THERAPEUTIC THERAPEUTIC PROTEIN ANALYSIS PROTEIN ANALYSIS

Edited by SudjadiEdited by Sudjadi

BiofarmaseutikalBiofarmaseutikal

merupakan biomolekul yang berguna merupakan biomolekul yang berguna sebagai obat dan dibuat dengan sebagai obat dan dibuat dengan bioteknologi modern: bioteknologi modern: – ProteinProtein– Asam nukleat Asam nukleat

Protein FarmasetikProtein Farmasetik

Protein obatProtein obat– InsulinInsulin

Protein yang digunakan untuk mensintesis Protein yang digunakan untuk mensintesis obatobat– Penisilin asilasePenisilin asilase

• Very large (mostly) and unstable molecules

• Structure is held together by weak non-covalent

forces

• Easily destroyed by relatively mild storage

conditions

• Easily destroyed/eliminated by the body

• Hard to obtain in large quantities

The Problem with Proteins

Elimination by B and T cells (antibodies)

Proteolysis by endo/exo peptidases

Small proteins (<30 kD) filtered out by the

kidneys very quickly

Unwanted allergic reactions may develop (even

toxicity)

Loss due to insolubility/adsorption

The Problem with Proteins

(in vivo - in the body)

Noncovalent Covalent

• Denaturation Deamidation

• Aggregation Oxidation

• Precipitation Disulfide

exchange

• Adsorption Proteolysis/

hydrolysis

The Problem with Proteins(in vitro - in the bottle)

Noncovalent

Noncovalent Covalent

• Denaturation Deamination

• Aggregation Oxidation

• Precipitation Disulfide

exchange

• Adsorption Proteolysis/

hydrolysis

The Problem with Proteins(in vitro - in the bottle)

At elevated temperature and extreme pH

Under air or potent oxidantsReplace by inert gas or antioxidant

oxidation

Disulphide exchange can result in aggregation

Disulphide exchange

Proteolytic degradation

Proteases belong to:

1.Serine proteases I or II2.Cysteine proteases3.Aspartic proteases4.Metalloproteases I or II

Protein with tight packing ~ less susceptible to protease attack

ExopeptidaseEndopeptidase

Analysis of final product

1.Protein-based contaminants- other proteins- viral particles- pyrogenic substances- DNA fragment- microorganism

2.Removal of altered form of protein interested3.Product potency

- bioassays

Diabetes - Insulin

Discovered in 1921 by Banting and BestDiscovered in 1921 by Banting and Best

Consist of Consist of AA & & BB chains linked by 2 disulfide chains linked by 2 disulfide bonds (plus additional disulfide in A)bonds (plus additional disulfide in A)

~ ~ ~ ~

A = 21 amino acids B = 30 amino acids

Comparison of Human Comparison of Human Insulins/AnalogsInsulins/Analogs

Insulin Onset of Duration ofPreparations Action Peak Action

Regular 30-60 min 2-4 h 6-10 h

NPH/lente 1-2 h 4-8 h 10-20 h

Ultralente 2-4 h Unpredictable 16-20 h

Lispro/aspart 5-15 min 1-2 h 4-6 h

Glargine 1-2 h Flat ~24 h

Gly ThrGlu Phe Tyr Pro Lys Thr

Gly ThrGlu Phe Tyr Asp Lys Thr

23 24 25 26 27 28 29 30

Insulin

Aspart

Primary Structure of Primary Structure of Asp(B28)-InsulinAsp(B28)-Insulin

ThrPhe Tyr Pro Lys Thr

25 26 27 28 29 30

InsulinB-chain

Glargine ThrPhe Tyr Pro Lys Thr Arg Arg

AsnLeu Glu Tyr Cys Gly

AsnLeu Glu Tyr Cys Asn

16 17 18 19 20 21

InsulinA-chain

Glargine

Primary Structure of Primary Structure of Gly(A21), Arg(B31), Arg(B32)-InsulinGly(A21), Arg(B31), Arg(B32)-Insulin

Gly ThrGlu Phe Tyr Pro Lys Thr

Gly ThrGlu Phe Tyr Pro Lys Thr

23 24 25 26 27 28 29 30

Insulin

Detemir

(CH(CH22))44

NHNH

COCO

RR

Primary Structure of Lys(B29)-N-Primary Structure of Lys(B29)-N---Tetradecanoyl, Des(B30)-InsulinTetradecanoyl, Des(B30)-Insulin

Amino acids essentialfor receptor binding

ala gly cys lys asn phe phe

cys ser thr phe thr

lys

trp

Dphe cys phe

throl

cys thr

lys

Dtrp

OctreotideHuman somatostatin

Somatostatin and Octreotide:Molecular Characteristics

Harris AG. Drug Invest. 1992;4(suppl 3):1-54.

