bpac meeting

BPAC Meeting - November 4, 2005 1

Characterization of ARALASTCompared to other A1PI

PreparationsHans Peter Schwarz, MD

Vice President, Global Preclinical R&D

Baxter


AgendaAgenda

1) Background Information

2) Modifications to primary structure of A1PI in all commercial products

3) Potential cause for microheterogeneity of A1PI protein in Aralast

4) Implications of microheterogeneity on protein structure and function


Aralast - Introduction

December 23, 2002: FDA approval of Aralast, developed and manufactured by Alpha Therapeutics (now: Grifols Biological Inc.)

Q2 2003: Baxter acquired Aralast and Aralast associated assets from Alpha Therapeutics


IEF Observations:IEF Observations:

IEF-gel provided by the FDA regarding their concerns of an anodal shift of M6 and M4 in ARALAST, suggesting that a population of AAT isoforms carry approximately one extra negative charge

1

plasma

Normal

2 3 4 5 6

pH 5

pH 4

M6M6M4M4M2M2

plasma

Aralast Prolastin

Anode Anode

CathodeCathode


2DIGE Analysis of Aralast and 2DIGE Analysis of Aralast and ProlastinProlastin

Fluorescence scan overlay4.0 pI pI 7.0

MW

hig

hlo

w

Anodal shift of spots derived from Aralast seen on 2DIGE analysisin the presence of urea

Aralast Prolastin

+ -


2DIGE Analysis of Aralast and 2DIGE Analysis of Aralast and ProlastinProlastin

Fluorescence scan overlay4.5 pI pI 5.5

MW

hig

hlo

w

Anodal shift of spots derived from Aralast seen on 2DIGE analysisin the presence of urea

Aralast Prolastin

+ -


AgendaAgenda






Modifications with Potential Modifications with Potential Impact on Protein ChargeImpact on Protein Charge

Secondary structure:

• Heterogeneity of glycosylation- Influence on the number of sialic acids

Primary structure:

• Deamidation (addition of a negative charge): - Non-enzymatic conversion of Asn116 or Asn314 to aspartic or

isoaspartic acid

• Cysteine modification:- Attachment (covalent) of another molecule to A1PI (a free available

cysteine exists in A1PI)

• Removal of terminally located charged amino acids- N-terminal truncation of 5 amino acids, loss of negatively charged

glutamic and aspartic acids M7, M8- C-terminal truncation, loss of positively charged lysine …des-Lys

A1PI


N-Glycan Analysis of A1PI N-Glycan Analysis of A1PI concentratesconcentratesHPLC Analysis

1600 1800 2000 2200 2400 2600 2800 3000 3200 3400m/z0

100

%

0

100

%

2223.40

1931.81

2078.17

2245.34

2880.65

2369.392589.092391.08 2735.49

3027.053048.31

2223.40

1931.81

2178.972078.62

2245.01

2880.29

2369.242589.032391.23 2735.28

3026.17

PROLASTIN

ARALAST

MALDI-TOF Analysis

No difference in the N-glycan profile between Aralast, Prolastinand Zemaira and the pattern is similar to that found for A1PIfrom human plasma


N-glycan pattern of all 3 A1PI concentrates used for treatment of Hereditary Emphysema is similar to that of plasma, therefore ethanol fractionation and downstream purification have no impact.


M6

M4

M2

M0

LH

0403

1A

LH

1001

A

LH

0300

8A

LH

0203

1A

PR

OL

AS

TIN

LH

0600

4A

Pla

sma

pI 4.9

pI 4.2 +

-

N-glycans are NOT responsible for the IEF pattern characteristic for Aralast

Aralast

Relation of N-glycan pattern to IEF Relation of N-glycan pattern to IEF bandsbands


Modifications with Potential Modifications with Potential Impact on Protein ChargeImpact on Protein ChargeSecondary structure:


Primary structure:


isoaspartic acid





A1PI


Deamidation AnalysisDeamidation Analysis

• Quantitative measurement of deamidation using an enzymatic assay(IsoQuant Kit, Promega) based on the methylation of iso-aspartate, a final product of Asn-deamidation

Deamidation as primary sequence modification occurs in all A1PI concentrates

Aralast (3 lots) 6%Zemaira (1 lot) 7%Prolastin (1 lot) 10%

% of molecules withone iso-aspartate

Product

• Confirmed qualitative detection of deamidation in all products by MS-analysis of two deamidation candidate tryptic peptides containing the sequence Asn-Gly (peptides 102-125 and 311-331)




Primary structure:


isoaspartic acid





A1PI


Cysteinylation of Cys232Cysteinylation of Cys232

Aralast, Prolastin, and A1PI from plasma all exhibit cysteinylation on Cys232, however this modification was not detected in Zemaira

pI4.9

pI4.2 +

M4

redu

ced

no

n red

.

redu

ced

no

n red

.

redu

ced

no

n red

.

redu

ced

no

n red

.

