A CZE Method for the Identification of Therapeutic Antibodies and Quality Control of Infusion Bags at
the Hospital
E. JACCOULET, M. TAVERNA, C. SMADJA Institut Galien Paris-Sud (France), UMR CNRS 8612
Team Protein and Nanotechnologies in Analytical Sciences
Hopital Europeen Georges Pompidou,
Paris, France
CE in the biotechnology and pharmaceutical industries, 17th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, September 20, 2015 - Thursday, September 24, 2015
Therapeutics antibodies
2
Hospitals stays, Treatments French national cancer Institute, Inca, 2014
mAbs is a growing class of cancer therapeutics:
French hospitals
Quality control of monoclonal antibodies for anticancer treatments at the hospital
• Anticancer mAbs - 14 Molecules FDA- or EMA- approved ,+ biosimilars
• Hospital activity - Chemotherapy (+10%)
- Ambulatory activity (short timeframe)
- Pharmaceutical compounding (EHGP: +61% in 4 years)
• Compounding anticancer drugs in Hospital - A specific area dedicated for compounding = aseptic production unit - Qualified staff for handling
4
Quality control of monoclonal antibodies for anticancer treatments at the hospital
Objectives Patient, Drug, Dose
Lean ( or just-in-time) production: Iatrogenic risk To secure drug supply : Quality control required
Quality control of coupounded therapeutic monoclonal antibodies
Quality control
– Currently in France (Europe): visual control and FTIR
What kind of QC method are we looking for?
– On line pre-delivery control
– Qualitative (ID) and quantitative information needed
– Fast , simple and automated analytical method
Analytical challenges
– Formulated products (excipients)
– Similar physico-chemical properties of mAbs.
5 Goal: to identify unambiguously mab present in infusion bags
1Martin et al., Bull Cancer, 2004 2Bazin et al., Ann. Pharm Fr, 2010
Compounded therapeutical monoclonal
antibodies
6
Capillary Zone Electrophoresis
High resolution automation
identification
quantitation
Ximabs
Zumabs
Rituximab (1 mg/mL) Cetuximab (1,5 mg/mL) Bevacizumab 0,5 mg/mL), Trastuzumab (1,2 mg/mL)
Development of a CZE method
o High Résolution ? counter electro-osmotique flow mode
o Avoid mAb adsorption to the capillary wall? Coating
7
Cationic coating / acidic electrolyte
pH < pI
Polybrene (hexadimethrine bromide)
-100
-50
0
50
100
150
2,5 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5 8 8,5 9 9,5 10
Ch
arge
pH
RituximabTrastuzumabCetuximabBevacizumab
Global theoretical charge of mAbs
- + Détector
RDPA 2015, Perugia
Development of CZE method
-4
0
4
8
12
16
20
24
0 5 10 15
C
Conditions: Phosphate buffer 75 mM (63,75 mM*) pH 3 – Injection : 0,5 psi, 10 sec – Voltage: 20 kV - Capillary : 50 µm ID, total lenght: 60 cm. Coating: Polybrene 0,2% - Mixture: rituximab (1 mg/mL), cetuximab (1,5 mg/mL), bevacizumab 0,5 mg/mL), Trastuzumab (1,2 mg/mL)
Various BGE composition (pH, ionic strength)
Sodium Formate 50 mM, pH 5
Sodium Phosphate 75 mM, pH 3
0
0,005
0,01
0,015
0,02
0 5 10 15 20
Phosphate 50 mM pH 7
E
Sodium Phosphate 50 mM, pH 7
BEVA
TRTU RTUX
Rituximab (1 mg/mL) Cetuximab (1,5 mg/mL) Bevacizumab 0,5 mg/mL), Trastuzumab (1,2 mg/mL)
Partial separation of Rituximab Trastuzumab and Bevacizumab
Jaccoulet et al., Electrophoresis 36, 2050–2056, 2015
Development of CZE methods
0
0,2
0,4
0,6
0,8
1
1,2
0 0,15 0,75 1,5
Rs
A
Effect of Perchloric acid on BGE
Concentration of perchloric acid (mM)
BEVA TRTU
RTUX
CTUX
BGE : perchloric acid 0.15 mM
Rituximab (1 mg/mL) Cetuximab (1,5 mg/mL)
Bevacizumab 0,5 mg/mL), Trastuzumab (1,2 mg/mL)
4 mAbs : resolution improvement Jaccoulet et al., Electrophoresis 36, 2050–2056, 2015
peaks 1 and 2;
peaks 2 and 3;
peaks 3 and 4
3,60E-04
3,70E-04
3,80E-04
3,90E-04
4,00E-04
4,10E-04
4,20E-04
µEOF
Phosphate Buffer pH 3, 75 mM
PB + Perchloric acid
cm2 V-1 S-1
Improvement of the resolution Why ??