Shelf Life of Recombinant Protein DrugsShelf Life of Recombinant Protein Drugs

NameName ProteinProtein Physical Physical FormForm Expiration Dating PeriodExpiration Dating Period

HumulinHumulin HIHI liquidliquid 2 years (2-8 C)2 years (2-8 C)

OrthocloneOrthocloneOKT3OKT3 MuMAbMuMAb liquidliquid 1 year (2-8 C)1 year (2-8 C)

Roferon-ARoferon-A IFN-a2aIFN-a2a solidsolid 3 years (2-8 C)3 years (2-8 C)

Intron AIntron A IFN-a2bIFN-a2b solidsolid 3 years (2-8 C)3 years (2-8 C)

ActivaseActivase TPATPA solidsolid 2.5 years (2-8 C)2.5 years (2-8 C)

ProtropinProtropin hGHhGH solidsolid 2 years (2-8 C)2 years (2-8 C)

Use Life of Reconstituted SolutionsUse Life of Reconstituted Solutions

NameName ProteinProtein Maximum Hold Maximum Hold TimeTime BacteriostatBacteriostat

Roferon-ARoferon-A IFN-IFN-2a2a 1 month at 2-8 C1 month at 2-8 C phenol (0.3 %)phenol (0.3 %)

Intron AIntron A IFN-IFN-2b2b 1 month at 2-8 C1 month at 2-8 C benzyl alcohol (0.9)%benzyl alcohol (0.9)%

ActivaseActivase TPATPA 14 days at 2-8 C14 days at 2-8 C m-cresol (0.3%)m-cresol (0.3%)

ProtropinProtropin22 hGHhGH 7 days at 2-8 C7 days at 2-8 C benzyl alcohol (0.9)%benzyl alcohol (0.9)%

HumatropHumatropee

hGHhGH 8 hours at 2-30 C8 hours at 2-30 C nonenone

Stability-Indicating Test Methods for Stability-Indicating Test Methods for Recombinant ProteinsRecombinant Proteins

MethodMethod Change that can be detectedChange that can be detected Example of UseExample of Use

SDS PageSDS Page fragmentationfragmentation IFN-IFN-

crosslinkingcrosslinking hGHhGH

oligomerizationoligomerization IFN-IFN-

RP-HPLCRP-HPLC deamidationdeamidation InsulinInsulin

crosslinkingcrosslinking InsulinInsulin

methionine oxidationmethionine oxidation IL-2IL-2

disulfide scramblingdisulfide scrambling IL-2IL-2

IEFIEF deamidationdeamidation IL-1IL-1

Potency DeterminationPotency Determination disulfide scramblingdisulfide scrambling IFN-IFN-

Stability of Recombinant TNF (Liquid Stability of Recombinant TNF (Liquid Formulation) Stored Under RefrigerationFormulation) Stored Under Refrigeration

(2-8°C)(2-8°C)

Time in Storage (months)Time in Storage (months) PotencyPotency Protein Purity by SDS PageProtein Purity by SDS Page

00 100 %100 % 100 %100 %

33 100 %100 % 100 %100 %

66 70 %70 % 100 %100 %

99 60 %60 % 100 %100 %

1212 50 %50 % 99 %99 %

Mengapa berbeda antara potensi dan SDS-PAGE?

ExampleExample

Haemoglobin Haemoglobin

Quaternary structure of 2 Quaternary structure of 2 and 2 and 2 monomers (monomers (each about 16 kDaeach about 16 kDa))

Gel-filtrationGel-filtration kDakDa

SDS-PAGESDS-PAGE kDakDa

(no difference if reduced)(no difference if reduced)

Detection of altered forms of protein:1. Denaturing (SDS) gel electrophoresis2. Non-denaturing gel electrophoresis3. 2D electrophoresis4. Isoelectric focusing5. Peptide mapping6. Amino acid Analysis7. Capilary Electrophoresis8. Sequencing9. HPLC10. GC-MS11. Potency and Immunology assay

AA are ampholyte

Anion, when at high pH

zwitterion, when at isoelectric point (pI)

Cation, when at low pH

NH2 CH COO-

RH

+

OH- NH3

+CH COO

-

RH

+

OH-

RCHCO2H

NH2

RCHCO2-

N+H3

H

NaOH

HCL

NOTE: peptide or protein also have both acid and base properties. They share the same property of being positively charged at low pH and negatively charged at high pH.

Isolectric Point (pI) of AAs

Anion in

basic

sol’n.

