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1 1 A Seminar On Tryptic Mapping Prepared By: Vinay Prajapati . M. Pharm Sem -II Roll no:13 Department of Quality Assurance Guided By: Ms. Parula B. Patel S.J.Thakkar Pharmacy College, Rajkot.

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2 CONTENTS : Introduction Reverse phase column for Tryptic mapping HPLC system for peptide mapping Mobile phase selection and operating conditions Uses and limitations References


1) INTRODUCTION Many therapeutically important peptides and proteins, e.g., insulin and growth hormone, are currently produced by the recombinant DNA (rDNA) technique. This sophisticated technology is based on insertion of foreign genetic material, coding for the substance of interest into a host cell. The host cells will then produce the desired substance in addition to their natural production . 3

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Biotechnological production of pharmaceuticals requires very rigorous quality control owing to the risk of undesirable protein modifications and the numerous possibilities for contamination of the product. The integrity of the amino acid sequence of the protein has to be confirmed for each production batch. Peptide mapping is an indispensable analytical method in biotechnology for quality control of rDNA -derived proteins 4

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A powerful method for the structural determination and confirmation of proteins, especially those obtained by r-DNA technology A peptide map may be viewed as a “FINGERPRINT” of a protein and is the end product of several chemical processes that provide a comprehensive understanding of the protein being analyzed Peptide mapping consists of fragmentation of the protein by enzymatic digestion or chemical cleavage, with subsequent separation of the fragments The fragmentation is in most instances performed by enzymatic digestion with trypsin 5

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Trypsin is by far most widely use proteolytic enzyme in peptide mapping because its having following desirable features : It cleaves at C-terminal side of lysine and arginine which is generally quantitative under proper conditions Trypsin also tolerates concentration of urea as high as 4M 6

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Two types of process : Enzymatic Chemical Four major steps : Isolation and Purification Selective Cleavage of Peptide Bonds Chromatographic Separation Analysis and Identification of Peptides 7

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PRINCIPLE: A reverse phase system consists of a Bondapack – fatty acid analysis column and an eluent which contains a hydrophilic ion - pairing reagent such as phosphoric acid It has two effect on chromatographic behaviour of a protein or peptide First, decrease in pH to approximately 2.2 results in ionic suppression of the carboxyl groups and also equilibrium to the right with a concomitant increase in polarity of the peptide because of formation of the amine cation R-NH2 + H+ R-+NH3 10

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In the addition, phosphoric acid acts as a source of dihydrogen phosphate anions which pair ionically with the counterion R-+NH3. The formation of this hydrophilic ion pair has the effect of greatly increasing the polarity of the sample , which results in decrease in retention time on reverse phase supports together with improved peak shape and resolution 11

Tryptic Digestion Procedure for Milligram Quantities: 

Tryptic Digestion Procedure for Milligram Quantities Sample protein (5 gm) in 2ml 8M urea Reduce with 10mM dithiothretol at 37 ̊C for 2 hrs Reaction with Iodoacetic acid for 10min in dark (pH 8.5) Dialyse overnight against 50mM NH4HCO3 Lyophilize to dryness Finally, residues redissolve in 5ml buffer and is digest with trypsin at an enzyme/substrate ratio of 1:100 for 4 hrs at 37 ̊C On completion, digestion is stop by adding 10% (V/V) phosphoric acid and stored at 4 ̊C until use 12

Tryptic Digestion Procedure for Subnanomole Quantities: 

Tryptic Digestion Procedure for Subnanomole Quantities It only differ in 2 steps: Low yield steps dialysis and lyophilization are replace by a dilution step to 2M urea in which trypsin remains active Iodoacetic acid is replace by Iodoacetamide which eliminates necessity of any pH adjustment 13

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14 Tryptic Map of Tissue Plasminogen Activator


2) COMMON COLUMN CHARACTERISTICS FOR TRYPTIC MAPPING 15 A. Column dimension : Conventional: 150-250 mm long , 4.6 mm i.d. Narrow-bore : 250 mm long , 2.1 mm i.d. B. Packing characteristics : Silica type : Pure , low silanophilic silica Particle diameter : 5-10 micron Pore size : 125-300 ˚A Bonded group : C4-C18 Parameter Typical range

