2. Biomanufacturing Defined
The production of pharmaceutical proteins using genetically engineered cells

3. Expression Choices
Cell type:
E. coli
Yeast
Mammalian
CHO

4. Expression Choices Parameter Bacteria Yeast Mammalian
Contamination risk
Low
High
Cost of growth medium
Product titer (concentration)
Folding
Sometimes
Probably
Yes
Glycosylation No
Yes, but different pattern
Full
Relative ease to grow
Easy
Difficult
Relative ease of recovery
Deposition of product
Intracellular
Intracellular or extracellular
Extracellular
Product
Often secreted into media
Secreted

5. Choice of host
Each host system has its own advantages and disadvantages, and it is important to consider these before final selection of host.
Expression in different parts of the cell will lead to varying amounts of cellular (contaminant) proteins that
will need to be removed to obtain a pure target protein.

7. Protein – The product of Biotech
USED IN THE TREATMENT OF:
Cell Production
Insulin
Diabetes
E. coli
Human growth hormone
Growth disorders
Granulocyte colony stimulating factor
Cancers
E. Coli
Erythropoietin
Anemia
CHO cells
Tissue plasminogen activator
Heart attack
Hepatitis B virus vaccine
Vaccination
Yeast
Human papillomavirus vaccine

8. The Value of Proteins Price Per Gram Bovine Growth Hormone $14 Gold
$48
Insulin
$60
Growth Hormone
$227,000
Granulocyte Colony Stimulating Factor (GCSF)
$1,357,000
*Prices in 2011 US Dollars

9. Induction
Biotech companies genetically engineer plasmids to place genes behind inducible promoters

10. Transcriptional Regulation in the pDHFR system
RNA Polymerase Z Y A
LacI
Effector (Lactose)
lac Operon
Transcriptional Regulation in the pDHFR system
Lactose
IPTG

11. 2 phases of growth

12. Recovery Separation of protein from other molecules Purification Separation of the protein of interest from other proteins

13. Protein Expression and Purification Series Workflow
Streak Cells
Overnight culture
Subculture, monitor, and induce
Harvest and lyse cells
Purify
Centrifugation or Instrumentation
Analyze

14. Steps in Recombinant Protein Purification
Design expression plasmid, transform, select
Grow culture of positive clone, induce expression
Lyse cells
Centrifuge to isolate protein-containing fraction
Column Chromatography—collect fractions
Assess purity on SDS-PAGE

15. Cell lysis Cell lysis: rupture cell wall / plasma membrane,
--> release contents (organelles, proteins…)
1. Physical means
2. Sonication
3. Osmotic shock

16. Chromatography Basics
Mobile phase (solvent and the molecules to be separated)
Stationary phase (through which the mobile phase travels)
paper (in paper chromatography)
glass, resin, or ceramic beads (in column chromatography)
Molecules travel through the stationary phase at different rates because of their chemistry.

17. Performing the chromatographic separation
Gravity Chromatography
Spin Column Chromatography
Chromatography Instrumentation
Small scale
Biomanufacturing scale
(bioreactors)

18. Protein purification – column chromatography
Protein mixture applied to column
Solvent (buffer) applied to top, flowed through column
Different proteins interact with matrix to different extents, flow at different rates
Proteins collected separately in different fractions

19. Column Chromatography
Molecules can be separated on the basis of:
SIZE—Gel filtration
CHARGE—Ion exchange
SPECIFIC BINDING—Affinity

20. Types of Column Chromatography
Ion Exchange (protein charge)
Size Exclusion (separates on size)
Hydrophobic Interaction (hydrophobicity)
Affinity:
Protein A
His-tagged
Glutathione-s-transferase ….
…..

21. Protein purification by chromatography

22. Gel filtration chromatography - separation by size
Beads have different size pores
As column flows:
large proteins excluded from pores
and therefore flow rapidly
small proteins enter pores and flow slowly

23. What is Affinity chromatography (AC) ?
Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure.
AC is designed to purify a particular molecule from a mixed sample.

