Downstream Processing in

Biopharmaceutical Fermentation
Alwani Hamad, ST, MSc.
Fakultas Farmasi
Universitas Muhammadiyah Purwokerto

Teknologi Fermentasi
MK: Bioteknologi
2
Course content
Persyaratan proses fermentasi
Kondisi dan variable fermentasi
Fermenter/ bioreaktor
Media dan optimasi media untuk fermentasi
Kinetika pertumbuhan bakteri untuk menghitung hasil
produk fermentasi
Downstream process hasil fermentasi (produk
biopharmacy)
Evaluasi : Ujian (closed book)
Buku rujukan:
G Rao.2007. Introduction to Biochemical Engineering. Tata Mc Graw- Hill Publishing
Company Limited
Stanbury, Whitaker and Hall, 2003. Principles of Fermentation Technology Butterworth



Typical Production Process Flow












(Feed 2)











(Feed 3)











(Feed 4)
Chrom 1
Chrom 3
Cryo-preservation
Concentration /
Diafiltration


Centrifuge
Viral Removal
Filtration












(Feed1)
Inoculum Expansion
(Spinner Bottles)
Ampule Thaw
Chrom 2
Media Prep
Working Cell
Bank
Sub-
Culture
Inoculum
Sub-
Culture
Sub-
Culture
Sub-
Culture
Sub-
Culture
Large Scale Bioreactor
Wave
Bag
Seed Bioreactors
Fermentation
150L
Bioreactor
750L
Bioreactor
5,000L
Bioreactor
26,000L
Bioreactor
Depth
Filtration
Collection
Centrifuge
Harvest/Recovery
Harvest
Collection
Tank

1,500L
Filter
Chromatography
Skid
Anion Exchange
Chromatography (QXL)
Column Eluate
Hold
Tank

8,000L
Eluate
Hold
Tank

6,000L
Filter
Chromatography
Skid
Protein A
Chromatography
Column
Chromatography
Skid
Column
Eluate
Hold
Tank

20,000L
Hydrophobic Interaction
Chromatography (HIC)
Eluate
Hold
Tank

20,000L
Viral
Inactivation
Eluate
Hold
Tank

5,000L
Filter
Chromatography
Skid
Anion Exchange
Chromatography
(QFF - Fast Flow)
Column
Post-viral
Hold
Vessel
3,000L
Viral Filtering Ultra Filtration
Diafiltration
Bulk
Fill
Purification
24 days 31 days
8 days
1 day
Upstream/Downstream Manufacturing Overview
Hasil proses fermentasi
Mikroorganisme
Product
Whole cells
Cell debris/fragments
Soluble and insoluble medium product
Proteins
Undissolved nutrient components
biomass
Downstream process dalam
Fermentasi
Langkah proses downstream merupakan salah satu langkah dalam proses
fermentasi yang sangat penting yaitu langkah setelah proses fermentasi
untuk menghasilkan bioproduk dengan menggunakan unit operation agar
diperoleh produk yang terpisah dari campuran yang lain
Dalam downstream process : cost 20 60% dari total cost proses. >90%
untuk produk rekombinant DNA
Downstream processing steps = operasi pemurnian produk hasil
fermentasi
Langkah langkah dalam downstream process
Initial isolation
Product recovery
Purification and concentration


Hal yang dipertimbangkan dalam rangka
pemilihan jenis unit operation dalam
downstream process
Lokasi produk : intracelluler atau ekstracelluler
Kestabilan terhadap panas
Jumlah produk di dalam broth
Cost product
Kegunaan produk
Minimal standar yang dapat diterima
Komposisi impuritas yang diijinkan dalam produk
Jenis unit operation yang digunakan
dalam downstream process
Lihat table 17.1 hal 158 Rao Introduction of biochemical engineering
Dapat dibagi sebagai berikut :
Pemisahan suspended solid
Filtrasi
Sedimentasi
Centrifugation
Foam separation
Precipitation
Cell Disruption
Pemisahan satu fasa
Ekstraksi
kromatografi


Cell disruption
Mechanical method
High speed agitation
Grinding with abrasives
High pressure pumping (homogenization)
Non-mechanical method
Osmotic shock
Treatment with solvent and detergents
Freezing and thawingf
Enzymatic digestion of cell walls
Know the Characteristics of
Your Protein
Green Fluorescent Protein
(GFP)
Sequence of Amino Acids
MSKGEELFTGVVPVLVELDGDVNGQKF
SVSGEGEGDATYGKLTLNFICTTGKL
PVPWPTLVTTFSYGVQCFSRYPDHM
KQHDFFKSAMPEGYVQERTIFYKDD
GNYKTRAEVKFEGDTLVNRIELKGID
FKEDGNILGHKMEYNYNSHNVYIMG
DKPKNGIKVNFKIRHNIKDGSVQLAD
HYQQNTPIGDGPVLLPDNHYLSTQS
ALSKDPNEKRDHMILLEFVTAARITH
GMDELYK

