# Presentation: 592935

## Characterization of ARALAST

**Characterization of ARALAST**
**Compared to other A1PI Preparations**

- Hans Peter Schwarz, MD
- Vice President, Global Preclinical R&D
- Baxter

## Agenda

- Background Information
- Modifications to primary structure of A1PI in all commercial products
- Potential cause for microheterogeneity of A1PI protein in Aralast
- 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-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**

## 2DIGE Analysis of Aralast and Prolastin

**Fluorescence scan overlay**

**4.0   pI**

**pI   7.0**

**MW**

**high**

**low**

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

**Aralast  ****     ****Prolastin**

- +

-  -

## 2DIGE Analysis of Aralast and Prolastin

**Fluorescence scan overlay**

**4.5   pI**

**pI   5.5**

**MW**

**high**

**low**

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

**Aralast  ****     ****Prolastin**

- +

-  -

## Agenda

- Background Information
- Modifications to primary structure of A1PI in all commercial products
- Potential cause for microheterogeneity of A1PI protein in Aralast
- Implications of microheterogeneity on protein structure and function

****

## Modifications with Potential Impact 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 concentrates

- _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.

**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.**

## Relation of N-glycan pattern to IEF bands

**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 bands**

## Modifications with Potential Impact 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**

****

## Deamidation 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

- _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)

## Modifications with Potential Impact 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**

****

## Cysteinylation of Cys232

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

## Modifications with Potential Impact 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**

****

## C-terminal Lys Truncation Analysis

- MS Analysis

**C-****terminal**** Lys Truncation Analysis**

**MS spectra showing C-terminal peptides of A1PI**

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

## Summary of A1PI Modifications

- ***1**  **S****ite 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**

**+**

*******1**

**no major difference**

**detectable**

**not observed (A1PI fully functionally active)**

******  ****A1PI in all products differs from A1PI found in plasma**

**+**

**+**

**-**

## Agenda

- Background Information
- Modifications to primary structure of A1PI in all commercial products
- Potential cause for microheterogeneity of A1PI protein in Aralast
- Implications of microheterogeneity on protein structure and function

****

## Carboxypeptidases

**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 Cohn Ethanol Fractionation

**Cohn Starting Material****  ****42 U/L Plasma****554 U/L Plasma**

**Fractionation I Supernatant**

**Fraction II+III Precipitate****  ****4 U/L Plasma****13 U/L Plasma**

**Fraction II+III Supernatant**

**IV****1**** Supernatant**

**IV****4**** Supernatant**

**IV****4 ****Precipitate**** ****20 U/L Plasma**** ****21 U/L Plasma**

**Albumin**

**Fraction III**
**Precipitate**

**Fraction III supernatant****< 2.5 U/L Plasma****21 U/L Plasma**

**Immunoglobulin**

**Alcohol 20%**

**Alcohol 40%**

**IV****1 ****Precipitate**** ****7 U/L Plasma**
**72 U/L Plasma**

_**CPN test method**_**: **
**Cleavage of hippuryl-L-Arg****measured by RP-HPLC.**
**1U CPN releases****1 μM hippuric acid/min**

_**ProCPU test method**_**: **
**Activation with Thrombin-****Thrombomodulin, cleavage of****hippuryl-L-Arg and measurement****with RP-HPLC.****ProCPU = (CPN+CPU) - CPN**

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

**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 CPN in absence and presence of ethanol

- Expressed as ratio: Lysine cleaved/internal standard

**rA1PI**

**Prolastin**

**Lys/IS**

## (rA1PI)

**(rA1PI)**

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

-  -

- +

**A      0       10      25    50   100   250**

**Aralast (A) LH02031A****Experimental Lot 900304B (IV,1)**

**CPM (mU/ml), after 40********10% EtOH treatment**

**pI 4.2**

**pI 4.9**

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

**M2***
**M4****M4***
**M6**

## A1PI Isoform Pattern in Human BAL

**1 ... PROLASTIN #PR4HA43A, 5 μg/ml****2 ... ARALAST #LH03002A, 5 μg/ml****3 ... Human BAL sample, 3.5 μg/ml****4 ... Human BAL sample, 6 μg/ml****5 ... 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 causing****the 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.”**

## Agenda

- Background Information
- Modifications to primary structure of A1PI in all commercial products
- Potential cause for microheterogeneity of A1PI protein in Aralast
- Implications of microheterogeneity on protein structure and function

****

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

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

## A1PI-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**

****

## Functional Relevance of C-terminal Truncation of A1PI

- 0

- 100

- 200

- 300

- 0%

- 10%

- 20%

- 30%

- 40%

- 50%

- 60%

- 70%

**% inhibition of porcine elastase**

**nM A1PI**

**   ****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:  438.7**

**r = 0.9850 **

**slope:  441.8**

**r = 0.9916**

**slope:  443.0**

**r = 0.9894**

**slope:  448.6**

**r = 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 ****C-terminal Truncation of A1PI**

## Higher Degree of C-terminal 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

## Conclusions

- A1PI in all products approved for augmentation therapy demonstrate at least one primary structure modification (deamidation, cysteine modification, and C-terminal lysine truncation)
- 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
- des-Lys is one of many known isoforms of A1PI that does not affect the inhibitor activity, immunogenicity or essential functions of A1PI

## REFERENCE SLIDES

## Molecular 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**

## HPLC isolated A1PI N-glycans

**1 ****PNGase F**

**2****Isolation****+****labelling**

**3****HPLC 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)**

## Cysteinylation of Cys-232: Electrostatic Surface Potential Maps

**Cysteinylation of Cys-232: ****Electrostatic Surface Potential Maps**

**A**

**B**

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

## Deamidation of Asparagine in Proteins

## Carboxypeptidase N, Ethanol Fractionation 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 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), 40********10% EtOH **

**0        10        20        50       100     200**

**pI 4.2**

**pI 4.9**

**M2***
**M4****M4***
**M6**

## Slide 41

## Airway Epithelium

- Interstitial Compartment

- Microscopic distribution of Aralast
- CENTRAL AIRWAY