Production of Pharmaceuticals and Therapeutic Proteins

ajay patidar

HIV Therapeutic Agents (e 2nd)

Acquired immune deficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). The target of HIV are the T helper cells (TH). TH cells play a pivotal role in the immune system by the release of cytokines which stimulate other immune cells. The gp120 glycoprotein of HIV binds to CD4 receptors of TH cells. The TH cells become infected with the virus and are destroyed, slowly shutting down the immune system.

Interaction of HIV with CD4

HIV Therapeutic Agents

HIV antiviral strategies may include: Production of antibodies to CD4 (will block CD4 receptors on TH cells and prevent infection by HIV). Production excess CD4 protein (react with gp120 protein therefore HIV cannot infect TH cells). Both strategies do not destroy HIV but only block infection. To stop HIV infection we need to develop strategies which will destroy HIV. 4

HIV Therapeutic Agents

One strategy which will protect TH cells and destroy HIV include the production of a fusion protein. The fusion protein will have 2 parts CD4 protein attached to the Fc portion of an immunoglobulin (CD4 immunoadhesion). The CD4 portion will attach to the gp120 protein of HIV or virus infected cells. The immunoglobulin portion will initiate a cytotoxic response to destroy the virus or virus infected cell. 5

CD4 Immunoadehesi on Fusion Protein

HIV Therapeutic Agents

Another strategy involves making a second fusion protein. The CD4 sequence is ligated to the sequence of Pseudomonas exotoxin A to form a fusion protein. HIV infected cells have gp120 proteins on their surfaces. The CD4 portion of the fusion protein will attach to the infected cells. The fusion protein will enter the cells and initiate the killing of the infected cell. Pseudomonas exotoxin A inactivates the protein synthesis by affecting elongation factor EF-2. This prevents further protein synthesis and eventually 7

CD4-Pseudomonas Exotoxin Fusion Protein

Protein Pharmaceuticals
Natural sources are often rare and expensive
Difficult to keep up with demand Hard to isolate product Lead to immune reactions (diff. species) Viral & pathogen contamination

Most protein pharmaceuticals today are produced recombinantly

Cheaper, safer, abundant supply

Recombinant Methods
Developed in 1970s &1980s Paul Berg (1973) restriction enzymes Herbert Boyer (1978) cloning human insulin into E. coli Genetech Two general approaches
Expression in isolated cells Expression in transgenic plants/animals

Six Step Process

Isolation of gene of interest Introduction of gene to expression vector Transformation into host cells Growth of cells through fermentation Isolation & purification of protein Formulation of protein product

Cloning Process
Gene of interest is cut out with restriction enzymes (RE) Host plasmid (circular chromosome) is cut with

same REs
Gene is inserted into plasmid and ligated with ligase New (engineered) plasmid inserted into bacterium (transform)

Cloning (Details)

Cloning (Details)

protein

Recombinant Protein Expression Systems

Escherichia coli Other bacteria Pichia pastoris Other yeast Baculovirus Animal cell culture Plants Sheep/cows/humans Cell free

Polyhedra

Expression System Selection

Choice depends on size and character of protein
Large proteins (>100 kD)? Choose eukaryote Small proteins (<30 kD)? Choose prokaryote Glycosylation essential? Choose baculovirus or mammalian cell culture High yields, low cost? Choose E. coli Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote

Which Vector?
Must be compatible with host cell system (prokaryotic vectors for prokaryotic cells, eukaryotic vectors for eukaryotic cells) Needs a good combination of
strong promoters ribosome binding sites termination sequences multi-enzyme restriction site

Plasmids and Vectors

Circular pieces of DNA ranging in size from 1000 to 10,000 bases Able to independently replicate and typically code for 1-10 genes Often derived from bacterial mini chromosomes May exist as single copies or dozens of copies (often used to transfer antibiotic resistance)

Key Parts to a Vector

Origin of replication (ORI) DNA sequence for DNA polymerase to replicate the plasmid Selectable marker (Amp or Tet) a gene, when expressed on plasmid will allow host cells to survive Inducible promoter Short DNA sequence which enhances expression of adjacent gene Multi-cloning site (MCS) Short DNA sequence that contains many restriction enzyme sites