Tertiary Structure
Contoh downstream process dalam
industri biopharmacy
Know the Characteristics of Your Protein
Green Fluorescent Protein (GFP)
MW (molecular weight = 27,000 Daltons (27 kD)
pI (isoelectric point) = 4.8
Hydropathicity (=hydrophobicity) =

Tissue Plasminogen Activator

MW 60 kD pI: 8.04 Hydrophobicity -.516
Human Serum Albumin
mkwvtfisll llfssaysrg vfrrdthkse iahrfkdlge ehfkglvlia fsqylqqcpf
61 dehvklvnel tefaktcvad eshagceksl htlfgdelck vaslretygd madccekqep
121 ernecflshk ddspdlpklk pdpntlcdef kadekkfwgk ylyeiarrhp yfyapellyy
181 ankyngvfqe ccqaedkgac llpkietmre kvltssarqr lrcasiqkfg eralkawsva
241 rlsqkfpkae fvevtklvtd ltkvhkecch gdllecaddr adlakyicdn qdtissklke
301 ccdkplleks hciaevekda ipenlpplta dfaedkdvck nyqeakdafl gsflyeysrr
361 hpeyavsvll rlakeyeatl eeccakddph acystvfdkl khlvdepqnl ikqncdqfek
421 lgeygfqnal ivrytrkvpq vstptlvevs rslgkvgtrc ctkpesermp ctedylslil
481 nrlcvlhekt pvsekvtkcc teslvnrrpc fsaltpdety vpkafdeklf tfhadictlp
541 dtekqikkqt alvellkhkp kateeqlktv menfvafvdk ccaaddkeac favegpklvw
601 stqtala




MW 69 kD pI 5.82 Hydrophobicity -.395

1 rrgarsyqvi crdektqmiy qqhqswlrpv lrsnrveycw cnsgraqchs vpvkscsepr 61
cfnggtcqqa lyfsdfvcqc pegfagkcce idtratcyed qgisyrgtws taesgaectn 121
wnssalaqkp ysgrrpdair lglgnhnycr npdrdskpwc yvfkagkyss efcstpacse 181
gnsdcyfgng sayrgthslt esgasclpwn smiligkvyt aqnpsaqalg lgkhnycrnp 241
dgdakpwchv lknrrltwey cdvpscstcg lrqysqpqfr ikgglfadia shpwqaaifa 301
khrrspgerf lcggilissc wilsaahcfq erfpphhltv ilgrtyrvvp geeeqkfeve 361 kyivhkefdd
dtydndiall qlksdssrca qessvvrtvc lppadlqlpd wtecelsgyg 421 khealspfys
erlkeahvrl ypssrctsqh llnrtvtdnm lcagdtrsgg pqanlhdacq 481 gdsggplvcl
ndgrmtlvgi iswglgcgqk dvpgvytkvt nyldwirdnm rp
Some Other Proteins of Interest
Clarification or
Removal of Cells and Cell Debris
Using Centrifugation
Using Depth Filtration
Control Panel
Cut-away view
Protective enclosure
Basic components of a centrifuge
Door
Rotor
Drive
shaft
Motor
Centrifugal force
Sedimentatio
n
path of
particles
Pellet
deposited
at an angle
C
e
n
t
e
r

o
f

r
o
t
a
t
i
o
n

r
minimum
r
average
r
maximum
Centrifuge
An instrument that generates centrifugal force.
Commonly used to separate particles in a liquid from the liquid.
Continuous Centrifugation
Media and Cells In & Clarified Media Out

Separation of particles from liquid by applying
a pressure to the solution to force the solution through a
filter. Filters are materials with pores.

Particles larger than the pore size of the
filter are retained by the filter.

Particles smaller than the pore size of the filter pass
through the filter along with the liquid.

Filtration
Traps contaminants larger than the pore size on the top surface of the membrane.
Contaminants smaller than the specified pore size pass through the membrane.
Used for critical applications such as sterilizing and final filtration.
Normal Flow Filtration
Depth Filtration:
Equipment
Depth Filtration: Cells and Cellular Debris
Stick to Ceramic Encrusted Fibers in Pads
PROTEIN of INTEREST
Uses crossflow to reduce build up
of retained components on the
membrane surface

Allows filtration of high fouling
streams and high resolution
Tangential Flow Filtration
vs. Normal Flow Filtration
Tangential Flow Filtration
vs. Normal Flow Filtration
Tangential Flow Filtration TFF
Separation of Protein of Interest
Using TFF with the right cut off filters, the protein of interest
can be separated from other proteins and molecules in the
clarified medium.
HSA has a molecular weight of 69KD. To make sure that the
protein of interest is retained, a 10KD cut-off filter is used.
After we concentrate or ultrafilter our protein, we can
diafilter, adding the phosphate buffer at pH 7.1 that we will
use to equilibrate our affinity column to prepare for affinity
chromatography of HSA.
How TFF Concentrates and Purifies
a Protein of Interest
Downstream Processing Equipment






