1. Analytical Biotechnology ביוטכנולוגיה אנליטית
Dr. Isam Khalaila

2. What is Biotechnology?
simple definition is the use of organisms by man.
Whereas modern biotechnology manipulates the genes of organisms and inserts them into other organisms to acquire the desired trait, traditional biotechnology uses the processes of organisms, such as fermentation.
Modern Biotechnology involves new technologies whose consequences are untested and should be met with caution. for e.g. stem cells, gene therapy, genetically modified organisms and tissue engineering.
The advancement tools and techniques is now allowing us to look at the universe of atoms.
Biotechnology is utilizing the sciences of biology, chemistry, physics, engineering, computers, and information technology to develop tools and products that hold great promise and concern.

3. What is Analytical Biotechnology?
New field since1980’s
Analytical Biotechnology field is the field of techniques for analysing biological Biotechnology products.
Specifically, Fusion of protein chemistry and analytical sciences
HPLC was the enabling technology followed by Mass Spectrometry in the early 1990’s

4. Analytical biotechnology is aiming to analyze biopolymer specifically proteins derived from Recombinant DNA
Recombinant Protein Production
Recombinant DNA synthesis
Vector Selection
Cell Type Selection
Manufacturing of recombinant proteins
Protein Purity: preparation
Affinity Chromatography
Size Exclusion Chromatography
Ion-exchange Chromatography
Reversed Phase – HPLC
Post-Translational Modifications
Phosphorylation
Acetylation
Glycosylation/Glycation
Proteolytic Cleavage
Product Formulation
Activity
Purity
Immunogenicity
Stability

5. Recombinant Protein Synthesis Vector cloning
Place recombinant DNA into a vector for replication
A vector is a vehicle for delivering genetic material.
Plasmid
Biochemistry; Mathews, C.K., van Holde, K.E., Ahern, K.G Eds. 3rd Ed. Benjamin/Cummings: San Francisco, CA pp 108,

6. Cell Type Selection
Determine cell type based on post-translational modifications
Microbial
Bacteria - high cell growth; no glycosylation
- Escherichia Coli (BL-21) – gram (-), most common
- Bacillus subtilus – gram (+), less protein production than E.coli
Yeast - Saccharomyces cerevisiae
- high cell growth
- incorrect glycosylation; high mannose production
Mammalian - slow cell growth; correct glycosylation
Chinese Hamster Ovary (CHO)
Baby Hamster Kidney (BHK-21)
Human Embryo Kidney (HEK-293)

7. Protein Characterization - Overview
Current technology enables comprehensive comparative characterization of all protein therapeutic products.
Characterization justifies abbreviated approval process for later products with same active chemical entity.
Extent of clinical program inversely related to extent of characterization.
Many protein products can be characterized so thoroughly as to eliminate need for clinical studies

8. Protein Characterization – Strategy
Characteristics of particular protein dictate characterization strategy.
Orthogonal methods – virtually every observable property of a protein is probed:
covalent structure, conformation, pI, aggregation, charge, mass, fragmentation, surface structure, hydrophobicity, spectrophotometric, magnetic resonance, fluorescence, light scattering, sedimentation, electrophoretic properties, immunological properties, enzyme activity, biological potency, etc.
Apply comprehensive array of sensitive and selective analytical methods.

9. What is the capability of current analytical technology?
Complete elucidation of covalent structure
Peptide mapping with high resolution LC/MS routinely locates every atom in peptide backbone
Sensitive methods for comparing higher order structure (fingerprint)
CD, HPLC, SEC, FTIR, fluorescence, NMR, light scattering, ELISA
Excellent:

10. What is the capability of current analytical technology?
Sensitive methods for measuring impurities
HPLC, SEC, IEC, SDS-PAGE, IEF
Comparison of products using multiple orthogonal analytical methods provides high level of assurance of pharmaceutical equivalence

11. Are there new technologies that hold promise for helping to characterize proteins?
Yes - multidimensional analytical methods (e.g., LC/MS, 2D NMR)
Multivariate mathematical methods for analyzing complex mixture data
provide greater information content than 1D methods
can reveal individual components that would otherwise be obscured

12. Is it possible to accurately predict safety and efficacy from analytical studies?
Or: Is it possible to ensure that two products have equivalent safety and efficacy from analytical studies?
Yes - modern analytical methods are much more sensitive indicators of product changes than are clinical studies. Analytical methods can readily detect minute batch-to-batch variations that are far too small to have any clinical consequence

13. How we analyze Biotechnology products?
First steps are: Separation and purification.
Primary structure analysis (sequencing).
Secondary, Tertiary and Quaternary structure analysis.
Post-translational modification.