RCHCO2H

NH2

RCHCO2-

N+H3

H+

RCHCO2H

N+H3

HO-

RCHCO2-

NH2

+++++++++

----

-

-

-

-

--

Zwitterion in pI sol’n. No move to either of Electrode. And with Lowest solubility

Cation in acidic sol’n.

pH<pIpH>pI

anode cathode

Steps in 2D GESteps in 2D GE

Sample preparationSample preparation

Isoelectric focusing (first dimension)Isoelectric focusing (first dimension)

SDS-PAGE (second dimension)SDS-PAGE (second dimension)

Visualization of proteins spotsVisualization of proteins spots

Identification of protein spotsIdentification of protein spots

Spot pattern evaluation/annotationSpot pattern evaluation/annotation

Isoelectric Focusing relies on the migration of charged proteins in

an electric field

IPG StripsIPG Strips

Strip Length 7.9 cm 11.8 cm 17.8 cm

Gel Length 7.3 cm 11.0 cm 17.1 cm

Strip Width 3.3 mm 3.3 mm 3.3 mm

Gel Thickness 0.5 mm 0.5 mm 0.5 mm

pH Gradients

Standard 3-10,4-7 3-10,4-7 3-10,4-7

Overlapping 3-6,5-8 3-6,5-8 3-6,5-8

R = weakly acidic or basic buffering group

CH2=CH-C-NH-R ||O

Acrylamido buffer

Narrow-Range IPG StripsNarrow-Range IPG StripspH 4 pH 5

pH 5 pH 6

pH 4 pH 9

IEF Phase of 2D GEIEF Phase of 2D GE

Rehydrate IPGstrip & apply

protein sample

Place IPG stripin IEF apparatus

and apply current

Steps in 2D GESteps in 2D GE

Sample preparationSample preparation

Isoelectric focusing (first dimension)Isoelectric focusing (first dimension)

SDS-PAGE (second dimension)SDS-PAGE (second dimension)

Visualization of proteins spotsVisualization of proteins spots

Identification of protein spotsIdentification of protein spots

Spot pattern evaluation/annotationSpot pattern evaluation/annotation

SDS PAGESDS PAGE ToolsTools

SDS-PAGE for 2D GESDS-PAGE for 2D GE

equilibration SDS-PAGE

Steps in 2D GESteps in 2D GE

Sample preparationSample preparation

Isoelectric focusing (first dimension)Isoelectric focusing (first dimension)

SDS-PAGE (second dimension)SDS-PAGE (second dimension)

Visualization of proteins spotsVisualization of proteins spots

Identification of protein spotsIdentification of protein spots

Spot pattern evaluation/annotationSpot pattern evaluation/annotation

Uses of SDS-PAGEUses of SDS-PAGE

Determine protein sizeDetermine protein size

Identify proteinIdentify protein

Determine sample purityDetermine sample purity

Identify existence of disulfide bondsIdentify existence of disulfide bonds

Quantify amounts of proteinQuantify amounts of protein

Stability-Indicating Test Methods for Stability-Indicating Test Methods for Recombinant ProteinsRecombinant Proteins

MethodMethod Change that can be Change that can be detecteddetected Example of UseExample of Use

SDS PAGESDS PAGE fragmentationfragmentation IFN-IFN-

crosslinkingcrosslinking hGHhGH

oligomerizationoligomerization IFN-IFN-

Polyacrylamide gel electrophoresis can occur under both denaturing and

reducing conditions

Gel Stains - SummaryGel Stains - Summary

Stain Sensitivity (ng/spot) Advantages

Coomassie R-250 50-100 Simple, fast, consistent

Colloidal Coomassie 5-10 Simple, fast

Silver stain 1-4 Very sensitive, awkward

Copper stain 5-15 Reversible, 1 reagent

negative stain

Zinc stain 5-15 Reversible, simple, fast

high contrast neg. stain

SYPRO ruby 1-10 Very sensitive, fluorescent

Electrophoresis based on MW: SDS-PAGERun under denaturing

conditions:

1. Proteins are mixed with SDS & -ME-containing buffer and boiled.

2. SDS-coated proteins migrate based solely on their MW.

Fig. 3-20

HPLCHPLC

Edited by SudjadiEdited by Sudjadi

Chromatographic ModeChromatographic Mode AcronyAcronymm Separation PrincipleSeparation Principle

Non-interactive modes of liquid chromatographyNon-interactive modes of liquid chromatographySize-exclusion Size-exclusion chromatographychromatography SECSEC Differences in molecular Differences in molecular

sizesize

--

Interactive modes of liquid chromatographyInteractive modes of liquid chromatographyIon-exchange Ion-exchange chromatographychromatography IECIEC Electrostatic interactionsElectrostatic interactions