Zemaira Prolastin Aralast Plasma

M6

IEF Analysis MS-Analysis




Primary structure:


isoaspartic acid





A1PI


MS Analysis

C-C-terminalterminal Lys Truncation Lys Truncation AnalysisAnalysis

MS spectra showing C-terminal peptides of A1PI

Des-Lys A1PI found in all products: Aralast (67%), Zemaira (6%) and Prolastin (2%)

Detail of the Aralast tryptic peptide map (detected at 214 nm) showing the region with the C-terminal truncated peptide and allowing quantitation of the modification.

Des-Lys C-terminalpeptide

Full length C-terminalpeptide

HPLC Analysis


Summary of A1PI Summary of A1PI ModificationsModifications

*1 Site specific N-Glycan patterns analyzed for the first time; detection of tetra-antennary structures and Lewis X structures on A1PI

*2 under investigation

Aralast Prolastin Zemaira Plasma/BAL

Glycoisoforms

Deamidation - qualitative - quantitative 6% 10% 7% not possible

Methionine oxidation

C-terminal Lys truncation 67% 2% 6% ? *2

Cys232 cysteinylation +

*1no major difference

detectable

not observed (A1PI fully functionally active)

A1PI in all products differs from A1PI found in plasma

+ +-


AgendaAgenda






CarboxypeptidasesCarboxypeptidases

Basic carboxypeptidases are enzymes that cleave COOH-terminal basic amino acids lysine and arginine from different peptides and proteins

They are involved in food digestion (CPB), modulation of peptide activity (CPM, CPN), pro-hormone processing (CPD, CPE), regulation of the plasminogen system (CPU)

- Carboxypeptidase B (pancreas) - Carboxypeptidase U

present in plasma as a pro-enzyme, pro-CPU synonyms: CPU = carboxypeptidase R = TAFIa (TAFI = pro-CPU) activated by thrombin, plasmin and trypsin potent inhibitor of fibrinolysis, possibly involved in inactivation of activated complement

proteins and anaphylatoxins

- Carboxypeptidase N plasma enzyme constitutively active in plasma, 30 µg/mL plasma inactivation of activated complement proteins C3a, C4a and C5a and bradykinin; maturation of

hormones

- Carboxypeptidase M GPI-anchored membrane protein, highly expressed in lung tissues


CPN and Pro-CPU Activity in CPN and Pro-CPU Activity in Cohn Ethanol FractionationCohn Ethanol Fractionation

Cohn Starting Material 42 U/L Plasma554 U/L Plasma

Cohn Starting Material 42 U/L Plasma554 U/L Plasma

Fractionation I SupernatantFractionation I Supernatant

Fraction II+III Precipitate 4 U/L Plasma13 U/L Plasma

Fraction II+III Precipitate 4 U/L Plasma13 U/L Plasma

Fraction II+III SupernatantFraction II+III Supernatant

IV1 SupernatantIV1 Supernatant

IV4 SupernatantIV4 SupernatantIV4 Precipitate

20 U/L Plasma 21 U/L Plasma

IV4 Precipitate 20 U/L Plasma 21 U/L Plasma

AlbuminAlbumin

Fraction IIIPrecipitate

Fraction IIIPrecipitate

Fraction III supernatant< 2.5 U/L Plasma

21 U/L Plasma

Fraction III supernatant< 2.5 U/L Plasma

21 U/L Plasma

ImmunoglobulinImmunoglobulin

Alcohol 20%

Alcohol 40%

IV1 Precipitate 7 U/L Plasma72 U/L Plasma

IV1 Precipitate 7 U/L Plasma72 U/L Plasma

CPN test method: Cleavage of hippuryl-L-Argmeasured by RP-HPLC.1U CPN releases1 µM hippuric acid/min

ProCPU test method: Activation with Thrombin-Thrombomodulin, cleavage ofhippuryl-L-Arg and measurementwith RP-HPLC.ProCPU = (CPN+CPU) - CPN


Ethanol Dependence of C-terminal Ethanol Dependence of C-terminal Lys Cleavage by CPNLys Cleavage by CPN

0

1

2

3

4

5

0 5 10 15 20 25

% EtOH (final)

Lys

/ in

tern

al s

tan

dar

dAralastProlastin

• At EtOH concentrations of >/=10% lysine cleavage showed a linear increase in dependence of the EtOH concentration

• The C-terminal Lysine of A1PI in both Aralast and Prolastin are susceptible to cleavage upon exposure to ethanol

• IEF analysis of these samples corroborates lysine truncation to anodal band shift