A Generic Vector

Gene Introduction (Bacteria)

Bacterial Transformation

Bacterial Transformation
Moves the plasmid into bacterial host Essential to making the gene actively express the protein inside the cell 2 routes of transformation
CaCl2 + cold shock Electroporation

Typical transformation rate is 1 in 10,000 cells (not very efficient) for CaCl2, but 1 in 100 for electroporation

Electroporator

25 microfarads = 2500 V @ 200 ohms for 5 ms

Electroporation
Seems to cause disruption in cell membrane Reconstitution of membrane leads to large pores which allow DNA molecules to enter Works for bacteria, yeast and animal cells

Bacterial Systems
Advantages Disadvantages

Grow quickly (8 hrs to produce protein) High yields (50-500 mg/L) Low cost of media (simple media constituents) Low fermentor costs

Difficulty expressing large proteins (>50 kD) No glycosylation or signal peptide removal Eukaryotic proteins are sometimes toxic Cant handle S-S rich proteins

Choice of Expression System

Growth pattern of cells (slow or fast) Cost of growing cells Level of expression of the product (high or low) Ease of product purification Post-translational modifications Proper folding and activity of protein product Availability of suitable vectors

Bacteria
E. coli most common strain Advantages Rapid growth on low-cost media Easy to scale-up from lab to production Disadvantages Proteins produced in E. coli are not glycosylated. Expressed protein may aggregate or fold improperly

Cloning & Transforming in Yeast Cells

Pichia pastoris

Pichia Pastoris
Yeast are single celled eukaryotes Behave like bacteria, but have key advantages of eukaryotes P. pastoris is a methylotrophic yeast that can use methanol as its sole carbon source (using alcohol oxidase) Has a very strong promoter for the alcohol oxidase (AOX) gene (~30% of protein produced when induced)

Pichia Cloning

Pichia Pastoris Cloning

Uses a special plasmid that works both in E. coli and Yeast Once gene of interest is inserted into this plasmid, it must be linearized (cut open so it isnt circular) Double cross-over recombination event occurs to cause the gene of interest to insert directly into P. pastoris chromosome where the old AOX gene used to be Now gene of interest is under control of the powerful AOX promoter

Pichia Systems
Advantages More advantages

Grow quickly (8 hrs to produce protein) Very high yields (505000 mg/L) Low cost of media (simple media constituents) Low fermentor costs

Can express large proteins (>50 kD) Glycosylation & signal peptide removal Has chaperonins to help fold tough prtns Can handle S-S rich proteins

Baculovirus Expression

Baculovirus Expression
Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs) Virus commonly infects insects cells of the alfalfa looper (small beetle) or armyworms (and their larvae) Uses super-strong promoter from the polyhedron coat protein to enhance expression of proteins while virus resides inside the insect cell

Baculovirus Expression

~12 days

Baculovirus Cloning Process

Transfer vector
Cloned gene 5

Cloned gene 5 3

Polyhedrin gene AcMNPV DNA Recombinant AcMNPV DNA

Baculovirus Successes
Alpha and beta interferon Adenosine deaminase Erythropoietin Interleukin 2 Poliovirus proteins Tissue plamsinogen activator (TPA)

Baculovirus Systems
Disadvantages Advantages

Grow very slowly (1012 days for set-up) Cell culture is only sustainable for 4-5 days Set-up is time consuming, not as simple as yeast

Can express large proteins (>50 kD) Correct glycosylation & signal peptide removal Has chaperonins to help fold tough prtns Very high yields, cheap

Mammalian Expression Systems

Mammalian Cells
Chinese hamster ovary cells (CHO) Baby hamster kidney cells (BHK) Hybridomas used in monoclonal antibody production Advantages Post-translational modifications (glycoslyation and folding) more closely resemble human Properly splices pre-mRNA Disadvantages Much slower growth than bacteria or yeast. (Doubling time = 18-24 h vs. 20-90 min bacteria) More difficult to culture More expensive to culture