Normal-phase Normal-phase chromatographychromatography NPCNPC Polar interactionsPolar interactions

Reversed-phase Reversed-phase chromtographychromtography RPCRPC Dispersive interactionsDispersive interactions

Hydrophobic interaction Hydrophobic interaction chromatographychromatography HICHIC Dispersive interactionsDispersive interactions

Affinity chromatographyAffinity chromatography ACAC Biospecific interactionBiospecific interaction

Chromatographic Modes of Protein Purification

(Christian G. Huber, Biopolymer Chromatography, Encylcopedia in analytical chemistry, 2000)

Stabilize SampleStabilize Sample

Control pHControl pH– Use appropriate bufferUse appropriate buffer

Control temperatureControl temperature– Keep samples on ice or work in cold roomKeep samples on ice or work in cold room– Prechill instrumentsPrechill instruments

Prevent frothing/foamingPrevent frothing/foaming– Handle gently.Handle gently.

Maintain concentrated sampleMaintain concentrated sample

Monitoring PurityMonitoring Purity

Total protein (mg)Total protein (mg)– Quantity of protein present in fractionQuantity of protein present in fraction

Total activity (units of activity)Total activity (units of activity)– Use a portion of sample to determine activity.Use a portion of sample to determine activity.

– Multiply activity by total volume to determine Multiply activity by total volume to determine total activity.total activity.

Monitoring Progress of Purification Monitoring Progress of Purification ProtocolProtocol

Specific activity (Specific activity (units of activity/mgunits of activity/mg))Total activityTotal activityTotal proteinTotal protein

% yield:% yield: measure of activity retained after each measure of activity retained after each step in procedure.step in procedure.

S.A. =

% yield = Total activity at particular stepTotal activity of initial extract

USP 36 Monograph for InsulinRAW MATERIAL FINISHED PRODUCT

Insulin Insulin Injection

Insulin Human Insulin Human Injection

Insulin Lispro Insulin Lispro Injection

Isophane Insulin Suspension

Prompt Insulin Zinc Suspension

Isophane Insulin Human Suspension

Extended Insulin Zinc Suspension

Human Insulin Isophane Suspension and Human Insulin Injection

Insulin Human Zinc Suspension

Insulin Zinc Suspension

Extended Insulin Human Zinc Suspension

4949

Insulin InjectionInsulin Injection

Insulin injection is an isotonic, sterile Insulin injection is an isotonic, sterile solution of insulin. It has a potency of not solution of insulin. It has a potency of not less than 95.0% and not more than less than 95.0% and not more than 105.0% of the potency stated on the label, 105.0% of the potency stated on the label, expressed in USP Insulin Units.expressed in USP Insulin Units.

5050

Insulin Injection SpecificationParameter Specification

Identification Positive (+)

Bacterial Endotoxin NMT 80 USP EU for each 100 USP IU

Sterility Sterile

pH 7.0 – 7.8

Particulate Matter Conform

Zinc Content 10 – 40 μg for each 100 USP IU

Limit of High Molecular Weight Protein NMT 2.0%

Assay 95.0 – 105.0 %

5151

Chromatographic system: Parameter Specification

Mode LC

Detector UV 276 nm

Column 7.8 mm x 30 cm, Packing L-20

Flow rate 0.5 mL/min

Injection volume 100 μL

Mobile phase Solution A, Acetronitrile, and Glacial Acetic Acid (65:20:15)Solution A: 1 mg/mL of L-arginine

5252

PRINCIPLE OF THE ELISA ASSAY

PROCESS 5353

Selection of Hydrophobic Interaction Selection of Hydrophobic Interaction ChromatographyChromatography

Fig. 2. Comparison of the hydrophobicities of BLA () and rHLA () in buffers containing different concentrations of ammonium sulfate in the presence of 100mM EDTA. Protein concentrations were 0.2 mg/ml. Excitation wavelength was set as 380nm (slit=5nm). Scan was executed at 20 nm/s speed and room temperature (25 ◦C).

Irma Antasionasti

Selection of Hydrophobic Interaction Selection of Hydrophobic Interaction ChromatographyChromatography

Fig. 3. Comparison of the hydrophobicities of BLA () and HLA () in buffers containing different concentrations of ammonium sulfate in the absence of EDTA. Protein concentrations were 0.2 mg/ml. Excitation wavelength was set as 380nm (slit=5nm). Scan was executed at 20 nm/s speed and room temperature (25 ◦C).

