Process Development Take a few minutes to think about what
steps would be necessary to develop a protein based medicine
product?
Slide 3
Fermentation Pathway A typical cell culture process comprises
vial thaw, seed expansion, and production stages.
Slide 4
Cell Culture Protein A Capture Column Anion Exchange Viral
filtration Fill and Finish Ultrafiltratio n/ Diafiltration Drug
Substance Viral Inactivation Cation Exchange Generic Mab Process
Centrifuge & Depth Filtration
Slide 5
General steps in Process Development for a Protein Based
Medicine Drug target selection Expression system and host (
R&D) Basic media and culture optimization, choose lead clone(s)
Fermentation process (Upstream Process) Clarification system
(Midstream process)
Slide 6
General steps in Process development Downstream isolation and
purification Formulation of final drug product and stability
testing Bioanalytical testing, Meet target product profile (TPP)
QA/QC, and regulatory affairs Other points, i.e. cell banking,
process robustness, viral clearance, toxicity studies, technology
transfer
Slide 7
Proteins as Biotechnology Products Making a Biotech Drug
Produced through microbial fermentation or mammalian cell culture
Complicated and time-consuming process Must strictly comply with
regulatory agencies at all stages of the process.
Slide 8
Recombinant proteins
Slide 9
Expression system and Host Related, need to know host to figure
out best expression system, bacteria, fungi, plant, mammalian, etc.
E. coli, bacteria A. niger, fungi mammalian ie CHO, NSO
Slide 10
Host selection Simple molecules can be expressed in bacteria
plants or fungus more efficiently and cheaper Complex molecules
like antibodies with glycosolation may need a mammalian host
Chinese Hamster Ovary (CHO) cells are the most frequently used,
grow fast and produce well. Plus have already had regulatory
approval! Human cells sometimes used, such as HEK 293 cells
(embryonic kidney)
Slide 11
Protein Modifications Glycosylation post-translational
modification wherein carbohydrate units are added to specific
locations on proteins. More than 100 post-translational
modifications can occur.
Slide 12
Glycosylation reactions in the Golgi Thorsten Marquardt Nature
Medicine 10, 457 - 458 (2004)
Slide 13
Different organisms have different Glycosylation patterns
Slide 14
Mammalian DMEM media COMPONENTS Many ( >80) and Well
defined! Inorganic Salts CaCl2, Fe(NO3)3 9H2O, MgSO4, KCl, NaHCO3,
NaCl, NaH2PO4 Amino Acids L-Alanyl, L-Glutamine, L-Arginine HCl, L-
Cysteine 2HCl, L-Glutamine, Glycine L-Histidine HCl H2O,
L-Isoleucine L-Leucine, L-Lysine HCl, L- Methionine,
L-Phenylalanine, L-Serine, L-Threonine, L- Tryptophan, L-Tyrosine
2Na 2H2O, L-Valine Vitamins Choline Chloride, Folic Acid,
myo-Inositol Niacinamide, D-Pantothenic Acid Ca, Pyridoxal HCl
Pyridoxine HCl, Riboflavin, Thiamine HCl Other D-Glucose, HEPES,
Phenol Red Na, Pyruvic Acid Na, Trace elements
Slide 15
General Fermentation Batch No feeding of the culture Fed Batch
Feeding of the culture Perfusion Simultaneous feeding and removal
of the culture Process for Antibody production reach 1-10 g/L Best
process typically not the most robust, manufacturers will take
10-20% reduction in productivity to achieve a reproducible
process
Slide 16
Necessary work before production Select molecule to produce
(DNA sequence) R&D Expression vector (Plasmid) R&D Host for
protein expression, bacteria, yeast, mammalian cells, etc. R&D
Pick lead clone to work with R&D and Fermentation Optimize
growth and feed media Fermentation defined media is best, no serum
if possible Optimize seed train, Fermentation typically 5x-10x
culture expansion each step
Slide 17
Consistency of Process is KEY! Need to get approval from
regulatory bodies, FDA, EMA, etc. The end process must meet cGMP
standards! Need consistent process for consistent product Need
stability testing of system. Start from Master Cell Bank (MCB)
Progress through seed train Then off to large scale
fermentation
Slide 18
Fermentation Time Table Ferm vesselVolume (L)Time (days)Total
time days MCB thaw0.0500 Shake flask 10.133 Shake flasks 20.436
Shake flasks 31.539 Wave bag 15312 Wave bag 220315 Reactor 1100318
Reactor 2500321 Reactor 32500324 Production reactor12500~13~37
Assumes cells with a 24 hour doubling time and ~ 4-5 x expansion
per step
Slide 19
Master Cell Bank (MCB) Why use MCB? Typically 250-500 vials
Store in Multiple locations Need consistency in process! Stability
of cell growth, productivity and product profile Heavily tested!!