24. The resin
Matrix
Affinity Ligand

25. Examples of tags and ligands
His-tag
FLAGTM peptide
Strep-tag
GST tag
Maltose binding protein fusion (MBP)
Calmodulin binding protein fusion
Transition metal ion
Monoclonal antibody
Biotin
Glutathione
Amylose
Ca2+
There are situations where you don’t need a tag.

26. Step 1. Loading affinity column.

27. Step 2.
Proteins sieve through matrix of affinity beads.

28. Step 3.
Proteins interact with affinity ligand with some binding loosely and others tightly.

29. Step 4.
Wash off proteins that do not bind.

30. Step 5.
Wash off proteins that bind loosely.

31. Step 6.
Elute proteins that bind tightly to ligand and collect purified protein of interest.

32. Affinity chromatography applied to recombinant proteins

33. Purity test
SDS-PAGE
Mass spectrometry
N-terminal sequencing, etc.

34. Downstream of protein purification
Biophysical characterization
Biochemical analysis of activities
Physiological relevance
Pathological mechanisms
etc.

36. Immobilized Metal Affinity Cromatography (His6 purification)
IMAC
Immobilized Metal Affinity Cromatography (His6 purification)
Metal-Chelate Affinity Chromatography (MCAC), also known as Immobilized Metal Affinity Chromatography (IMAC), was first successfully demonstrated in 1975 by Porath and collaborators for human serum proteins.

37. IMAC commonly utilizes zinc (Zn2+), nickel (Ni2+) or copper (Cu2+)
to form stable complexes with histidine, tryptophan and cysteine residues within proteins.
Proteins with a high affinity for given metal ions bind through open coordination sites and are retained on the column whilst other proteins without a high affinity for immobilised metal ion elute from the column during the wash.
The protein can be eluted with imidazole, which competes with the polyhistidine tag for binding to the column, or by a decrease in pH (typically to 4.5), which decreases the affinity of the tag for the resin.
Strong chelators such as EDTA at the end of elution are used to strip metal ions from the column.

38. Group 8 transition metal ions such as cobalt and nickel have six orbitals available for binding to histidine.
But, you need six histidine residues close together.

39. Key Parameters for the Operation of IMAC
The pH is critical for initial binding and subsequent elution of bound proteins. Typically, binding occurs at neutral or slightly alkali pH ( ), whereas elution generally occurs under acidic environments (< 6.0).
Chelating agents, such as ethylenediaminetetracetic acid (EDTA) and ethylene glycolbis(β-aminoethyl ether) N, N, N’, N’,-tetraacetic acid (EGTA), must be excluded from all solutions because they will strip the metal ions from the matrix.
(1)For most MCAC applications, including His-Tagged protein purification (2) It is important to utilize compatible buffer systems that maintain their pH accurately at all temperatures experienced during the bindingand elution of the proteins.

40. The His-tag can be placed on either the N- or C-terminus.
Optimal binding and, therefore, purification efficiency is achieved when the His-tag is freely accessible to metal ion support.

43. Columns or Proteins can be eluted in 3 ways!
1. Lower the pH.
2. Add excess imidazole
3. Add EDTA to remove metal ion from purification resin.

44. His tags His and imidazole structure similarities
Imidazole competes with His for Ni2+ sites
Histidine
Imidazole
N3H+
-OOC

51. IMAC ADVANTAGES
the small size of the affinity ligand. Due to the small size, it has minimal effects on the folding of the protein.
if the His-tag is placed on the N-terminal end of the protein, it can easily be removed using an endoprotease.
Another advantage of using His-tag purification methods is that polyhistidine tags can bind proteins under both native and denaturing conditions.
The use of denaturing conditions becomes important when proteins are found in inclusion bodies and must be denatured so they can be solubilized.

52. IMAC DISADVANTAGES potential degradation of the Histag
when a few histidine residues are proteolytically degraded, the affinity of the tagged protein isgreatly reduced leading to a decrease in the protein yield.
dimer and tetramer formation,
once a protein has a His-tag added to its structure, it has the potential to form dimers and tetramers in the presence of metal ions. While this is often not a large problem, it can lead to inaccurate molecular mass estimates of the tagged protein.
coelution of other histidine-containing proteins.
coelution of proteins that naturally have two or more adjacent histidine residues.