14. Protein Structure
Primary structure is sometimes called the "covalent structure" of proteins.
In contrast, the higher orders of proteins structure (i.e. secondary, tertiary and quartenary) involve mainly noncovalent interactions.
because, with the exception of disulfide bonds (see below), all of the covalent bonding within proteins defines the primary structure.

15. Protein sequence determination
Determining the amino acid sequences
The two major direct methods of protein sequencing are:
Edman degradation reaction.
Mass spectrometry
It is also possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is known

16. How are proteins isolated, purified and tested for purity, activity, stability, and immunogenicity?
FDA regulation
Requires: Validates manufacturing facility, processes and detailed analytical methods to characterize.
Classical chromatographic methods used to discriminate impurities. However, single component versus consistent components: “Well characterized”
FDA Guidelines based on small molecule drugs.

17. Analysis of Protein in Final Drug Product
Activity
Is the protein in the drug product doing what it is supposed to?
Purity
Is there any other molecules present in the final drug product that may interfere with the efficacy of the drug?
Immunogenicity
Does the drug cause an unwanted immune response?
Stability
Does the drug have the same Activity, and Purity after it has been stored and been exposed to certain condition changes?

18. Activity
Proteins containing certain enzymes that indicate it’s activity.
Enzymes react with certain substrate and cleave at known spots to create fluorescent product.
Reacted product is read on an assay plate alongside a control with a known activity. +
Protein Sample
Substrate
Protein Bound to substrate
Fluorescent Product
Fluorescent Source
Detector
Protein Bound to substrate

19. Purity
Quickest, most common and most cost efficient way of checking protein purity = slab gel electrophoresis.
Separation of proteins according to mass via SDS-PAGE – Western Blot.
Separation according to isoelectric point by isoelectric focusing.
Rodriquez-Diaz, R., Wehr, T., Tuck, S., Analytical Techniques for Biopharmaceutical Development, Taylor and Francis, Boca Raton, 2005.
Rupesh Naidu, Shire Human Genetic Therapies, 2006

20. Immunogenicity and Stability
Little or no immunogenicity is desired.
Determined by testing for the presence of an anti-drug antibody.
ELISA performed utilizing reagent that contains antibody known to react with anti-drug antibody.
Stability is the test for a change in activity or purity over time.
Sterility as well as changes in visual appearance are also tested.
Effect of long term storage is accelerated by altering temperature, humidity, and pH conditions.
Rodriquez-Diaz, R., Wehr, T., Tuck, S., Analytical Techniques for Biopharmaceutical Development, Taylor and Francis, Boca Raton, 2005.

21. Sample Preparation

23. Helpful information

24. Detergents used for protein solubilization

25. Purification Process:
Primary Isolation of Protein Products
Precipitation: Separation based on solubility (Am Sulf/Ethanol)
Centrifugation: Separation based on migration properties/size
Filtration: Separation based on size
Contaminants: Foreign DNA, Viruses, Prions, Endotoxins, Other Proteins
Biosimilars: PTMs, Aggregation, Sequence Modifications, Degradation

26. Agents used to precipitate proteins
Agent class
Example
Mechanism
Polar solvent
Water
Salting-in
(descrease in ionic strength)
Salt
Ammonium sulfate
KCl
Salting-out
(increase in ionic strength)
Polymer
Polyethylene glycol
Polyethyleneimine
Temperature
Heat
Denaturation and aggregation pH
Acid
Reduce charge of protein
Non-polar Solvent
Ethanol
Acetone
Reduce activity of water
Water activity: It is defined as the vapor pressure of water above a sample divided by that of pure water at the same temperature; therefore, pure distilled water has a water activity of exactly one.

30. Centrifugation

33. Proteins extraction and fractionation

34. Protein Determination
Lowry ( most cited reference in biology) Color assay
A280– Intrinsic absorbance – Relies on aromatic amino acids
BCA – Modification of Lowry: increased sensitivity and consistency
Bradford – Shifts Amax of dye from 465nm to 595nm

35. Protein Determination
Warburg-Christian Method (A280)
This method is a direct spectrophotometric method.
Measures absorbance at 280 nm.
This method is also very sensitive to tyrosine and tryptophan. When might one use this method?
Absorbance at 260 nm also used to correct for nucleic acid concentration.
Wharton and McCarty, 1980

36. A280 Uses intrinsic absorbance
Detects aromatic residues– Resonating bonds
Depends on protein structure, native state and AA composition
Retains protein function

37. A280

38. Fluorescence Method Advantage: more sensitive than UV absorption.
Tyrosine and Tryptophaneare fluorescent compounds.
Excite the amino acids at 280 nm.
Measure emission at 348 nm.
Advantage: more sensitive than UV absorption.