The concentration of EtOH determines the amount of Lys-truncation


C-terminal lysine cleavage of A1PI by C-terminal lysine cleavage of A1PI by CPN in absence and presence of ethanolCPN in absence and presence of ethanol

Expressed as ratio: Lysine cleaved/internal standard

0

0,5

1

1,5

2

2,5

3

prolastin® recombinant

Lys

/IS

;

rA1PIProlastin

Lys

/IS

0

0,5

1

1,5

2

2,5

3


0% ethanol

15 % ethanol

0

0,5

1

1,5

2

2,5

3


0% ethanol

15 % ethanol

ethanol

ethanol

0 %

15 %


Removal of C-terminal lys as function of time

0

5

10

15

20

25

0 500 1000 1500 2000

Time (sec)

St

(uM

)(rA1PI)


-

+

A 0 10 25 50 100 250

Aralast (A) LH02031AExperimental Lot 900304B (IV,1)

CPM (mU/ml), after 4010% EtOH treatment

pI 4.2

pI 4.9

Generation of Anodal Isoforms of Generation of Anodal Isoforms of A1PI by Treatment with rCPM A1PI by Treatment with rCPM

M2*

M4M4*

M6

0

20

40

60

80

100

0 10 25 50 100 250U CPM/mL

% (

M6

+ M

4* =

100

%)

M6M4*

M6 M4* shift

0

20

40

60

80

100

0 10 25 50 100 250CPM in U/mL

M4M2*

M4 M2* shift

% (

M4

+ M

2* =

100

%)

M7*

M7

M8*

M8


A1PI Isoform Pattern in Human BALA1PI Isoform Pattern in Human BAL

1 ... PROLASTIN #PR4HA43A, 5 µg/ml2 ... ARALAST #LH03002A, 5 µg/ml3 ... Human BAL sample, 3.5 µg/ml4 ... Human BAL sample, 6 µg/ml5 ... Human BAL sample, 2.7 µg/ml

A highly sensitive IEF gel was used to detect A1PI in BAL samples from subjects not on A1PI augmentation therapy

A1PI was detected in all BAL samples, and the IEF pattern resembles the A1PI shift observed for Aralast

This suggests that an isoform shift can naturally occur, possibly induced by CPM

+

- 1 2 3 4 5


CP Effect on A1PI: Summary

• All basic CPs cleave C-terminal lysine from A1PI

• Cleavage of C-terminal Lys occurs in absence of ethanol

• Ethanol enhances the reaction CPN (20-fold effect of 15% ethanol on kcat/Km)*

• CPN is the most likely candidate causingthe C-terminal Lys cleavage in plasma

* Similar to findings reported by Folk et.al. JBC 1962, vol. 237 pg. 3105 “Kinetics of Carboxypeptidase B Activity - Effects of alcohol.”


AgendaAgenda






Implications of Higher Degree of C-terminal Implications of Higher Degree of C-terminal Truncation in Aralast: InvestigationTruncation in Aralast: Investigation

Structural bioinformatics analysis In vitro function: anti-elastase activity Pharmacokinetics Tissue distribution and diffusion


A1PI-des-Lys394 – Structural Bioinformatics AnalysisA1PI-des-Lys394 – Structural Bioinformatics Analysis

• There is no conservation between species of the C-terminal Lys in A1PI Lys394 is unlikely to play a major structural or functional role

• 3D analysis of available structures for A1PI and its complex with protease do not support a major structural role for Lys394

• C-terminal loop region is stabilized by an H-bond network in which Lys394 is not involved

Lys394 plays no major structural role in A1PI when uncleaved and cleaved as well as when forming complexes with a protease

Results


0

100

200

300

0% 10% 20% 30% 40% 50% 60% 70%

% inhibition of porcine elastase

nM

A1

PI

Prolastin (n = 6 lots / 22 dilution series) Zemaira (n = 2 lots / 7 dilution series) ARALAST (n = 15 lots / 21 dilution series) 100%-des-Lys-A1PI (n = 1 lot / 3 dilution series)

slope: 438.7r = 0.9850

slope: 441.8r = 0.9916

slope: 443.0r = 0.9894

slope: 448.6r = 0.9974

No difference in activity of Aralast, Prolastin, Zemaira & des-Lys A1PI

A1PI dependent inhibition of porcine elastase:Response of different A1PI concentrates

Functional Relevance of Functional Relevance of C-terminal Truncation of A1PIC-terminal Truncation of A1PI


Higher Degree of C-terminal Higher Degree of C-terminal Truncation has no Impact on:Truncation has no Impact on: Metabolic clearance in rats

Comparability of A1PI-preparations with differing degrees of C-terminal lys-truncation

Distribution to lung (rat)