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Optimization of Hydrophobic Interaction Optimization of Hydrophobic Interaction ChromatographyChromatography

Irma Antasionasti

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Optimization of Hydrophobic Interaction Optimization of Hydrophobic Interaction ChromatographyChromatography

Fig. 4. Elution profile in hydrophobic interaction chromatography. Conditions: Column: Butyl Sepharose 4 FF (60mm×10mmi.d., CV=5ml). Sample: 10 ml recombinant whey containing 0.8Mammonium sulfate (protein concentration: 3mg/ml). System: ÄKTA explorer 100. Elution buffer: Buffer D (20mMsodium phosphate, pH 7.0).

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Fig. 7. Fig. 7. HPRPC chromatograms of whey protein standards (A–B), HPRPC chromatograms of whey protein standards (A–B), acid whey (C) andacid whey (C) and purified rHLA product (D). Injection volume was purified rHLA product (D). Injection volume was 30l, protein concentrations were30l, protein concentrations were 1 mg/ml (BLA, HLA) or 3mg/ml 1 mg/ml (BLA, HLA) or 3mg/ml (acid whey) or 0.5 mg/ml (purified rHLA). Other(acid whey) or 0.5 mg/ml (purified rHLA). Other conditions for conditions for HPRPC were as described in Section 2 of the text.HPRPC were as described in Section 2 of the text.

Purification Process

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Ion Exchange Chromatography

Ion Exchange Chromatography

– Ion exchange chromatography – binding Ion exchange chromatography – binding and separation of proteins based on and separation of proteins based on charge-charge interactionscharge-charge interactions

– Proteins bind at low ionic strength, and are Proteins bind at low ionic strength, and are eluted at high ionic strengtheluted at high ionic strength

++

+

+

++

+ ++

+

-

- -

-

++

+

+

+

++

+

+

+-

- -+

Positively charged(anionic) ion

exchange matrix

Net negatively charged (cationic)

protein at selected pHProtein binds to matrix

1. BAGAIMANA PROTEIN DAPAT TERPISAH ?2. BILA DIGUNAKAN CM, PROTEIN yang mana?

Ion Exchange ChromatographyIon Exchange Chromatography

Low salt High salt

Ion Exchange (IEX) Chromatography

Affinity chromatography chromatography

Affinity chromatographyAffinity chromatography

Binding of a protein to a matrix via a Binding of a protein to a matrix via a protein-specific ligandprotein-specific ligand– Substrate or product analogueSubstrate or product analogue– AntibodyAntibody– Inhibitor analogueInhibitor analogue– Cofactor/coenzymeCofactor/coenzyme

Specific protein is eluted by adding Specific protein is eluted by adding reagent which competes with bindingreagent which competes with binding

Affinity chromatographyAffinity chromatography

Matrix Spacer arm

Affinity ligand

+

Active-site-bound enzyme

1. Substrate analogue affinity chromatography

Matrix Spacer arm

Antibody ligand

+

Antibody-bound enzyme

2. Immunoaffinity chromatography

Protein epitope

Enzyme

Affinity Chromatography

Gel Filtration Chromatography

Gel permeation chromatography Gel permeation chromatography (GPC)(GPC)

Also known as Also known as ‘‘size exclusion chromatographysize exclusion chromatography’’ and and ‘‘gel filtration chromatographygel filtration chromatography’’

Separates molecules on the basis of molecular sizeSeparates molecules on the basis of molecular size

Separation is based on the use of a porous matrix. Separation is based on the use of a porous matrix. Small molecules penetrate into the matrix more, and Small molecules penetrate into the matrix more, and their path length of elution is longer.their path length of elution is longer.

Large molecules appear first, smaller molecules laterLarge molecules appear first, smaller molecules later

GEL FILTRASI/GEL EKSKLUSI

Gel Filtration (GF) Chromatography

Stability-Indicating Test Methods for Stability-Indicating Test Methods for Recombinant ProteinsRecombinant Proteins

Method Change that can be detected Example of Use

SDS Page fragmentation IFN-

crosslinking hGH

oligomerization IFN-

RP-HPLC deamidation Insulin

crosslinking Insulin

methionine oxidation IL-2

disulfide scrambling IL-2

IEF deamidation IL-1

Potency Determination disulfide scrambling IFN-

Stability of Recombinant TNF (Liquid Stability of Recombinant TNF (Liquid Formulation) Stored Under RefrigerationFormulation) Stored Under Refrigeration

(2-8°C)(2-8°C)

Time in Storage (months) Potency Protein Purity by SDS Page

0 100 % 100 %

3 100 % 100 %

6 70 % 100 %

9 60 % 100 %

12 50 % 99 %