Mammalian Cell-line Expression

Sometimes required for difficult-toexpress proteins or for complete authenticity (matching glycosylation and sequence) Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line Vectors usually use SV-40 virus, CMV or vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene

Mammalian Expression
Gene initially cloned and plasmid propagated in bacterial cells Mammalian cells transformed by electroporation (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression & integration of DHFR and the gene of interest

Methotrexate (MTX) Selection

Gene of interest DHFR

Transfect dfhr- cells

Grow in Nucleoside Free medium

Culture a Colony of cells

Grow in 0.05 uM Mtx

Culture a Colony of cells

Methotrexate (MTX) Selection

Grow in 0.25 uM Mtx

Culture a Colony of cells

Grow in 5.0 uM Mtx

Culture a Colony of cells

Foreign gene expressed in high level in CHO cells

Mammalian Systems
Disadvantages Advantages

Selection takes time (weeks for set-up) Cell culture is only sustainable for limited period of time Set-up is very time consuming, costly, modest yields

Can express large proteins (>50 kD) Correct glycosylation & signal peptide removal, generates authentic proteins Has chaperonins to help fold tough prtns

Mammalian Cell Successes

Factor IX Factor VIII Gamma interferon Interleukin 2 Human growth hormone Tissue plamsinogen activator (TPA)

Cell Growth Needs

A sterile carbon, nitrogen, hydrogen and oxygen source (air and H2O) + trace metals (Zn, Fe, Cu, Ca, Mg, Mn) A sterile energy source (light, sugar, acetate, methanol, ethanol) A constant (or near constant) temperature above 20 oC A growth regulating chemical (antibiotic)

Prototrophs vs. Auxotrophs

Protrophic cells (bacteria, plants) can produce all essential amino acids, nucleic acids, carbohydrates and lipids from simple nutrients (water, oxygen, nitrogen or ammonia, CO2) Auxotrophic cells (yeast, insect cells, mammalian) need vitamins, essential amino acids (His, Cys), sugars, lipids, etc. to grow because they have lost this ability through evolution (bacterial symbiosis)

Features
Cell wall Cell membrane Growth Rate O2 Requirement Nutritional Rqmt CO2 Requirement

Microbes
Generally present Present 10-50% per hour High Usually simple Sometimes

Animal Cells
Generally absent Present 1-5% per hour Low Complex Key for buffering

Environmental FX
Size Seeding density Growth density

Less affected
100-2000 nm 1 cell 109-1010 cells/mL

Very susceptible
10000-100000 nm 105 cells/mL 106 cells/mL

Shake Flask Incubators

Sometimes called environmental chambers Heavily insulated, heated with thermoregulation to keep temperature within 0.5 oC of set-pt. Rotatable platform to spin up to 500 rpm to facilitate aeration (dissolves N2 and O2 needed for growth) Designed for small-scale growth

Fermentors & Bioreactors

Larger scale, sustained growth requires bioreactors & fermentors Fermentors have been used for centuries primarily for brewing alcohol and making vinegar Modern technology and chemical engineering principles continue to improve fermentor design

Date
3000 BC to 1900 1900 to 1940 1940 to present 1960 to present 1979 to present

Products Vessels Process Control

Alcohol Vinegar Glycerol Citric acid Acetone Antibiotics Nucleotides Amino acids Native proteins & enzymes Recombinant proteins & enzymes Wooden Copper Trays Steel with mechanical stirring Sterile mechanical aerated Pressure jet vessels Fermentors (all 4 types) Thermometer heat exchanger pH control Temp control pH & O2 electrodes Temp control

Culture Method
Batch Methods Batch and Fed-Batch Fed-Batch and continuous

Quality control
Nil

Almost Nil

Very important Very important Very important

Computer Continuous linked control culture with loops recycler Computer Batch, Fed linked control Batch & pH, O2 , Temp Continuous

Fermentors & Bioreactors

Four basic bioreactor designs
Stirred tank reactors (mechanical agitation for aeration) Bubble column reactors (bubbling air into media for aeration) Internal loop airlift reactors (air and media circulate together) External loop airlift reactors