Comparability of A1PI-preparations with differing degrees of C-terminal lys-truncation including a 100% -Lys A1PI in a rat BAL study

Diffusion from vasculature into interstitium (guinea pig)

Comparability of A1PI-preparations with differing degrees of C-terminal lys-truncation including a 100% -Lys A1PI in a guinea pig suction blister model

Confocal studies to assess diffusion and lung tissue distribution

Comparability of A1PI-preparations with differing degree of C-terminal lys-truncation


ConclusionsConclusions

1) A1PI in all products approved for augmentation therapy demonstrate at least one primary structure modification (deamidation, cysteine modification, and C-terminal lysine truncation)

2) The des-Lys A1PI is induced by carboxypeptidases, and the ubiquitous presence of carboxypeptidases in plasma and in lung tissue (CPM) will likely result in exposure and hence tolerance to the des-Lys394 form of A1PI

3) des-Lys is one of many known isoforms of A1PI that does not affect the inhibitor activity, immunogenicity or essential functions of A1PI


REFERENCE SLIDESREFERENCE SLIDES


Molecular characteristics of A1PIMolecular characteristics of A1PI

Single chain glycoprotein consisting of 394 AA

Carries a high negative charge because of sialic acid residues on three complex glycans attached to Asn46, Asn83 and Asn247

Exhibits multiple bands reflecting microheterogeneity upon isoelectric focusing (M1 [M0] anodal-low pI to M8 cathodal–high pI) Two minor cathodal isoforms, M7 and M8, are truncated at the N-terminus lacking five AA (1-5) leading to an additional cathodal shift due to the loss of negatively charged glutamic and aspartic

acid

1 single Cysteine residue in position 232 covalently bound to either free Cys or Glutathione via a disulfide bridge

Asn116 and Asn314 are susceptible to deamidation (Asn Asp) due to sequence as followed by Gly

pI

4.2

4.9

M0/M1

M2

M4

M6

M7

M8


Ser

xxx

Asn

xxx

Ser

xxx

Asn

xxx

1 PNGase F

2Isolation

+labelling

3HPLC and MS

Isolated A1PI N-glycans are labelled with a fluorescent dye

Different structural isomers can be separated and characterized by HPLC retention time before and after treatment with specific exoglycosidases

* Other for glycan analysis widely used fluorophores are e.g. 2-aminobenzamide (AB)

Ser

xxx

Asp

xxx

Ser

xxx

Asp

xxxPA *


Cysteinylation of Cys-232: Cysteinylation of Cys-232: Electrostatic Surface Potential Electrostatic Surface Potential MapsMaps

A B(A) cysteinylated cys-232; (B) free cys-232


Deamidation of Asparagine in Deamidation of Asparagine in ProteinsProteins


Carboxypeptidase N, Ethanol Fractionation Carboxypeptidase N, Ethanol Fractionation and Aralast and Aralast

Carboxypeptidase N (CPN) is the most likely candidate causing the IEF pattern of Aralast:

Plasma does not contain CPU (only proCPU or TAFI)

Trace amounts of of plasmin/thrombin/thrombomodulin could activate some CPU

Experiments with the basic carboxypeptidase inhibitor Mergetpa support CPN; however, Mergetpa also inhibits CPU, but at a lower potency ( Ki=2nM for CPN and 750 for CPU)

Mergetpa = DL-2-mercapto-methyl-3-guanidino-ethylthiopropanoic acid


A1PI Isoform Shift Induced by A1PI Isoform Shift Induced by Treatment with CPU (TAFIa) Treatment with CPU (TAFIa)

-

+

A1PI from IV,1

Enyzme: CPU (0.5 U/ml)A1PI (+4°C, 2mg/ml) + 96% EtOH (-20°C), 20 min at –20°C; + 10 mM TRIS/HCl, pH 8.8 (+4°C) + CPU (4°C); incubated for 60 min at +37°C

A1PI + CPU (in mU/ml), 4010% EtOH

0 10 20 50 100 200

pI 4.2

pI 4.9

M2*

M4M4*

M6

M6 M4* shift

0

20

40

60

80

100

0 10 20 50 100 200CPU U/mL

% (

M6

+ M

4* =

100

%)

M6M4*

0

20

40

60

80

100

0 10 20 50 100 200

% (

M4

+ M

2* =

100

%)

M4M2*

M4 M2* shift

CPU U/mL


Airway Epithelium

Interstitial Compartment

Airway Epithelium

Microscopic distribution of Aralast

CENTRAL AIRWAY

bpac meeting - november 4, 20051 characterization of aralast compared to other a1pi preparations...

Documents

dige analysis of aralast

aralast introduction

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lys a1pi slide

primary structure of

nglycan analysis of

a1pi concentrates