BIOPROCESSING
www.integrabiosciences.com
BIOMAN 2009 - K. Lampe, MC3
Protein Biochemistry - Part I
Background
Up-Stream Processing 
Scale-up
Production
Down-Stream Processing 
Isolation
Purification
Characterization
Finishing / Fill
BIOMAN – 2011
K. Lampe, MC3
BIOMAN 2011 - K. Lampe, MC3
Proteins are EVERYTHING
genes are just a means to an end
BIOMAN 2009 - K. Lampe, MC3
Proteins => Traits
Single-celled
organism
characteristics
Simple multi-cellular
organism
characteristics
Complex multi-cellular
organism
characteristics
Organ characteristics
Tissue characteristics
Set of cellular structural and functional characteristics
Set of cellular products
Genes
BIOMAN 2009 - K. Lampe, MC3
Proteins => Targets
• Nearly ALL therapeutic compounds,
manufactured or natural, exert their effects
by interacting with one or another form of
protein…
Enzymes
Receptors
Channels
Transporters
Antigens
BIOMAN 2009 - K. Lampe, MC3
Protein Basics - 1
• 1 Gene
1 protein
• Translation  polypeptide
• Maturation / Modification  protein
BIOMAN 2009 - K. Lampe, MC3
Protein Basics – 2a
• Maturation / Modification  protein
– Folding (1o -> 2o -> 3o agg -> 4o)
• Largely spontaneous
– Hydrophobic/hydrophilic interactions
– H-bonding
– R-group ionic interactions
– Modification of in-chain A.A.’s
– Cleavage
– Addition of non-protein groups
• Glycosylation, acylation, phosphorylation, etc.
BIOMAN 2009 - K. Lampe, MC3
Protein Basics – 2b
• Sorting / Transport / Insertion
• Activation / Inactivation
• Degradation
• Structure determines function
BIOMAN 2009 - K. Lampe, MC3
What is Protein Biochemistry ?
-- After the molecular biology -• Expression / Synthesis / PTranslM / Sort &
Transport / Activate or Inactivate / Degrade
• Structure / Function
• Methods
• Identify / isolate / purify / modify
• Characterize
BIOMAN 2009 - K. Lampe, MC3
Why do Protein Biochemistry ?
A) Biotech. / Biopharm. / Manufacturing
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Product isolation
Product purification
Product modification
Product characterization
Product stability / storage
BIOMAN 2009 - K. Lampe, MC3
Why do Protein Biochemistry ?
B) Research
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Product isolation
Product purification
Product modification
Product characterization
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Product stability / storage
BIOMAN 2009 - K. Lampe, MC3
Methods for Identifying & Localizing
• Study mutants
• Ligand binding
• In situ hybridization
• Chimeric (tagged) proteins (made GFP famous)
BIOMAN 2009 - K. Lampe, MC3
Methods for Isolating & Purifying -1
• Primary difference between industrial and
research procedure is simply scale (methods and steps
generally comparable)
• Difficult at best… May be impossible (want to isolate
in native/active form)
• Scheme depends upon location and character of
protein
BIOMAN 2009 - K. Lampe, MC3
Methods for Isolating & Purifying – 2a
• Scheme depends upon location and
character of protein
– Location, e.g…
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Cytoplasmic free
Cytoplasmic inclusions/vesicles
Membrane-bound
Cell wall
Secreted
BIOMAN 2009 - K. Lampe, MC3
Methods for Isolating & Purifying – 2b
• Scheme depends upon location and
character of protein
– Character, e.g…
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Hydrophobic / hydrophilic
Globular / fibrous
Large / small
Non-protein components ?
Secreted
BIOMAN 2009 - K. Lampe, MC3
Methods for Isolating & Purifying – 2c
• Denaturing
– Is it likely to occur ?
– Does it matter at this point ?
BIOMAN 2009 - K. Lampe, MC3
Methods for Isolating & Purifying – 2d
• Basic Steps
– #1 Crude preparation
– Cell homogenate
– Cell media
– #2 Initial fractionation / “clarification” / concentration
– #3 Subsequent purification / “polishing”
BIOMAN 2009 - K. Lampe, MC3
#1 - Crude Preparation
• Cell homogenate (cell lysate / lysed cells)
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Freeze thaw
Lysozyme
Sonication
Homogenization
Osmotic Pressure / Pressure
Shearing
Detergent / Organic Solvent
• Cell media
– Centrifugation / filtration
BIOMAN 2009 - K. Lampe, MC3
#2 - Fractionation / Clarification /
Concentration
• Differential precipitation / de-solublization
– Salts vs. organics
• Differential re-solublization
• Filtration
• Direct to chromatography
BIOMAN 2009 - K. Lampe, MC3
#3 - Subsequent Purification
• Filtration / Dia-filtration
• Chromatography (elaboration to follow)
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Size (GF / SEC)
Hydrophobic Interaction (HIC)
Ion Exchange (IEC)
Affinity (AC)
• Ligand
• Antibody
• Antigen
BIOMAN 2009 - K. Lampe, MC3
How Pure ?
• “Protein Biochemistry – Part II”
– QC and Characterization
• How pure does it need to be ?
