Deamidation - Amazon AWS

Compatibility Assessment of Model Monoclonal Antibody Formulation in Glass and Blow Fill Seal (BFS) Plastic Vial Delivery Formats. ABSTRACT

catalent pharma solutions Dipesh Shah, Gregory Bleck and Ian J. Collins Woodstock, IL and Madison, WI

Amount (mg/mL)

Model mAb ( Mol. Wt. 144kDa)

10

Polysorbate 80

0.7

Sodium Citrate

6.5

Sodium Chloride

9.0

pH

6.5

Table 4: % Modification -Methionine Oxidation and Deamidation Type

Oxidation

Deamidation

Figure 4: % High Molecular weight species vs Time (5°C)

storage to 6M at 5°C. II. Complement Dependent Cytotoxic Assay: The potency was determined using responsive cell line in a complement dependent cytotoxic assay using a fluorescence read out. Comparison of a dilution series with standard, formulation in glass and the Advasept™ Vials were generated (Figure 1).

Figure 1: Dose Response Curve for model mAb formulation

Figure 8: Magnified Chromatograms Time 0 a) Glass Vial

b) Advasept™ Vial

Sample

% Modification

Glass vial

10.8

Advasept™ Vial

4.5

Glass vial

7.2

Advasept™ Vial

7.6

The Glass and Advasept™ vial samples show no major difference in UPLC/UV chromatograms from both trypsin and ASP-N digested peptides, with 100% sequence coverage with MS detection. Observed deamidation levels in MS are comparable between Glass and Advasept™ Vials. The oxidation levels were higher in Glass relative to Advasept™ Vials and could be attributed to mAb’s surface interaction with glass surface causing more oxidation than in plastic Advasept™ Vials.

Graph#1

Results and Data Analysis: The SEC Data shows comparable % high molecular weight

3000

species between the Glass and Advasept™ Vials. 2000

2. SDS-PAGE Analysis:

0 0.001

Results and Data Analysis: The chromatograms showed no comparable charge

Figure 5: SDS-PAGE (Non-Reduced)

1000

0.01

0.1

1

distribution between the Glass and Advasept™ Vial. 4. Peptide Mapping:

10

x axis y = ( (A - D)/(1 + (x/C)^B ) ) + D: Advasept 2 (Advasept 2: Conc vs MeanValue) Glass vial 2 (Glass Vial 2: Conc vs MeanValue) Advasept 1 (Advasept 1: Conc vs MeanValue) Glass vial 1 (Glass vial 1: Conc vs MeanValue)

A 3574.447 3511.099 3534.978 3568.287

B 1.618 1.729 1.711 1.728

C 0.373 0.482 0.391 0.46

D 247.764 325.305 320.817 338.779

Aged Glass and Advasept™ Vials (4M/5°C) were subjected to peptide mapping by

R^2 0.996 0.998 0.998 0.999

initially denaturing and reduction of the mAb with DTT, alkylation with Iodoacetamide, followed by clean-up on a column, and digestion with Trypsin and ASP-N. The peptides

Table 3: mAb potency values (6M/5°C)

were separated on a UPLC column and UV and MS detectors were employed.

Container

Average EC50 (mcg/mL)

Parameters

Method

Target Range

pH

USP 791

6.5 + 0.3

Appearance

Visual Inspection

Report Results

UV (280 nm)

T =0 ± 10%



4) Stability:

Activity

Report EC50



1. SEC Analysis

Size-Exclusion Chromatography (SEC)

Report % Monomer and % High and low molecular weights

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Report % Area (HC +LC)

Capillary Isoelectric focusing (cIEF)

pI (% of each peak)

Peptide Mapping**

% Chemical Modification

Bacterial Endotoxin*

USP 85

Report Results

Bioburden*

USP 61