Downstream Processing
in Biopharmaceutical Manufacturing
Harvest and Clarification
Tangential Flow Filtration (UF/DF)
Low Pressure Liquid Column Chromatography
Know the Characteristics of Your Protein
Human Serum Albumin
Sequence of Amino Acids
MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE
EHFKGLVLIA FSQYLQQCPFDEHVKLVNEL TEFAKTCVAD
ESHAGCEKSL HTLFGDELCK VASLRETYGMADCCEKQEP
ERNECFLSHK DDSPDLPKLK PDPNTLCDEFKADEKKFWGK
YLYEIARRHP YFYAPELLYYANKYNGVFQE CCQAEDKGAC
LLPKIETMRE KVLTSSARQR LRCASIQKFG ERALKAWSVA
RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR
ADLAKYICDN QDTISSKLKECCDKPLLEKS HCIAEVEKDA
IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR
HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL
KHLVDEPQNL IKQNCDQFEKLGEYGFQNAL IVRYTRKVPQ
VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL
NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY
VPKAFDEKLF TFHADICTLPDTEKQIKKQT ALVELLKHKP
KATEEQLKTV MENFVAFVDK CCAADDKEACFAVEGPKLV
WSTQTALA
Tertiary Structure
Know the Characteristics of Your Protein
Human Serum Albumin:
• MW (molecular weight = 69,000 Daltons (69 kD)
• pI (isoelectric point) = 5.82
• Hydropathicity (=hydrophobicity) = -.395
Typical Production Process Flow
Ampule Thaw
Inoculum Expansion
(Spinner Bottles)
(Feed1)
(Feed 2)
Centrifuge
(Feed 3)
(Feed 4)
Concentration /
Diafiltration
Cryo-preservation
Chrom 3
Chrom 2
Viral Removal
Filtration
Chrom 1
LSCC Mfg Process Overview
Large Scale Bioreactor
1 day
Media Prep
Seed Bioreactors
Working Cell
Bank
150L
Bioreactor
SubCulture
SubCulture
SubCulture
SubCulture
SubCulture
26,000L
Bioreactor
Centrifuge
Depth
Filtration
Wave
Bag
Collection
Harvest/Recovery
Fermentation
Inoculum
24 days
31 days
Filter
Harvest
Collection
Tank
750L
Bioreactor
5,000L
Bioreactor
Column
Chromatography
Skid
Eluate
Hold
Tank
Viral
Inactivation
Eluate
Eluate Hold
Hold Tank
Tank
20,000L
20,000L
Column
Chromatography
Skid
8,000L
1,500L
Anion Exchange
Chromatography (QXL)
Hydrophobic Interaction
Chromatography (HIC)
Purification
Filter
Column
Chromatography
Skid
Filter
Eluate
Hold
Tank
Post-viral
Hold
Vessel
3,000L
6,000L
Protein A
Chromatography
Viral Filtering
Column
Chromatography
Skid
5,000L
Anion Exchange
Chromatography
8 days
Eluate
Hold
Tank
(QFF - Fast Flow)
Ultra Filtration
Diafiltration
Bulk
Fill
Clarification or
Removal of Cells and Cell Debris
Using Centrifugation
(Using Depth Filtration)
Continuous Centrifugation
Media and Cells In & Clarified Media Out
Filtration
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.
Tangential Flow Filtration
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 – 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.
Overview of TFF SOP
• Prepare buffer:
Sodium phosphate buffer pH 7.1
• Set up the apparatus-CAUTION Stored in NaOH
• Flush with water-CAUTION Stored in NaOH
Adjust flow rate to 30-50ml/min
Flush retentate line
Flush permeate line
• Precondition with buffer (just the permeate line)
•
•
•
•
Perform TFF
Prepare cleaning solution (NaOH)
Flush with water
Flush with NaOH to clean and store
Downstream Processing Equipment
Lab-Scale TFF System
Large-Scale TFF System
Lab-Scale TFF Filter = Pall’s Pellicon
How TFF Concentrates and Diafilters
the Protein of Interest
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.