– Depends on use
– Depends on type of impurity(ies)
• FDA allows some level of impurity in biopharm.
product
BIOMAN 2009 - K. Lampe, MC3
GFP as a model for classroom
activities
• GFP is ideal
– Easily expressed
– Easily scaled up
– Relatively easy to “purify”
– Stable
– Amendable to further study (struct. / funct.)
– Looks very cool !
BIOMAN 2009 - K. Lampe, MC3
Chromatography
• Separation of molecules from masses of others based
on differences in characteristics of the molecules in the
mixture
• With chromatography… Everything is RELATIVE
– Many variations, but all are based on same concept
• Force mix of molecules to “race” along a narrow, restrained path
• Molecules interact with the path differentially… Some move along
the path faster than others
• Allow the molecules to move along the path far/long enough so that
the one you want is well separated from (most of) the rest
• Collect the molecule of interest
BIOMAN 2009 - K. Lampe, MC3
Chromatography Variations
• The Path (“Stationary Phase”)
– A column (tube) filled (packed) with some retentive material
(“resin”)
– A glass plate covered with a “thin layer” of retentive material
– Paper
• The Carrier (“Mobile Phase”)
– Liquid (“LC”) vs. Gas (“GC”)
• Flow Rate and Pressure of “Mobile Phase”
– Gravity vs. Low Pressure (“LP”) vs. High Pressure (“HP”)
• Properties of the retentive material
BIOMAN 2009 - K. Lampe, MC3
Chromatography Variations
• Properties of the retentive material – continued
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Sieve size (“size-exclusion” / “gel-filtration”)
Charge attraction (“ion exchange”)
Hydrophobic / Hydrophilic
“Affinity”; a specific size, shape, charge “fit” between the molecule of
interest and another (natural or synthetic)
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Antibody – Antigen
Receptor – Ligand
Enzyme – Substrate
Molecule of Interest – Synthetic “binder”
• Properties of retentive material and those of mobile phase are
customized (matched) to optimize separation of molecule of interest
from the rest in the mixture
BIOMAN 2009 - K. Lampe, MC3
Chromatography Principle
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Properties of retentive material and those of mobile phase are customized (matched)
to optimize separation of molecule of interest from the rest in the mixture
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With Chromatography, everything is RELATIVE
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With SEC…
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Pore size of resin is selected to either allow molecule of interest to pass through resin
“beads” (move relatively slowly through stationary phase) or go around resin (move relatively
quickly through stationary phase)
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Because some molecules will be larger, and others smaller than the molecule of interest, a
single pass through a SEC resin can not “purify” the molecule of interest
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A single mobile phase can be run continuously through / over the stationary phase
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Molecule of interest is collected as it washes off of (“elutes” from) the stationary phase
BIOMAN 2009 - K. Lampe, MC3
Chromatography Principle
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With IXC, HIC, and AC…
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Molecules in the mixture have a relative affinity (attractiveness) for the stationary phase and
the mobile phase
• Higher affinity for stationary phase – move along path slowly
• Higher affinity for mobile phase – move along path quickly
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Conditions may be established such that the molecule of interest is selectively (and
relatively) retained on the stationary phase OR be allowed to pass freely through the
stationary phase with the mobile phase
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Load  Wash  Elute
BIOMAN 2009 - K. Lampe, MC3
Chromatography Abbreviations
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STATIONARY PHASE
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MOBILE PHASE
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GC
LC
FLOW RATE / PRESSURE
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TLC
PC
CC
LP (e.g. LPLC)
HP (e.g. HPLC)
RETENTIVE MATERIAL PROPERTY
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SEC / GFC
IEC (IXC) / AEC (AXC) / CEC (CXC)
HIC
AC
BIOMAN 2009 - K. Lampe, MC3
Chromatography Abbreviations
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STATIONARY PHASE
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GC
LC
gas chromatography
liquid chromatography
FLOW RATE / PRESSURE
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thin layer (plate) chromatography
paper chromatography
column chromatography
MOBILE PHASE
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TLC
PC
CC
LP
HP
low pressure (e.g. LPLC)
high pressure (e.g. HPLC)
RETENTIVE MATERIAL PROPERTY
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SEC / GFC size exclusion / gel filtration chromatography
IEC (IXC) / AEC (AXC) / CEC (CXC) ion exchange chromatography
HIC hydrophobic interaction chromatography
AC
affinity chromatography
BIOMAN 2009 - K. Lampe, MC3
Gravity-Flow Liquid Column
Chromatography
• Mini columns packed with resin (stationary
phase)
– AX resin
– CX resin
– HI resin
• Mobile phase(s) depends upon particular
stationary phase
• Load sample
• Allow mobile phase to run through column by
gravity to “wash away” unwanted molecules
• “Elute” molecule of interest
BIOMAN 2009 - K. Lampe, MC3
Size Exclusion / Gel Filtration Chromatography
http://www.science.fau.edu/chemistry/Mari/biochemlab/05_012.jpg
BIOMAN 2009 - K. Lampe, MC3
• Preparative vs. analytical
– LPLC usually preparative
– HPLC usually analytical
– Isocratic, step gradient, continuous gradient
BIOMAN 2009 - K. Lampe, MC3