1.

UPSTREAM PROCESS
1.1 Cell Production Chinese Hamster Ovary cells
The Chinese Hamster Ovary (CHO) cells are the mammalian cells that are being widely used in
the production of therapeutic proteins. In this review, production of growth hormones from CHO
will be the topic of interest. CHO or Cricetulus griseus cells are epithelial cells that grow as a
monolayer on an articifical substrate. The first CHO cell line was obtained from the parental
CHO cell line via biopsy of an ovary of a female Chinese hamster. It was first discovered by T.T
uc! in "#$% (Ham &'., "#($).
These CHO cells need proline, an amino acid that is a constituent of most proteins, in the
medium of growth. In order to achieve complete growth medium, ")* of fetal bovina serum is
added to the CHO cells.
In sub culturing, the culture medium will discarded and rinsed wit ).+$* Trypsin,).$- m.
/0T1 solution to get rid of serum traces that contains trypsin inhibitor. Once the serum traces
are removed, -.) m2 of Trypsin,/0T1 solution will be added to a flas!. The CHO cells are
observed under an inverted microscope until dispersion occurs. The thorough dispersion of the
cells will ta!e around $ to "$ minutes. 0uring the dispersion stage, the cells must not be agitated
by any means to avoid clumping. Cells are dispersed at -%
)
C. Then, about ( to 3 m2 of the
complete growth medium prepared in the previous stage will be pipetted into the dispersed cells.
The mi4ture is then transferred to new culture vessels and it is further incubated at -%
)
C(5ao 6T.,
"#(3).
6or the purpose of avoiding contamination, the culture medium will be renewed at most twice
during the sub culturing stage. The storage medium is li7uid nitrogen vapour phase (5ao 6T.,
"#(3).


1.! Mammalian Cell E"#ression Systems
The CHO cells are widely used in the production of recombinant proteins with native
mammalian glycosylation patterns. The CHO cells can be modified genetically to produce
growth hormones with mammalian post,translational modifications. Through gene
amplifications, high level of growth hormones can be produced. Two gene amplification
systems used in CHO cells are the dihydrofolate reductase ( 0H6&) system using
methotre4ate (.T8) resistance and the glutamine synthetase ('9) system using methionine
sulfo4amine (.98) resistance. 6or the growth hormone production, 0H6& is used (:oseph et
al., +)))).
The first step is delivering the recombinant 0;1 into the host cell nucleus for chromosomal
integration. 1 few methods li!e electroporation, lipofection and retroviral transfection are
commonly used with optimi<ed protocols. =hen the 0;1 enters the nucleus, the integration
site of the vector is random and the e4pression of the transgene is dictated by the surrounding
chromosomal structure and associated features. High transcriptional activity will occur at the
locus of 0;1 integration. 1 mutant 0H6& gene with reduced en<ymatic activity is used as a
stringent selection for higher transgene e4pression. 1fter the 0;1 is delivered, a pool of cells
stably e4pressing the co,transfected 0H6& en<yme are selected using low level of .T8 and
cultivating it in the absence of glycine, hypo4anthine and thymidine. 1t this stage, ma>ority
of cells that did not successfully integrate the vector 0;1 are !illed and the remaining cells
that e4pressing the protein of interest ( growth hormone) are recovered at the end. The pool is
then e4posed to high concentrations of .T8 (amplified) in the range of ).$ , "?.. This
increases the selection pressure and in order to survive, CHO cells typically undergo genomic
arrangements and amplification of the locus of 0;1 integration, which leads to increased
numbers of 0H6& and the growth hormone (:ayapal et al., +))%).
=hen the number of 0H6& and the growth hormone increases, isolation of a pool of cells
enriched for clones with a high specific yield will occur. This pool is heterogeneous,
containing cells with different integration sites and varying specific productivities. The
growth hormones produced need to be isolated (:ayapal et al., +))%).
$i%ure & Several common #ath'ays (rom sta)le #ool to clone scale*u#
1.& $ermentation in +ioreactor
Chinese hamster ovary cells start its fermentation in stirred,tan! bioreactors from the smaller
$ 2 vessel and finishing up in the larger $) 2 vessel. These vessels are e7uipped with a
pitched,blade impeller, with a porous macro,sparge and all the necessary tubing,filters and
connectors. The $ 2 culture is conducted in a batch style and the $) 2 culture is completed as
a fed,batch (Capone., +)"+).
6or the $ 2 bioreactor inoculation, CHO cells are used to inoculate a "+$ m2 sha!e flas! that
contains -) m2 of serum,free CHO medium added with 3m. 2,glutamine and "* enicilin,
9treptomycin. The initial sha!ing culture is e4panded to " 2 sha!e flas! containing +@) m2.
