CE in the biotechnology and pharmaceutical industries ... · CV (MT) and (RMT) mAbs (n=6) TM TMR RMT: RSD (%) RMT Identification (SI U) Tm (Time (min) Mabs identification Jaccoulet

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)



22 % Cetuximab effective mobility

8% Electroosmotic flow

11




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


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


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


-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



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


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


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

Documents

CE in the biotechnology and pharmaceutical industries ... · CV (MT) and (RMT) mAbs (n=6) TM TMR RMT: RSD (%) RMT Identification (SI U) Tm (Time (min) Mabs identification Jaccoulet