
Ph.s. II'
I

Percent

..
Purity
99.9

... --- .".----


....
-
99 Gll002
= Gll008
Gll015
Phase I

1979 1980 1981 1982 1983 1984


Jan. Jan. Jan. Jan. Jan. Jan.

Figure 15 Purity of different hGH preparations. The approximate purity of various met-hGH
preparations used clinically is plotted versus time. The clinical trials that were run with each
preparation are indicated by the arrows. The purity of the GIl 002 was estimated by 35S-labeled
process validations and the Gll008 and Gll015 preparations using the immunoradiometric ECP
assay.
18 • Recombinant DNA Synthesis of Human Growth Hormone 255

2.2. Summary
Three different preparations of met-hGH have been tested by Genentech
in human clinical trials. Each new process was significantly purer than its
predecessor and required the development of new analytical tools before that
new level of purity could be achieved (Fig. 15). Our experience has led to the
conclusion that the different preparations of met-hGH behave very differently
in the clinic. Consequently, it is very important to differentiate between
manufacturers and between different processes from the same manufacturer
when examining clinical results. We must rely on rigid control of a specific
manufacturing process to guarantee the safety of the product.
ACKNOWLEDGMENTS. We thank Matt Field, Vince Anicetti, Kathy McKeown,
Greg Bennett, Baron Reed, and Michele Sanda for their excellent technical
assistance and Sara Wright for help in preparing this manuscript.

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