The inoculum is then grown for @ days until the viable cell density reached -."( 8 ")
(
cells A
m2 with a viability of ##.-*. On inoculation day, the bioreactor vessel is removed from its
sterile pac!aging and the heat blan!et is wrapped outside the vessel. The vessel containing
the cell culture medium is connected to one of the bioreactor vesselsB inlet lines using a 7uic!
connect. 1ppro4imately + 2 of sterile CHO serum,free medium is pumped into the vessel and
warmed to -%
)
C. =hen the growth medium achieved -%
)
C, the traditional polarographic
probe (0O probe) is calibrated to an electronic <ero and then spanned after the agitation is set
at $) rpm. The airflow is set to "))* at " 92. for nearly +) minutes. The optical pH
calibration is performed using the pH probe raw data and an offline sample is ta!en to re,<ero
the medium within the bioreactor. 0issolved o4ygen and pH are calibrated through the
touchscreen controller and all process set points are entered on the touchscreen of the
controller. Once the parameters are set, the inoculum flas!s are connected to the addition line
in a sterile manner using a 7uic! connect. Cells are then pumped into the bioreactor vessel for
a total volume of + 2 with an inoculation density of ).- 8 ")
(
cells A m2 (Capone., +)"+).
Once $ 2 bioreactor achieved sufficient density, the inoculum is connected to one of the $) 2
bioreactor vesselsB inlet lines using a 7uic! connect. This portion of the process is a fed batch
process with a starting volume less than +) 2, so only "%.3 2 of the medium is pumped into
the bioreactor vessel. +)* ;aOH is added for pH control. The polarographic probe on the $)
2 vessel is calibrated to an electronic <ero once the growth medium stabili<es to -%
)
C and
then spanned when the agitation is set at $) rpm and the airflow is set to "))* at %.$ 92.
for nearly +) minutes. =hen all the parameters are set as the previous stage, the calculated
".+ 2 of high density CHO cells are pumped in for a total volume of "# 2 with a final starting
cell density of ).- 8 ")
(
cells A m2. The $) 2 vessel is then fed with an additional +" litres of
pre,warmed CHO serum,free on day $ to support high cell growth and viability( Capone.,
+)"+).
!. ,O-.STREAM PROCESS
!.1 Protein /solation
In this stage of the growth hormone production, the growth hormone is present in the broth
and ready to be harvest. However, there is still high concentration of cells, nutrients, by
product and water present in the broth. Hence, it is important to first remove these substances
to further concentrate the growth hormone in the broth to ease the purification process later
stage. In most cases, mammalian cell produces e4tracellular product where the protein which
is the growth hormone in this case were secreted by the cells in the broth. This further ease
the recovery process as there is no need of cell lysis to obtain the protein present inside the
cell for intracellular product.
In the removal of suspended solid in the broth, a solid,li7uid separation is done before
removal of the cell. 9olid,li7uid separation can be done due to the difference in density and
si<e of the content. 1 dead end filtration (0/6) is usually chosen by the industries for this
purpose as dead end is simple and disposable compare to 0ead /nd Cltrafiltration (0/C6)
which is also widely available. 1 dead end filtration is cheaper and is easier to be cleaned as
there is no tremendous cleaning needed compared to 0/C6. 9ince growth hormone is
considered as large protein comparing to drugs li!e aspirin, 0/6 is used instead of 0/C6.
$i%ure 0 ,ead End $iltration Mechanism 1S#ectrum 2a)s !31&4
In the later stage, the broth is further concentrated by removing the cell via microfiltration.
Cells are usually much larger than the growth hormone and hence the cells can be easily
removed using microfiltration. There is many benefits of using microfiltration in the
industries as this filtration is highly selective, the separation without the need of any au4iliary
material, operation at ambient temperature, low capital cost and small unit si<e. (ele!a et. 1l
+))$) In microfiltration, the pore diameter of the membrane can be specified to be the si<e all
the micro molecules in the broth.
$i%ure 5 Micro(iltration Mechanism 1$umatech n.d4
2astly, growth hormone is concentrated by reducing the water content in the broth. 1
concentrated broth will ease the purification step as less chromatography column needed to
achieve a higher purity protein. This is important as it helps in saving operation cost in the
industries.
!.! Puri(ication
!.!.1 The Three Phase Strate%y
rotein purification is the most comple4 and time consuming stage for a recombinant protein
production. In order to achieve efficient protein purification, appropriate techni7ues are
chosen in a logical manner to suit the re7uirements and combine them in a manner to
ma4imi<e the yield with minimum number of steps. Chromatography is an important tool
re7uired for protein purification and various techni7ues combined together results in an
effective purification of proteins. The aim of a purification process is not only to remove the
unwanted contaminants, but also to attain the concentration of the desired protein enable its
transfer to an environment where it remains stable and suitable for the intended application.
