59
A biochemical analysis was conducted to investigate the
purity profile of Berinert
®
, 2 other commercially availab-
le plasma-derived pasteurized virus filtered (nanofiltered)
C1-INH concentrates (C1-INH A and B) and 1 recombinant
human transgenic C1-INH concentrate (rhC1-INH) (Ta-
ble 17).
113
The molecular weight identification with pro-
tein gel electrophoresis revealed a main protein band
at
≈
105 kDa for all plasma-derived C1-INH concentrates
and at
≈
98 kDa for the rhC1-INH concentrate (Figure
30). For C1-INH A and B, an additional prominent band
at
≈
64 kDa, identified as alpha-1-antichymotrypsin, was
found to be the main contaminant. Size exclusion chro-
matography revealed the highest purity profile for the
rhC1-INH concentrate (99%). It is, however, not identical
to the human C1-INH protein and has a considerably
shorter half-life. Among the plasma-derived products,
Berinert
®
C1-INH A C1-INH B rhC1-INH
Specific activity (U/mg)
a
11.59
10.42
9.01
12.14
C1-INH antigen (mg/mL)
13.59
26.85
31.29
55.01
Ratio antigen/activity
b
1.24
1.22
1.42
1.67
Purity (%)
95
89
90
99
C1-INH=Human plasma-derived C1-inhibitor concentrate;
rhC1-INH=Recombinant human C1-inhibitor concentrate; U=Units.
a
For Berinert
®
, the potency of C1-INH is expressed in international units (IU).
b
C1-inhibitor activity converted from U/mL to mg/mL, 1 U corresponding to 220 µg.
Values for Berinert
®
, C1-INH B, and rhC1-INH are mean values of 2 analyzed batches.
Table 17 – Biochemical Properties of
Different C1-INH Concentrates
113
Production
Figure 29 – Simplified Flow Diagram of the Manufacture of Berinert
®
1. Cryo-
precipitation
2. Diethylamino-
ethyl (DEAE)
ion exchange
chromatography
3. Quaternary
aminoethyl (QAE)
adsorption
Human
Plasma
C1-INH
Eluate
C1-INH
Precipitate
C1-INH
Pasteurized
C1-INH
Highly
Purified
C1-INH
Concentrate
Berinert
®
4. Ammonium
sulphate
precipitations
5. Stabilization with
sucrose/glycin
6.
Pasteurization in
solution for 10 h
at 60°C
Validated virus reduction steps are highlighted in green.
7. Ammonium
sulphate
precipitations
8.
Hydrophobic
interaction
chromatography
(HIC)
9.
20N/15N virus
filtration
10.Diafiltration
11.Ultrafiltration
12.Formulation
13.Sterile filtration
14.Filling
15. Lyophilization
[kDa]
250
148
98
64
50
36
22
16
6
Non-reduced
Berinert
®
Berinert
®
rhC1-INH
rhC1-INH
Batch 1
Batch 1
Batch 1
Batch 1
Batch 1
Batch 1
Batch 1
Batch 1
Marker
Marker
Batch 2
Batch 2
Batch 2
Batch 2
Batch 2
Batch 2
A
A
B
B
C1-INH
C1-INH
Reduced
Protein Gel Electrophoresis
(8% to 16% Tris Glycine; 5 mOD/lane)
OD=Optical density.
C1-INH=Other plasma-derived C1-INH concentrate.
rhC1-INH=Recombinant human transgenetic C1-INH concentrate.
Berinert
®
has the highest purity profile, with less con-
comitant proteins than C1-INH A and B (Table 17).
The impact of impurities on efficacy or on possible side
effects has not been evaluated yet.
Figure 30 – Protein Gel Electrophoresis
(8% to 16% Tris Glycine; 5 mOD/lane)
113