Lab-Scale TFF System






Large-Scale TFF System
Low Pressure Production
Chromatography
The System: Components and Processes
The Media: Affinity, Ion Exchange,
Hydrophobic Interaction Chromatography
and Gel Filtration
LP LC Components
Mixer for Buffers, Filtrate with Protein of
Interest, Cleaning Solutions
Peristaltic Pump
Injector to Inject Small Sample (in our case for
HETP Analysis)
Chromatography Column and Media (Beads)
Conductivity Meter
UV Detector





Peristaltic Pump
Creates a gentle
squeezing action to
move fluid through
flexible tubing.
Liquid Column Chromatography
Lonza, Portsmouth, NH
A Commercial LP LC
Chromatography Column
Downstream Processing Equipment
Lab Scale
Chromatography System
Large Scale
Chromatography System
Overview of LP LC Chromatography
The molecules of interest, in our case proteins, are adsorbed or
stuck to beads packed in the column. We are interested in the
equilibrium between protein free in solution and protein bound
to the column. The higher the affinity of a protein for the bead
the more protein will be bound to the column at any given time.
Proteins with a high affinity travel slowly through the column
because they are stuck a significant portion of the time.
Molecules with a lower affinity will not stick as often and will
elute more quickly. We can change the relative affinity of the
protein for the column (retention time) and mobile phase by
changing the mobile phase (the buffer). Hence the difference
between loading buffers and elution buffers. This is how proteins
are separated.
The most common type of adsorption chromatography is ion
exchange chromatography. The others used in commercial
biopharmaceutical production are affinity, hydrophobic
interaction and gel filtration.
Liquid Chromatography
Protein solution is applied to a
column
Column filled with matrix (stationary
phase) + liquid phase (mobile phase)
Proteins separated based on
differing affinity for the stationary
and mobile phases
1 2 3 4
Column Chromatography
Separates molecules by their chemical and
physical differences
Most common types:
Size exclusion (Gel filtration): separates by
molecular weight
Ion exchange: separates by charge
Affinity chromatography: specific binding
Hydrophobic Interaction: separates by
hydrophobic/hydrophilic characteristics


Component
Culture Harvest
Level
Final Product
Level
Conventional
Method
Therapeutic Antibody 0.1-1.5 g/l 1-10 g/l UF/Cromatography
Isoforms Various Monomer Chromatography
Serum and host proteins 0.1-3.0 g/l < 0.1-10 mg/l Chromatography
Cell debris and colloids 10
6
/ml None MF
Bacterial pathogens Various <10
-6
/dose MF
Virus pathogens Various
<10
-6
/dose (12
LRV)
virus filtration
DNA 1 mg/l 10 ng/dose Chromatography
Endotoxins Various <0.25 EU/ml Chromatography
Lipids, surfactants 0-1 g/l <0.1-10 mg/l Chromatography
Buffer Growth media Stability media UF
Extractables/leachables Various <0.1-10 mg/l
UF/
Chromatography
Purification reagents Various <0.1-10mg/l UF
Common Process Compounds and Methods of Removal or Purification
Biopharmaceutical Production Overview
Typical Process Flow












(Feed 2)











(Feed 3)











(Feed 4)
Chrom 1
Chrom 3
Cryo-preservation
Concentration /
Diafiltration


Centrifuge
Viral Removal
Filtration












(Feed1)
Inoculum Expansion
Ampoule Thaw
Chrom 2
What Will Change During Scale-up?
Process Development Considerations
Utility requirements
Water requirement
Cleaning/Sanitizing solution requirements
Buffer prep
Number of steps in cell culture scale up
Harvest techniques
Column packing; distribution of introduced liquid at
large columns
Equipment bubble trap
Automation of process
Data collection
Sample load

How to survive when scale up process?
Understanding the physics, chemistry and biology of the
chromatographic system and the binding of the protein of
interest to the chromatographic matrix or beads (Science)
Understanding the design and operation of chromatography
components and of the chromatographic process (Technology
and Engineering).
Understanding the calculations needed to run the
chromatographic system (column volume) and the
measurements on chromatograms needed to calculate the
HETP, number of theoretical plates, retention time, and
resolution (Mathematics).
Actual BioLogic System
Complex System
Not easy to see
interaction of components
Students use virtual
system to prepare to use
actual system
Use virtual system for
BIOMANonline
System same as industrial
chromatography skid
Further reading
G Rao.2007. Introduction to Biochemical
Engineering , Chapter 17



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