Conditions: Phosphate buffer 75 mM (63,75 mM*) pH 3, perchloric acid (0.15 mM) – Injection : 0,5 psi, 10 sec – Voltage: 20 kV - Capillary : 50 µm ID, total lenght: 60 cm. Coating: Polybrene 0,2% cetuximab (1,5 mg/mL)., Eof marker (thiourea 1 g/L)
Polybrene (hexadimethrine bromide)
Development of CZE methods
22 % Cetuximab effective mobility
8% Electroosmotic flow
11
Development of CZE methods
Polybrene (hexadimethrine bromide)
Polybrene (hexadimethrine bromide)
Increase of the double Layer thickness
Separation improvement by Perchloric Acid
Interaction with Mabs by
ion pairing
0
1
2
3
4
5
6
7
Beva Cetux Ritux Trastu
Conditions: Phosphate buffer 75 mM pH 3 (with tween 80 + HClO4 1 mM) – Injection : 0.5 psi/10 sec – Voltage : 20 kV - EOF marker = Thiourea 1 g/L
% change of the EOF before and
after Mab runs (n=10)
Eof variation
-mAbs Residual adsorption - Influence of excipients
in therapeuticals mabs ??
%EO
F ch
ange
Development of CZE methods
Bevacizumab Trastuzumab Rituximab Cetuximab
Polysorbate 20 Polysorbate 20 Polysorbate 80 Polysorbate 80
Dihydrate trehalose
Dihydrate trehalose
Sodium Citrate Glycine
Sodium phosphate
Histidine NaCl, NaOH Citric acid, NaOH
L-Histidine
Therapeutics antibodies : Excipients
Development of CZE methods
Influence of Polysorbate 80 (0.001%) (Tween 20) in the BGE
Cetuximab Rituximab Bevaxizumab
eof (rsd)
Without PS 80 2,67 6,30 2,06
With PS 80 1,93 2,89 1,53
Development of CZE methods
-0,006
-0,001
0,004
0,009
0,014
0 5 10 15
PS 80 0.01%
-0,002
0
0,002
0,004
0,006
0,008
0,01
0 5 10 15
PS 80 0,001%
Rituximab (1 mg/mL) Cetuximab (1,5 mg/mL)
Bevacizumab 0,5 mg/mL), Trastuzumab (1,2 mg/mL)
Influence of various concentrations of Polysorbate 80 (Tween 20) in BGE
Conditions: Phosphate buffer 75 mM (63,75 mM*) pH 3 – Injection : 0,5 psi, 10 sec – Voltage: 20 kV - Capillary : 50 µm ID, total lenght: 60 cm. Coating: Polybrene 0,2% - Mixture: rituximab (1 mg/mL), cetuximab (1,5 mg/mL), bevacizumab 0,5 mg/mL), Trastuzumab (1,2 mg/mL)
Improvement of the resolution, 4 mabs separation
Jaccoulet et al., Electrophoresis 36, 2050–2056, 2015
Development of CZE methods
o Coating : positively charged
o Background Electrolyte optimisation:
16
Low buffer pH and high ionic strength
Conditions: Phosphate Buffer 75 mM (63,75 mM*) pH 3 – Injection : 0,5 psi, 10 sec – Voltage: 20 kV - Capillary: 50 µm ID, total length: 60 cm. Coating: Polybrene 0,2% - Mixture: rituximab (1 mg/mL), cetuximab (1,5 mg/mL), bevacizumab 0,5 mg/mL), Trastuzumab (1,2 mg/mL)
1
Optimized BGE :
- Sodium Phosphate 75 mM, pH 3.0
- HClO4 0.15 mM
- Polysorbate 80 0.01% (m/v)
3
Addition of a Surfactant
Addition of an ion pairing agent
2
Development of a CZE method
Jaccoulet et al., Electrophoresis 36, 2050–2056, 2015
o Internal standart (IS)
17
Conditions: Phosphate Buffer 75 mM (63,75 mM*) pH 3 A cide Perchloric Acid (0,15 mM) – Polysorbate 80 (0,01%) – Injection : 0,5 psi, 10 sec – Voltage V= 20 kV - Capillary : 50 µm ID, Tl: 60 cm. Coating: Polybrene 0,2% - Mix: rituximab (R) at 1 mg/mL, cetuximab (C) at 1,5 mg/mL, bevacizumab (B) at 0,5 mg/mL, Trastuzumab (T) at 1,2 mg/mL, Glutamine (IS).