The Three hase 9trategy aims at development of protein purification by combining various
such chromatographic techni7ues in Capture, Intermediate urification and olishing stages.
The capture phase aims to isolate, concentrate and stabilise the target product, while the
intermediate purification phase deals with the removal of bul! impurities such as other
nucleic acids, endoto4ins and viruses. In the final polishing phase the ob>ective is to achieve
high purity by removing any remaining trace impurities or closely related substances. It is to
be noted that the three phase strategy does not mean that all of the stages must necessarily
have three purification steps. In some cases, the capture and intermediate purification is
achievable in a single step and the number of steps will depend upon the purity re7uirements
and safety demands. 9ince a protein such as growth hormone is aimed for therapeutic use it
must be e4tremely pure and purification must then be done in several subse7uent steps.
2.2.2 Capture Step
The most common separation techni7ue at the capture stage is the ion e4change
chromatography (I/8), which ta!es place through electrostatic interactions that occur
between proteins and a charged stationary phase. The column to be used is selected based on
the protein type and the nature and strength of the ionic charge on the protein. The binding of
a protein to I/8 must be determined by trial and error, using solvents with a range of pH
values, to determine the optimum pH for protein retention. This techni7ue offers different
selectivity using either anion or cation e4changers as it varies according to the surrounding
pH conditions and when it is usually above the isoelectric point (pI), a protein will bind to a
positively charged anion e4changer, whereas below its pI, it will bind to a negatively charged
cation e4changer. The pH conditions of the separation can be modified in order to change the
charge characteristics of the protein molecules and due to this advantage, I/8 is used more
than once in the purification strategy, either for capture, intermediate purification or
polishing.
Typically, conditions for binding to one type of ion e4change will be more suitable for a
particular protein than the other and in case of growth hormone, anion e4change
chromatography has been found to be more suitable. The anion e4change resins have a
positive charge and they retain and separate negatively charged proteins. In this case it is
necessary to ma!e sure that pH of the buffer used should be atleast " unit higher than the pI
of the protein so that the protein molecules ac7uire negative charged and thereby bind to the
positively charged resin. /4amples of anion e4changers include the strong anion e4changer D
(7uaternary resin), and the wea! anion e4changer 0/1/ (diethylaminoethane). In the elution
stage, the proteins are eluted from the I/8 coloumn by increasing the concentration of a non,
buffering salt, such as ;aCl.These ions compete with the protein for binding sites on the resin
thereby removing the more wea!ly charged proteins at lower salt concentrations and the more
strongly charged proteins are eluted at higher salt concentrations (0onald,:.,+)")).
!.!.& /ntermediate Sta%e
Hydrophobic interaction chromatography (HIC) separates proteins based on their
hydrophobicity, and is often used as an intermediate step in a purification scheme. roteins
are bound to a stationary phase in a high ionic strength buffer and this method is typically
performed immediately after ion e4change chromatography with no buffer e4change or
dilution re7uired.
!.!.0 Polishin% Sta%e
Size Exclusion chromatography or gel fltration is ideal for the fnal polishing
steps when protein sample volumes have been reduced. This technique
separates proteins based on diferences in the molecular size and is therefore
not dependent on the adsorptive property of the molecules. This method is
frequently used in the fnal stages of protein purifcation and has been found to
be successful in the case of growth hormone due to its simple performance and
some features not found in other techniques. The principal advantage of gel
fltration is its gentle noninteraction with the sample protein thereby enabling
high preservation of biological activity. !s growth hormones are usually intended
as an in"ectable drug# it requires a high purity and therefore $%&' step should be
used near the end of the purifcation process to achieve high product purity
()ames# *. +,,,-.
urification of growth hormone is typically performed using combinations of
chromatography techni7ues including ion e4change, hydrophobic interaction, and metalE
chelate with a final gel filtration step principally for si<ing and removal of aggregate. It is a
highlighted fact that most of the downstream process applied to purify growth hormone
involves chromatographic methodology. However ion e4change chromatography is the most
fre7uently employed chromatographic method for purification of proteins such as human
growth hormones. 9everal studies suggests five to si4 purification steps re7uirement to
achieve the e4pected purity and the most common method used is repetitive ion,e4change
chromatography followed by gel filtration. 0uring purification and subse7uent storage, many
processes could occur which affects the protein 7uality such as protein unfolding,
aggregation, degradation, and there by leading to loss of function. Therefore it is very
important to have a successful purification scheme carefully planned to purify protein as
7uic!ly as possible under the most stabili<ing conditions.
