A CZE Method for the Identification of Therapeutic Antibodies and Quality Control of Infusion Bags at the Hospital

Transcription

1 CE in the biotechnology and pharmaceutical industries, 17th Symposium on the Practical Applications for the Analysis of Proteins, Nucleotides and Small Molecules, September 20, Thursday, September 24, 2015 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 Ho pital Europe en Georges Pompidou, Paris, France

2 Therapeutics antibodies French hospitals Hospitals stays, Treatments mabs is a growing class of cancer therapeutics: French national cancer Institute, Inca,

3 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 Lean ( or just-in-time) production: Iatrogenic risk To secure drug supply : Quality control required Objectives Patient, Drug, Dose 4

5 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. 1 Martin et al., Bull Cancer, Bazin et al., Ann. Pharm Fr, 2010 Goal: to identify unambiguously mab present in infusion bags 5

6 Compounded therapeutical monoclonal antibodies Capillary Zone Electrophoresis Rituximab (1 mg/ml) Cetuximab (1,5 mg/ml) Bevacizumab 0,5 mg/ml), Trastuzumab (1,2 mg/ml) Ximabs Zumabs High resolution automation quantita tion identific ation 6

7 Charge Development of a CZE method o High Résolution? counter electro-osmotique flow mode Détector - + o Avoid mab adsorption to the capillary wall? Coating Rituximab Trastuzumab Cetuximab Bevacizumab Cationic coating / acidic electrolyte ,5 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5 8 8,5 9 9,5 10 ph ph < pi -100 RDPA 2015, Perugia Global theoretical charge of mabs Polybrene (hexadimethrine bromide) 7

8 Development of CZE method Various BGE composition (ph, ionic strength) Rituximab (1 mg/ml) Cetuximab (1,5 mg/ml) Bevacizumab 0,5 mg/ml), Trastuzumab (1,2 mg/ml) 0,02 E Phosphate 50 mm ph C BEVA TRTU RTUX 0, , , Sodium Phosphate 50 mm, ph 7 Sodium Formate 50 mm, ph 5 Sodium Phosphate 75 mm, ph 3 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) Jaccoulet et al., Electrophoresis 36, , 2015 Partial separation of Rituximab Trastuzumab and Bevacizumab

9 Rs Development of CZE methods Effect of Perchloric acid on BGE Rituximab (1 mg/ml) Cetuximab (1,5 mg/ml) Bevacizumab 0,5 mg/ml), Trastuzumab (1,2 mg/ml) 1,2 1 0,8 0,6 peaks 2 and 3; A peaks 3 and 4 BEVA TRTU RTUX 0,4 0,2 peaks 1 and 2; CTUX 0 0 0,15 0,75 1,5 Concentration of perchloric acid (mm) BGE : perchloric acid 0.15 mm Jaccoulet et al., Electrophoresis 36, , mabs : resolution improvement

10 Development of CZE methods Improvement of the resolution Why?? cm 2 V -1 S -1 4,20E-04 4,10E-04 Polybrene (hexadimethrine bromide) 4,00E-04 3,90E-04 Phosphate Buffer ph 3, 75 mm 3,80E-04 3,70E-04 3,60E-04 µeof 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) PB + Perchloric acid 8% Electroosmotic flow 22 % Cetuximab effective mobility

11 Development of CZE methods Separation improvement by Perchloric Acid Polybrene (hexadimethrine bromide) Increase of the double Layer thickness Interaction with Mabs by ion pairing 11

12 %EOF change Development of CZE methods % change of the EOF before and after Mab runs (n=10) 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 Eof variation -mabs Residual adsorption - Influence of excipients in therapeuticals mabs??

13 Development of CZE methods Therapeutics antibodies : Excipients 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

14 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

15 Development of CZE methods Influence of various concentrations of Polysorbate 80 (Tween 20) in BGE Rituximab (1 mg/ml) Cetuximab (1,5 mg/ml) Bevacizumab 0,5 mg/ml), Trastuzumab (1,2 mg/ml) 0,01 0,014 0,008 0,006 PS 80 0,001% 0,009 PS % 0,004 0,002 0, , , ,006 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, , 2015

16 o Coating : positively charged o Background Electrolyte optimisation: Development of a CZE method 1 Low buffer ph and high ionic strength 2 Addition of an ion pairing agent 3 Addition of a Surfactant 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) Optimized BGE : - Sodium Phosphate 75 mm, ph HClO mm - Polysorbate % (m/v) Jaccoulet et al., Electrophoresis 36, ,

17 CV (%) Absorbance (mau) Mabs identification o Internal standart (IS) Glutamate (pi = 5,65) Relative Migration time (RMT) P2 IS Tm (is) Tm (P2) SI Time (min) 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). Jaccoulet et al., Electrophoresis 36, , ,6 0,5 0,4 0,3 0,2 0,1 0 CV (MT) and (RMT) mabs (n=6) CTUX BEVA TRTU RTUX RMT: RSD (%) RMT Identification TM TMR 17

18 Absorbance (mau) Mabs identification Internal standard = Glutamine Relative Migration time (RMT) 0,014 0,012 0,01 0,008 0,006 0,004 Glutamine (IS) Citric acid Glycine Histidine 0, ,002 Time (min) Polybrene coating BGE : 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 MMB Submitted

19 Mabs identification : validation 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) Jaccoulet et al., Electrophoresis 36, ,

20 CTUX3 CTUX1 CTUX2 A23 A21 A22 BEVA1 BEVA2 BEVA3 RTUX1 RTUX2 RTUX3 TRTU1 A31 TRTU3 A32 TRTU2 A33 Dissimilarité Mabs identification 14 Blind test o 5 infusion bags (4 mabs + NaCl 0.9%) o 3 randoms : A2, A3, A4 o CZE : A2 et A3 Peaks o A4 no Peak Agglomerative hierarchical clustering (AHC) oaggregation: Ward ocriteria: RMT AHC: 4 groups= 4 mabs A2 Bevacizumab (BEVA) A3 Trastuzumab (TRTU) Results A2 = Bevacizumab A3 = Trastuzumab A4 = NaCl 0.9% (pas de pics) Jaccoulet et al., Electrophoresis 36, ,

21 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

22 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