Stirred Tank Fermentor/Bioreactor

Four Bioreactor Designs

Airlift Reactors

Stirred Tank Reactor

Fermentor Scale Up
Cant start cell culture in 100000 L, must do repeated, scaled inoculations Start with stock culture (5-10 mL) Then shaker flask (200-500 mL) Then seed fermentor (10L to 100 L) Then production fermentor (1000L to 100,000 L)

Cell Isolation/Harvesting
Cells

Cell Suspension

Membrane

Cell Concentrate

Cross Flow Filtration

Cell-free culture medium

Cell-free culture medium

Dead End Filtration

Protein Isolation & Purification

After cells (or media) are harvested proteins may be purified/isolated Intracellular (inside cell) proteins are harder to purify
Require cell disruption, separation, removal of cell debris, DNA, RNA, lipid

Extracellular (outside cell) proteins are easier to purify

No cell disruption needed, just isolate

Cell Disruption Methods

Vigorous Methods

Sonication Glass bead disruption Enzymatic Gentle Methodslysis Detergent lysis Freeze-thaw Osmotic lysis

Protein Isolation Methods

Differential salt precipitation Differential solvent precipitation Differential temperature precipitation Differential pH precipitation Two-phase solvent extraction (PEG) Preparative electrophoresis Column chromatography

Most purifications require combinations of 2-3 steps

Column Chromatography
Most common (and best) approach to purifying larger amounts of proteins Able to achieve the highest level of purity and largest amount of protein with least amount of effort and the lowest likelihood of damage to the protein product Standard method for pharma industry

Column Chromatography
Can be done either at atmospheric pressure (gravity feed) or at high pressure (HPLC, 500-2000 psi) Four types of chromatography:
Affinity chromatography Gel filtration (size exclusion) chrom. Ion exchange chromatography Hydrophobic (reverse phase) chrom.

Affinity Chromatography
Adsorptive separation in which the molecule to be purified specifically and reversibly binds (adsorbs) to a complementary binding substand (a ligand) immobilized on an insoluble support (a matrix or resin) Purification is 1000X or better from a single step (highest of all methods) Preferred method if possible

Affinity Chromatography

Step 1: Attach ligand to column matrix

Step 2: Load protein mixture onto column

Affinity Chromatography

Step 3: Proteins bind to ligands

Step 4: Wash column to remove unwanted material, elute later

Affinity Chromatography
Used in many applications Purification of substances from complex biological mixtures Separation of native from denatured forms of proteins Removal of small amounts of biomaterial from large amounts of contaminants

Affinity Chromatography
The ligand must be readily (and cheaply) available Ligand must be attachable (covalently) to the matrix (typically sepharose) such that it still retains affinity for protein Binding must not be too strong or weak Ideal KD should be between 10-4 & 10-8 M Elution involves passage of high salt or low pH buffer after binding

Ligand
AMP

Specificity
Enzymes with NAD cofactors an ATP dependent kinases

Arginine
Cibacron Blue Dye

Proteases such as prothrombin, kallikrein, clostripain Serum Albumin, Preablumin Growth factors, cytokines, coagulation factors Fc region of immunoglobulins

Heparin
Protein A Calmodulin

Calmodulin regulated kinases, cylcases and phosphatases EGTA-copper Proteins with poly-Histidine tails

Size Exclusion Chrom.

Molecules are separated according to differences in their size as they pass through a hydrophilic polymer Polymer beads composed of crosslinked dextran (dextrose) which is highly porous (like Swiss cheese) Large proteins come out first (cant fit in pores), small proteins come out last (get stuck in the pores)

Size Exclusion Chromatography (SEC)

Sephadex Structure

Ion Exchange Chromatography (IEC)

Principle is to separate on basis of charge adsorption Positively charged proteins are reversibly adsorbed to immobilized negatively charged beads/polymers Negatively charged proteins are reversibly adsorbed to immobilized positively charged beads/polymers

Ion Exchange Chromatography

Has highest resolving power Has highest loading capacity Widespread applicability (almost universal) Most frequent chromatographic technique for protein purification Used in ~75% of all purifications