BIOMAN 2009 - K. Lampe, MC3 U P S T R E A M D O W N S T R E A M B I O P R O C E S S I N G ...

BIOMAN 2009 - K. Lampe, MC3

U P S T R E A M

D O W

N S T R

E A M

B I O P R O C E S S I N G

www.integra-biosciences.com

http://www.pharmaceutical-technology.com/contractors/purification/amersham/


Protein Biochemistry - Part I

Background

Up-Stream Processing Scale-up

Production Down-Stream Processing

IsolationPurification

CharacterizationFinishing / Fill

BIOMAN – 2011

K. Lampe, MC3


Proteins are EVERYTHING

genes are just a means to an end


Proteins => Traits

Single-celled Simple multi-cellular Complex multi-cellular organism organism organismcharacteristics characteristics characteristics

Organ characteristics

Tissue characteristics

Set of cellular structural and functional characteristics

Set of cellular products

Genes


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


Protein Basics - 1

• 1 Gene 1 protein

• Translation polypeptide

• Maturation / Modification protein


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.


Protein Basics – 2b

• Sorting / Transport / Insertion

• Activation / Inactivation

• Degradation

• Structure determines function


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


Why do Protein Biochemistry ?

A) Biotech. / Biopharm. / Manufacturing

• Product isolation• Product purification• Product modification• Product characterization• Product stability / storage


Why do Protein Biochemistry ?

B) Research

• Product isolation• Product purification• Product modification• Product characterization• Product stability / storage


Methods for Identifying & Localizing

• Study mutants

• Ligand binding

• In situ hybridization

• Chimeric (tagged) proteins (made GFP famous)


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


Methods for Isolating & Purifying – 2a

• Scheme depends upon location and character of protein– Location, e.g…

• Cytoplasmic free• Cytoplasmic inclusions/vesicles• Membrane-bound• Cell wall• Secreted


Methods for Isolating & Purifying – 2b

• Scheme depends upon location and character of protein– Character, e.g…

• Hydrophobic / hydrophilic• Globular / fibrous• Large / small• Non-protein components ?• Secreted


Methods for Isolating & Purifying – 2c

• Denaturing– Is it likely to occur ?– Does it matter at this point ?


Methods for Isolating & Purifying – 2d

• Basic Steps

– #1 Crude preparation– Cell homogenate

– Cell media

– #2 Initial fractionation / “clarification” / concentration

– #3 Subsequent purification / “polishing”


#1 - Crude Preparation

• Cell homogenate (cell lysate / lysed cells)– Freeze thaw– Lysozyme– Sonication– Homogenization– Osmotic Pressure / Pressure– Shearing– Detergent / Organic Solvent

• Cell media– Centrifugation / filtration


#2 - Fractionation / Clarification / Concentration

• Differential precipitation / de-solublization– Salts vs. organics

• Differential re-solublization

• Filtration

• Direct to chromatography


#3 - Subsequent Purification

• Filtration / Dia-filtration

• Chromatography (elaboration to follow)– Size (GF / SEC)– Hydrophobic Interaction (HIC)– Ion Exchange (IEC)– Affinity (AC)

• Ligand• Antibody• Antigen


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


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 !


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


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


Chromatography Variations

• Properties of the retentive material – continued

– 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)• 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


Chromatography Principle

• 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

• With Chromatography, everything is RELATIVE

– With SEC…

– 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)

– 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

– A single mobile phase can be run continuously through / over the stationary phase

– Molecule of interest is collected as it washes off of (“elutes” from) the stationary phase


Chromatography Principle

• With IXC, HIC, and AC…

– 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

– 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

– Load Wash Elute


Chromatography Abbreviations

• STATIONARY PHASE– TLC – PC– CC

• MOBILE PHASE– GC– LC

• FLOW RATE / PRESSURE– LP (e.g. LPLC)– HP (e.g. HPLC)

• RETENTIVE MATERIAL PROPERTY– SEC / GFC– IEC (IXC) / AEC (AXC) / CEC (CXC)– HIC– AC


Chromatography Abbreviations

• STATIONARY PHASE– TLC thin layer (plate) chromatography– PC paper chromatography– CC column chromatography

• MOBILE PHASE– GC gas chromatography– LC liquid chromatography

• FLOW RATE / PRESSURE– LP low pressure (e.g. LPLC)– HP high pressure (e.g. HPLC)

• RETENTIVE MATERIAL PROPERTY– SEC / GFC size exclusion / gel filtration chromatography– IEC (IXC) / AEC (AXC) / CEC (CXC) ion exchange chromatography – HIC hydrophobic interaction chromatography– AC affinity chromatography


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


Size Exclusion / Gel Filtration ChromatographySize Exclusion / Gel Filtration Chromatographyhttp://www.science.fau.edu/chemistry/Mari/biochemlab/05_012.jpghttp://www.science.fau.edu/chemistry/Mari/biochemlab/05_012.jpg


• Preparative vs. analytical– LPLC usually preparative– HPLC usually analytical

– Isocratic, step gradient, continuous gradient

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BIOMAN 2009 - K. Lampe, MC3 U P S T R E A M D O W N S T R E A M B I O P R O C E S S I N G ...

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