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A) COLUMN DIMENTIONS: COLUMN LENGTH (L) For complex maps high-efficiency columns are require. Column efficiency α column length COLUMN DIAMETER (dc) As such dc itself doesn’t affect resolution and analysis time But narrow bore column increase mass sensitivity significantly because of lower sample dilution However, resolution is less than conventional column due to difficulty in packing of narrow bore columns . 16

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B) COLUMN PACKING CHARECTERISTICS SILICA TYPE : Should with low silanophilic activity Commonly use column for mapping are : Bondapack C18 Synchropack Lichrosorb RP-18 or RP-8 Aquapore RP300 PARTICLE DIAMETER (dp) : Plate height (H) α dp Low dp lower H higher column efficiency 19

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PORE SIZE : Studies indicates the pore diameter of support should be at least 3 to 5 times larger than the Stoke’s diameter of solute to prevent “restricted” diffusion, which causes band broadening This criterion is much less important for tryptic mapping because tryptic fragments are small peptides BONDED GROUPS : C4, C8, C18 and to a lesser extent, phenyl and cyano groups are use in peptide separation Most commercial bonded phases are “end-capped” with trimethylsilane to reduce the level of residual silanols 21


FUTURE TRENDS IN COLUMN Organic polymers Fast peptide mapping 3-micron porous materials 2-micron nonporous materials 22

a) Organic polymers : e.g. : cross linked poly(styrene dvb), poly(alkyl methacrylate) : 

a) Organic polymers : e.g. : cross linked poly(styrene dvb ), poly(alkyl methacrylate ) Advantages: more versatile (available in different particle size, pore size and bonded groups) mechanically stronger more efficient extremely symmetrical peak shape than silica because of absence of silanophilic interactions Disadvantages: Lower pressure limits, lower column efficiency and higher material cost 23

b) Fast peptide mapping: : 

b) Fast peptide mapping: Routinely at lab peptide mapping takes 1-2 hrs per sample Fast mapping can be achieved by reducing particle diameter of the packing materials which allows use of shorter column lengths without reducing column efficiency By using 3-micron porous and 2-micron nonporous materials 24

c) 3-micron porous and 2-micron nonporous material : 

c) 3-micron porous and 2-micron nonporous material 25


26 3) HPLC SYSTEM FOR PEPTIDE MAPPING HPLC instrumental conditions for peptide mapping System Biocompatible Low dispersion Pump Good mixing and gradient reproducibility Low pulsation Microbore and semipreparative flow capacity Column oven Detector ( UV absorbance detector ) Dual channel detector (215 and 280 nm) High sensitivity Rapid scanning Other detectors fluoroscence and electrochemical, etc.

Dual wavelength detection: 

Dual wavelength detection 27

Operating parameter for Tryptic Mapping: 

Operating parameter for Tryptic Mapping 28 Parameter Typical range Column temperature Ambient to 25-50 ̊ Mobile phase composition Organic solvent Acetonitrile pH 2 - 3 Ion pair reagent and buffer 0.05 – 0.1 % TFA or 50mM phosphate Gradient conditions Gradient time ( t G ) 30 – 120 min Flow rate ( F ) 0.5 – 2 ml/min Initial and final solvent strength 0 – 40 % ACN Gradient shape Linear or multisegment 4) MOBILE PHASE SELECTION AND OPERATING CONDITIONS

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5) TRYPTIC MAPPING : USES AND LIMITATIONS USES : It’s one of the most demanding application of HPLC It’s the primer method for structural determination of newly discovered proteins Use to locate glycosylation sites and disulfide linkages Provides vital information on lot-to-lot product consistency, expression errors , mutation and deamination sites Today it is increasingly use in biotechnology for quality control of recombinant proteins 30

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LIMITATIONS : Because of large number of peaks in a tryptic map, it would be difficult to identify peptide fragments contributed by a contaminant protein at a level of 1-5% A high level of column resolution and system precision is require to accurately reproduce the maps 31


6) REFERENCES Michael W. Dong, The perkin- Elmer corporation, Norwalk, connecticut, Ch.2 The United States Pharmacopoeia, Official Revision Bulletin, July 1, 2009, (1055) Biotechnology derived article W. S. Hankock and C. A. Bishop, Analytical Biochemistry 89,203-212 R. C. Chloupek, R. J. Harris, Journal of Chromatography, 463 (1989) 375-396 Michael W. Dong and D.Tran, Journal of Chromatography, 499 ,125-l 39 32

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33 Thank You