Glutamate (pI = 5,65)
0
0,1
0,2
0,3
0,4
0,5
0,6
CTUX BEVA TRTU RTUXC
V (
%)
CV (MT) and (RMT) mAbs (n=6)
TM
TMR
RMT: RSD (%) RMT
Identification
SI
Ab
sorb
ance
(m
AU
)
Time (min)
Mabs identification
Jaccoulet et al., Electrophoresis 36, 2050–2056, 2015
Relative Migration time (RMT)
Tm (iS) Tm (P2)
IS
P2
-0,002
0
0,002
0,004
0,006
0,008
0,01
0,012
0,014
-1 1 3 5 7 9 11 13 15
Histidine
Glutamine
(IS) Citric acid
Glycine
Time (min)
Polybrene coating – BGE : 63.75 mM Phosphate / 0.15 mM HClO4 pH 3 with 0.01% Tween 80 - Sample:
Mix of 4 mabs (Bevacizumab, cetuximab, rituximab and trastuzumab) + Glutamine (IS). PeakMaster 5.3
(Takeshi) for theoretical position of Citric acid, Glycine,and Histidine). G
Ab
sorb
ance
(m
AU
)
Internal standard = Glutamine
Mabs identification
Relative Migration time (RMT)
MMB Submitted
• Repetability
• Intraday RSD < 0.32% (n=6)
• Intermediate precision < 1.3 % (n=18)
• Specificity
• No interference with excipients
• Discrimination • RMT (p < 0.023) (ANOVA test)
19 Jaccoulet et al., Electrophoresis 36, 2050–2056, 2015
Mabs identification : validation
Blind test
o 5 infusion bags (4 mAbs + NaCl 0.9%)
o 3 randoms : A2, A3, A4
20 Jaccoulet et al., Electrophoresis 36, 2050–2056, 2015
AHC: 4 groups= 4 mAbs A2 Bevacizumab (BEVA) A3 Trastuzumab (TRTU)
Results A2 = Bevacizumab A3 = Trastuzumab A4 = NaCl 0.9% (pas de pics)
CTU
X3
CTU
X1
CTU
X2
A2
3
A2
1
A2
2
BEV
A1
BEV
A2
BEV
A3
RTU
X1
RTU
X2
RTU
X3
TRTU
1
A3
1
TRTU
3
A3
2
TRTU
2
A3
3
0
2
4
6
8
10
12
14
Dis
sim
ilari
té
o CZE : A2 et A3 Peaks
o A4 no Peak
Agglomerative hierarchical clustering (AHC)
oAggregation: Ward
oCriteria: RMT
Mabs identification
Conclusions
o Identification of the 4 mAbs by CZE Hospitals
o Fast, simple, automation
o RMT
o discrimination
o Futur:
o Quantification
o Application to other mAbs
o Spectroscopic analysis
21
Aknowledgments
o PNAS - Institut Galien Paris Sud - CNRS-UMR 8612 (Pr M. Taverna)
o Dr E. Jaccoulet
o Pr M. Taverna
o European Hospital Georges Pompidou (Pharmacy) o Pr. Patrice Prognon
22