Alpha1-antitrypsin Deficiency


Table 1: Functional Activity of Prolastin-C (Mean ± 1 Standard Deviation)


Test


Functional activity (mg/ml)* Total protein content (mg/ml) Alpha1


Prolastin-C (n = 6 lots)


-PI antigen content (mg/ml)


Specific activity (functional activity/protein content) Active content (functional activity/antigen content)


*Anti-neutrophil elastase capacity.


Figure 1: SDS-CGE (A) and SDS-PAGE (B) of Prolastin-C under Non-reducing Conditions


Time (sec)


44 42 40 38 36 34 32


30 28 26 24 22 20 18 16


AB


The bands appearing above and below the prominent alpha1 band in SDS-CGE (A) represent the internal standards used in the SDS-CGE method.


SDS-CGE = sodium dodecyl sulfate capillary gel electrophoresis. directly proportional to the quantity of elastase present and inversely


proportional to the quantity of functional alpha1-PI. Specific activity was calculated as the ratio of functional alpha1-PI (mg) to total protein (mg); total protein concentration was determined by the Biuret method.


Functional alpha1-PI content (active content) was calculated as the ratio of functional alpha1-PI (mg) to the alpha1-PI antigen content (mg). Alpha1-PI content was determined by immunonephelometry using the Dade Behring BN II Nephelometer.


Purity


Capillary zone electrophoresis (CZE) was performed on a P/ACE™ MDQ (Beckman Coulter) unit. The running buffer was 200mM borate at pH 9.8. Samples were injected using a 0.5-psi pressure injection for 20 seconds and run with an applied voltage of 10kV and detection at 214nm.


The mass distribution was evaluated by size-exclusion high-performance liquid chromatography (SE-HPLC) using both ultraviolet and ight-scattering detection. SE-HPLC was performed with a TSK-GEL Super


28 kDa


54.3 ± 3.1 55.2 ± 1.7 52.1 ± 3.5 0.98 ± 0.03 1.04 ± 0.04


SW3000 (TosoHaas) column capable of separating proteins of masses from 10,000–500,000Da. The molecular weight and size of the analyte was determined post-ultraviolet detection using the PD2000DLS Plus (Precision Detectors).


Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-capillary gel electrophoresis (SDS-CGE) were performed under non-reducing conditions. SDS-PAGE used Invitrogen 4–12% Bis-Tris polyacrylamide gels in a 3-(N-morpholino)propanesulfonic acid buffer. Gels were stained with SimplyBlue. SDS-CGE was performed on an Agilent 2100 Bioanalyzer.


Reversed-phase HPLC (RP-HPLC) was performed using an Agilent 1100 100 60 Alpha1 -PI 50 40 30 20 15


Matrix-assisted laser desorption ionization (MALDI) was performed on the Voyager DE STR (Applied Biosystems). Samples analyzed by MALDI


mass spectrometry were cleaned up using C4 ZipTips (Millipore). Approximately 0.5µl of a saturated sinapinic acid solution in 50% acetonitrile (ACN) was spotted on the MALDI plate followed by 0.5µl of the cleaned protein solution. Spectra were recorded in the linear mode, using bovine serum albumin (BSA) as external calibrant.


Glycan Profile


Isoelectric focusing (IEF) was performed using a pre-cast Novex® IEF gel (Invitrogen Corporation) with an ampholyte range of pH 3–7. Gels were either stained with SimplyBlue dye or used for Western blotting. Western blots were performed using a SNAP i.d.™ system


(Millipore) with an antialpha1-PI monoclonal primary antibody (QED Bioscience) and a Thermo ImmunoPure Goat Antimouse Immunoglobulin G (IgG) (H+L) (Thermo Fisher Scientific) secondary antibody. The Western blot was stained using bromo-chloro-indolyl phosphate/nitroblue tetrazolium substrate.


Impurities


Plasma protein impurities were assessed by immunonephelometry using a Dade Behring BN II Nephelometer. The 16 proteins tested were albumin, alpha-2 macroglobulin, alpha-1 acid glycoprotein, apolipoprotein A-1, apolipoprotein B, antithrombin III, ceruloplasmin, fibrinogen, fibronectin, haptoglobin, IgA, IgG, IgM, plasminogen/plasmin, prealbumin, and transferrin.


Results Potency


The mean functional activity of six final container lots of Prolastin-C manufactured at commercial scale was 54.3mg/ml (see Table 1), representing approximately twice the functional activity of Prolastin at approximately 25mg/ml. The higher functional activity of Prolastin-C was associated with a mean specific activity of 0.98mg/mg and active content of 1.04mg/mg (see Table 1).


US RESPIRATORY DISEASE


system and a 2.1mm x 150mm Waters Symmetry® C4 column. A gradient elution using 0.1% trifluoroacetic acid in water and increasing amounts of 0.1% trifluoroacetic acid in acetonitrile were used, with detection at 214nm. The column eluate was subjected to electrospray mass spectrometry using an Agilent LC/MSD single quad mass spectrometer. Spectra deconvolution to assess the glycosylation heterogeneity was carried out using MassLynx (Waters) software.


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