54. Glutathione S-transferase (GST) Gene Fusion System
The Glutathione S-transferase (GST) Gene Fusion System is a versatile system for the expression, purification, and detection of fusion proteins produced in Eschericia coli.
The system is based on inducible, high-level expression of genes or gene fragments as fusions with Schistosoma japonicum GST.
GST occurs naturally as a Mr protein that can be expressed in E. coli with full enzymatic activity
Expression in E. coli yields fusion proteins with the GST moiety at the amino terminus and the protein of interest at the carboxyl terminus.
The protein accumulates within the cell’s cytoplasm.

56. Gene encoding affinity tag-glutathione S tranferase (GST)
pGEX plasmid:
Gene encoding affinity tag-glutathione S tranferase (GST)
Spacer between genes - encodes protease cleavage site (thrombin)
Ptac promoter-induce with IPTG
Ribosome binding site
Figure 1: Diagram of the pGEX expression vector.

57. Cloning the gene or gene fragment into a pGEX expression vector pGEX vectors GST fusion proteins are constructed by inserting a gene or gene fragment into the multiple cloning site of one of the ten pGEX vectors.
Expression is under the control of the tac promoter, which is induced by the lactose analog isopropyl b-D thiogalactoside (IPTG).
All pGEX vectors are also engineered with an internal lacIq gene.
The lacIq gene product is a repressor protein that binds to the operator region of the tac promoter, preventing expression until induction by IPTG, thus maintaining tight control over expression of the insert.

58. Ezyme cut site
GST
POI
POI
GST

59. Affinity chromatography separation by biological binding interactions
wash
porous
bead
glutathione
elute
GST
apply sample
thrombin site
protein of interest
Example: GST - Glutathione
GST-tagged proteins bind to gluthatione on beads
Non-specifically or weakly bound proteins washed off
GST-tagged proteins eluted with glutathione (competitor) or thrombin (protease)

62. GST fusion proteins are purified from bacterial lysates by affinity chromatography using immobilized glutathione.
GST fusion proteins are captured by the affinity medium, and impurities are removed by washing.
Fusion proteins are eluted under mild, non-denaturing conditions using reduced glutathione.
The purification process preserves protein antigenicity and function.
If desired, cleavage of the protein from GST can be achieved using a site-specific protease whose recognition sequence is located immediately upstream from the multiple cloning site on the pGEX plasmids.

72. The Strep-tag® II is a short peptide (8 amino acids, WSHPQFEK), which binds with high selectivity to Strep-Tactin®, an engineered streptavidin.
The binding affinity of Strep-tag® II to Strep-Tactin® (Kd = 1 µM) is nearly 100 times higher than to streptavidin.
This technology allows one-step purification of almost any recombinant protein under physiological conditions, thus preserving its bioactivity.
The Strep-tag® system can be used to purify functional Strep-tag® II proteins from any expression system including baculovirus, mammalian cells, yeast, and bacteria.

74. After application of the crude extract on a Strep-Tactin® column and a short washing step, gentle elution of purified recombinant protein is performed by addition of low concentrations (2.5 mM) desthiobiotin.

76. FLAG TAG AspTyrLysAspAspAspAspLys
FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a polypeptide protein tag that can be added to a protein using recombinant DNA technology, having the sequence motif DYKDDDDK.
 It has been used for studying proteins in living cells and for protein purification by affinity chromatography.
AspTyrLysAspAspAspAspLys

77. it may be used in tandem, commonly the 3xFLAG peptide:
DYKDHDG-DYKDHDI-DYKDDDDK (with the final tag encoding an enterokinase cleavage site).
A fold increased detection enhancement has been shown using 3X FLAG fusions.

78. Some commercially available antibodies (e. g
Some commercially available antibodies (e.g., M1/4E11) recognize the epitope only when it is present at the N-terminus.
However, other available antibodies (e.g., M2) are position-insensitive.

81. Instrumentation BioLogic LP Demo
BioLogic DuoFlow™

82. Scaling up of the process developed during research and development
Biomanufacturing