Viruses, growth, product quality, etc. Some groups then create
Working Cell Bank (WCB) Once again typically ~250 - 500 vials
Slide 20
Growth Curves Plot the total viable cells at any time in the
culture Typically expressed as 10e6 viable cells/mL IVCD (integral
of viable cell density) Area under the curve Want to maximize IVCD
of culture, Why? Viable cell curveCalculating area under the
curve
Slide 21
Specific Productivity Each clone produces a characteristic
amount of product (ie antibody) Expressed as Picogram
(protein)/cell/day (PCD) When choosing a lead clone, want to
balance good specific productivity (PCD) with good growth (Max
IVCD) PCD X IVCD= mg/L of protein produced (for antibodies)
Example: 40 PCD and 50 IVCD = final culture with 2000 mg/L antibody
concentration
Slide 22
Bioreactor Basics
Slide 23
Seeding Production Reactor To few cells takes to long with lag
phase growth To many cells have carry over of metabolites and leads
to lag phase too. For CHO system optimal inoculation is ~10-20 % of
final culture volume. Typically seed between 0.15 and 0.4 x 10e6
Viable cells /mL For a bacterial system only need ~1% of final
culture volume for seeding
Slide 24
Bioreactor Parameters Dissolved Oxygen needed for cell growth
and metabolism To little and cells will be oxygen starved, reducing
growth and impacts on product characteristics To much leads to
oxygen stress of the cells and oxygen radicals and increased DNA
damage Typical setting range from 20-70% dissolved oxygen Measure
this on line with DO probes in bioreactor
Slide 25
Bioreactor Parameters Agitation Need to keep the cells in
suspension and well mixed for access to food and oxygen To little
agitation and cells can settle and can not adequately access food
and may start to clump or have growth inhibition. To much agitation
leads to mechanical shearing and damages cells. Adding certain
chemicals can help minimise shear effect ie. Pluronic F-68 at 0.1%.
Agitation rate needed depends on size of impeller used Slower RPM
with larger diameter
Slide 26
Bioreactor Parameters Temperature Need to optimize temperature
for the system For E. coli and most mammalian systems temperature
optimum is ~37 C Can temperature shift lower at times (32-35 C)
once cell numbers nearly reach the plateau to change from cell
growth to protein production. Temperature shift can lengthen
culture life (increase IVCD) by arresting cells in G1 phase of cell
cycle.
Slide 27
Bioreactor Parameters pH optimizing pH can have critical
impacts on productivity Typically in pH 6.8 7.2 range Measure on
line with probe in bioreactor Lowering pH can slow down cell growth
and productivity Increasing pH can raise productivity and increase
cellular metabolism as well as waste products Can alter pH at
various times in the culture to optimize protein production
Slide 28
Fed batch fermentation Fed Batch - feeding additional
components to the bioreactor during the course of the fermentation.
This typically improves cell numbers and product yield, but adds
complication to process. Main additives for mammalian system
Glucose Glutamine Starting media concentrations Glucose 6-8 g/L
Glutamine 6-10 mM
Slide 29
Fed batch fermentation Glucose or glutamine starvation. If
starved, cells stop growing, reducing product production Reaching 0
g/L glucose or 0 mM glutamine is very bad! Do not want to over feed
or cells will metabolize extra glucose and glutamine, leading to a
build up of waste products, ie. lactate and ammonia which inhibits
cell growth and product production. Over 8-10 g/L glucose and 8-10
mM glutamine leads to problems with cell growth and viability
Slide 30
Media Analysis Need to check various paramaters of your media
pH, Glucose, Glutamine, Ammonia, Lactate, Dissolved O2, CO2,
Potassium, Phosphate, Antibody conc. etc, Typically done with
automated analysers NOVA flex, BGA, etc.
Slide 31
Typical pH and lactate profiles in a fed-batch process
Slide 32
Assessing Cell numbers Manual counting methods, Hemacytometer
Automated methods Vi-Cell, Countess, CEDEX, etc Need to know cell
numbers to understand ferment HemacytometerVi-Cell
Slide 33
Assessing cell viability Want to keep cell viability as high as
possible for as long as possible Trypan blue assay dye exclusion
method (Vital dye) Live cells with intact membranes exclude dye
Dead cells take up dye and are stained dark blue Know health of
culture Trypan blue
Slide 34
Genentech video
http://www.dnalc.org/view/15503-Tour-of-Genentech-
David-Ebersman.html
http://www.dnalc.org/view/15503-Tour-of-Genentech-
David-Ebersman.html
Slide 35
Stainless Steel vs Disposable Stainless advantages Can use over
a long time, years Can make at any scale Stainless disadvantages
Cleaning costs Slow turn around time Minimal flexibility once
installed Disposable advantages No cleaning and validation costs
Rapid turn around time Flexible footprint Disposable disadvantages
Greater cost in long run Limited size (up to ~ 2000 L) at current
time
Slide 36
Conclusions Recombinant proteins can be made in a number of
different hosts. Some proteins need mammalian cells for proper
function (glycosylation) Drug target selection can be difficult
Lots of risk Expression systems vary and you need to know host and
product for informed choice
Slide 37
Conclusions II Need to select a good lead clone for
manufacturing, so you need good ones to choose from!!! Sounds
simple, but not always the case! Need to optimize pH, Temperature,
Oxygen levels, agitation for optimal process Need to optimize media
and feed parameters in a fed batch process. Need to constantly
monitor cell culture and media for best results.
Slide 38
Pharmatopia lab Next you will apply your gained knowledge in a
fermentation practical simulation in Pharmatopia Biotechbasic:
https://www.pharmatopia.monash.edu/
monashunity/bio/https://www.pharmatopia.monash.edu/
monashunity/bio/ Biotech advanced:
https://www.pharmatopia.monash.edu/biotech/
https://www.pharmatopia.monash.edu/biotech/