UV Detector
Detects proteins coming out of the column
by measuring absorbance at 280nm
Conductivity Meter
• Measures the amount of salt in the buffers –
high salt or low salt are often used to elute
the protein of interest from the
chromatography beads.
• Also measures the bolus of salt that may be
used to determine the efficiency of column
packing (HETP)
Liquid Column Chromatography Process
Purge Air from System with Equilibration Buffer
Pack Column with Beads (e.g. ion exchange,
HIC, affinity or gel filtration beads)
Equilibrate Column with Equilibration Buffer
Load Column with Filtrate containing Protein of
Interest in Equilibration Buffer
Wash Column with Equilibration Buffer
Elute Protein of Interest with Elution Buffer of
High or Low Salt or pH
Regenerate Column or Clean and Store
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.
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
Gel Filtration Chromatography
Ion Exchange Chromatography
Ion Exchange Chromatography relies on charge-charge
interactions between the protein of interest and charges on a
resin (bead).
Ion exchange chromatography can be subdivided into cation
exchange chromatography, in which a positively charged
protein of interest binds to a negatively charged resin; and
anion exchange chromatography, in which a negatively
charged protein of interest binds to a positively charged
resin.
One can manipulate the charges on the protein by knowing
the pI of the protein and using buffers of different pHs to
alter the charge on the protein.
Once the protein of interest is bound, the column is washed
with equilibration buffer to remove unattached entities.
Then the bound protein of interest is eluted off using an
elution buffer of increasing ionic strength or of a different pH.
Either weakens the attachment of the protein of interest to
the bead and the protein of interest is bumped off and eluted
from the resin.
Ion exchange resins are the cheapest of the chromatography
media available and are therefore almost always used as a
step in biopharmaceutical protein production purification.
Isoelectric Focusing or IEF
Once you know the pI of your
protein (or the pH at which
your protein is neutral), you
can place it in a buffer at a
lower or higher pH to alter its
charge. If the pH of the buffer
is less than the pI, the protein
of interest will become
positively charged. If the pH of
the buffer is greater than the
pI, the protein of interest will
become negatively charged.
pH < pI < pH
+
0
-
Affinity Chromatography
Affinity chromatography separates the protein of
interest on the basis of a reversible interaction
between it and its antibody coupled to a
chromatography bead (here labeled antigen) .
With high selectivity, high resolution, and high
capacity for the protein of interest, purification levels
in the order of several thousand-fold are achievable.
The protein of interest is collected in a purified,
concentrated form. Biological interactions between
the antigen and the protein of interest can result
from electrostatic interactions, van der Waals' forces
and/or hydrogen bonding. To elute the protein of
interest from the affinity beads, the interaction can
be reversed by changing the pH or ionic strength.
The concentrating effect enables large volumes to be
processed. The protein of interest can be purified
from high levels of contaminating substances.
Making antibodies to the protein of interest is
expensive, so affinity chromatography is the least
economical choice for production chromatography.
Affinity Chromatography
Abs 280nm
Time (min)
Hydrophobic Interaction Chromatography (HIC)
HIC is finding dramatically increased use in
production chromatography. Antibodies
are quite hydrophobic and therapeutic
antibodies are the most important proteins
in the biopharmaceutical pipeline. Since
the molecular mechanism of HIC relies on
unique structural features, it serves as a
non-redundant option to ion exchange,
affinity, and gel filtration chromatography.
It is very generic, yet capable of powerful
resolution. Usually HIC media have high
capacity and are economical and stable.
Adsorption takes place in high salt and
elution in low salt concentrations.
Common Process Compounds and Methods of Purification or Removal
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
106/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
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
Conductivity Meter
UV Detector
Injector Valve
Column
Buffer Select
Mixer
Peristaltic
Pump
The Virtual Chromatography Laboratory
http://ATeLearning.com/BioChrom/