2.3 Quality Control (QC) & Quality Assurance (QA):
In a pharmaceutical industries, Duality Control (DC) and Duality 1ssurance (D1) is very
important to ensure that the growth hormone produce meets the standard and 7uality
approved by the ;ational harmaceutical Control Fureau (;CF) from .alaysia .inistry of
Health. Duality Control (DC) includes purity test, identity test, 7uantity test and contaminant
test of the product. .eanwhile, Duality 1ssurance (D1) includes design control, production
G process control, e7uipment G facilities control and material control. The main reason D1
is ta!en place in the facilities is to ensure all drug has the same 7uality and uniform. Hence,
production practices such as 'ood .anufacturing ractice ('.), 'ood 2aboratory ractice
('2) and 'ood Clinical ractice ('C) is practices throughout the entire facilities.
1ll the facilities which follow '. will ensure that all processes is documented and
validated to ensure that there are no errors in the production of the drug. Fesides that, the
facilities must ensure that all e7uipment is under good condition and also ensure that the
facilities is always clean and safe. This can be achieved by carrying out regular audits to
chec! on the wor!ers and the facilities.
.eanwhile, '2 includes carrying out purity, identity, 7uantity and contaminant test on raw
material and product. 1ll of these tests is done in the laboratory using a High erformance
2i7uid Chromatography (H2C). This device helps in identifying the compound of the
product and the 7uantity of all compound present in the product.
$i%ure 6 Hi%h Per(ormance 2i7uid Chromato%ra#hy 1HP2C4
In addition to that, 'C ensures the patient safety, data integrity and ensuring results are
properly recorded. 'C is the most important measures where the safety of consumer health
is prioritise. Hence, certification from national body such as ;CF and 6ood and 0rug
1dministration (601) is important to approve that the drug produce from the facilities is
safe.
2astly, safety and cleanliness is another important aspect to discuss. 9afety and cleanliness is
classified into personnel, process and facilities. ersonnel safety and cleanliness states mainly
on the rule on the dress code in the facilities. One must always wear laboratory coat and
covered shoes when entering the facilities. Fesides that, one must follows the facilities rules
and report any accident happen in the facilities.
In process safety and cleanliness, the process e7uipment should undergoes sterilisation with
suitable cleaning solution or steam. 1lso, microbiological monitoring is done on the utilities
such as the water, steam and air to measure and identify the present of bacteria. .eanwhile,
in facilities safety and cleanliness, measure in this aspect is includes facility floor plan,
material and people flow and the design of the facilities.
&.Re(erences
Capone, :, 5ohlstrom, ; and 9ha, . +)"+, H9ingle,Cse 9calability I CHO Cell Culture using
$ to $) 2 ;ew Frunswic! Celli'en JF2C Fenchtop 9tirred,Tan! FioreactorsB, Eppendorf
Inc., /nfield, CT ,C.9.1
0onald,:. (+)")). Ion chromatography. Liquid chromatography. "$ (+), (,"$.
6umatech n.d, Microfiltration, viewed # 9eptember +)"-, httpIAAwww.fumatech.com
Ham &'. Clonal growth of mammalian cells in a chemically defined, synthetic medium.
roc. ;atl. 1cad. 9ci. C91 $-I +33,+#-, "#($.
:ayapal, .5, =laschin, 5.6, Hu, =.9, Kap, '.9.. +))%, H &ecombinant rotein
Therapeutics from CHO cells E +) years and countingB, CHO Consortium- SE Special
Section ECniversity of .innesota and Fioprocessing Technology Institute, Fiomedical
9ciences Institute, C.9.1
:ames, 6. (+)))). Ion chromatography. !nion chromatography. ( (-), ")+,""3.
:oseph, C +))), H Chinese Hamster Ovary Cells for the roduction of &ecombinant
'lycoproteinsB, !rt to Science "ournal, vol. "#, no. "
5ao 6T, uc! TT. 'enetics of somatic mammalian cells, LII. Induction and isolation of
nutritional mutants in Chinese hamster cells. roc. ;atl. 1cad. 9ci. C91 ()I "+%$,"+3", "#(3
ele!a, /, 6anidou, ., .avros, G .atis, 5 +))$, ! hy#rid flotation $ microfiltration cell
for solid%liquid separation& operational characteristics, /lsevier, Thessaloni!i, 'reece.
9pectrum 2abs +)"-, 'angential (lo) *s +ead End (iltration, viewed # 9eptember +)"-,
httpIAAwww.spectrumlabs.com