39. Protein Determination
Biuret Reaction
In alkaline solutions, Cu2+ complexes with the C-N bonds in protein. The result is a purple color.
This method is relatively insensitive.
Tris buffer and other substances often interfere.
Wharton and McCarty, 1980

40. Biuret reaction
Estimate in 560nm
Cu2+
C-N
N-C
Protein

41. Protein Determination
Lowry Method
This method relies upon both the biuret reaction and the reduction of arsenomolybdate reagent (Folin reagent) by tryptophan and tyrosine. Consequently,
what type of proteins will give higher absorbances?
Tris buffer and reducing compounds often interfere.
Wharton and McCarty, 1980

42. Lowry method A  Biuret reaction B  Folin-Ciocalteu reaction
(phosphomolybdate and phosphotungstate)
Estimate in nm

43. Standard Curve Protein standards: Protein (mg) A750 100 0.601 80 0.480
0.372 40
0.254 20
0.120
0.000

44. y = ax + b Using Standard Curve Protein UNKNOWN: Protein (mg) A750 33
0.200 92
0.550
y = ax + b

45. Protein Determination
The BCA (Bicinchoninic Acid) Method
Uses a similar principle as that described in the biuret reaction except that BCA is included and sensitivity is increased.
This process is a two-step reaction.
Pierce Protein Assay
Technical Handbook, 1999

46. The BCA method
Estimate in 562nm

47. Protein Determination
Coomassie® Blue Dye Method (The Bradford Method)
This method relies on the binding of protein to Coomassie® Brilliant Blue G-250 which causes an absorbance shift from 465 nm to 595 nm.
Pierce Protein Assay
Technical Handbook, 1999

48. Protein Determination
The Coomassie® dye binds primarily with basic and aromatic side chains. The interaction with arginine is very strong and less strong with histidine, lysine, tyrosine, tryptophan, and phenylalanine.
About 1.5 to 3 molecules of dye bind per positive charge on the protein.

49. Protein Determination
Method
Detection range
Wavelength nm
Interference
UV280
0.1-1 mg/ml
280
Nucleic acid
detergents
cofactors
Phenolic compounds
Pigments
Biuret reaction
mg/ml
560
Glucose
Ammonium sulphate
Sulfhydryl compounds
phosphate buffers
Lowry method
mg/ml
655
Reducing agents
Bradford method
g/ml
595
Detergents,
basic buffers

50. Chromatography Overview of Complicated, Orthogonal Process:
Utilize different protein
characteristics to separate
impurities.
Chromatography
Bhikhabhai et al., Journal of Chromat. A, 2005, 1080, 83-92

51. Affinity Chromatography

52. Ion Exchange Chromatography

53. Size Exclusion/Gel Filtration

54. Gel Filtration: Gel types
Sephacryl
Sephadex Gel
Sepharose / Agarose

55. Uses of Gel filtration Protein purification (last step)
Determination of molecular weight / size
Removing low MW materials (desalting, non-incorporated monomers)
Buffer change

56. Determination of mw by gel filtration

57. Ion Exchange Chromatography
Steps in ion exchange chromatography
Equilibration
Sample Application
Elution
Wash

58. How we analyze Biotechnology products
Spectroscopic methods have provided perhaps the most widely used tools for the elucidation of the structure of molecular species as well as the quantitative and qualitative determination of both organic and inorganic compounds.

59. Classification of spectroscopic methods
Electromagnetic spectroscopy involves interactions with electromagnetic radiation, or light. NMR is an example
Electronic spectroscopy involves interactions with electron beams.
Mass spectroscopy involves the interaction of charged species with magnetic and/or electric fields, giving rise to a mass spectrum. The term "mass spectroscopy" is deprecated in favour of mass spectrometry, for the technique is primarily a form of measurement, though it does produce a spectrum for observation.

60. Protein electrophoresis
Native / Denaturated
Size / Charge
1 D / 2 D
Gel / buffer composition
Sample buffer
When to stop
Visualization / Analysis

61. Native or Denaturing Buffers
Native gels contain buffers that are compatible with folded active macromolecules
Denaturing gels disrupt intra and interchain interactions molecules migrate as individual chains
– Proteins denatured by SDS, disulfide bonds disrupted by reducing agents
– SDS negative charge overwhelms the natural charge variation of proteins to promote predictable behaviour.

62. Continuous or discontinuous gel systems
Many protein gel systems are discontinuous
– that is there are 2 separate gel concentrations and 2 or 3 different buffer compositions
– Stacking gels are designed to compress a tall column of protein into a thin band at the interface with a
– Separating gel - the composition of this gel is varied to match the resolution desired
DNA gels are almost always continuous, same gel composition and buffer throughout.

63. “Stacking” Gels

64. Native Gel Electrophoresis