Development of an ADC Process with Single-Use Membrane ...€¦ · World ADC 2019 Cambridge, UK -...
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Development of an ADC Process with Single-Use Membrane Technology Juan Carlos Cordova 1 , Sheng Sun 1 , Jieyu Zhou 1 , Jesse Novak 1 , Jeffrey Bos 1 , Srinath Thirumalairajan 1 , Ian Schwartz 2 , Sanjeevani Ghone 1 1. Abzena, 360 George Pa�erson Blvd. Bristol, PA 19007, United States 2. Sartorius North America, 565 Johnson Avenue. Bohemia, NY 11716, USA Membrane chromatography is r ou�nely used to remove host cell proteins, viral par�cles, and aggregates during an�body downstream processing. The applica�on of membrane chromatography to the field of an�body-drug conjugates (ADCs) has been applied in a limited capacity and in only specialized scenarios. Here, we u�lized the characteris �cs of the membrane adsorbers, Sartobind® S and Phenyl, for aggregate and payload clearance whilst polishing the ADC in a single chromatographic run. The Sartobind® S membrane was used in the removal of excess payload while the Sartobind® Phenyl was used to polish the ADC by clearance of unwanted DAR species and aggregates. The Sartobind® S membrane reproducibly achieved log-fold clearance of free payload with a ten membrane volume wash. Applica�on of the Sartobind® Phenyl decreased aggregates and higher DAR species while increasing DAR homogeneity. The Sartobind® S and Phenyl membranes were placed in tandem to simplify the process in a single chromatographic run. With the op�mized binding, washing and elu�on condi�ons, the tandem membrane approach was performed in a shorter �mescale with minimum solvent consump�on with high yield. The applica�on of the tandem membrane chromatography system presents a novel and efficient purifica�on scheme that can be realized during ADC manufacturing. The development of ca�on-exchange and hydrophobic inter ac�on membrane chromatography for site-specific ADC process is described using an engineered cysteine-mAb with Pyrrolobenzodiazepine(PBD)-dimer as a model conjuga�on system. Key process parameters such as product yield, efficiency of free PBD- dimer and aggregate removal were evaluated. Abstract Process Diagrams Figure 1. Process diagram. (A) Bind/Elute using Sartobind® S to remove residual payload, and (B) Bind/Elute using Sartobind® Phenyl to remove aggregate in stand-alone model; (C) Simplified process to purify target ADC with Sartobind® S and Phenyl in tandem model. A. B. C. Results C/C0 = 0.19%, 24.13 ng/mL C/C0 = 0.15%, 19.63 ng/mL B. C. D. A. Figure 2. (A) Removal of residual PBD using Sartobind® S in stand-alone model. The quenched reac�on mixture (gram scale) was loaded to a 150 mL Sartobind® S membrane, then washed with 10 mv binding buff er prior to elute. (B) HIC and (C) SEC pr ofiles showing the removal of free PBD dimer. (D) LC-MS method showing PBD dimer removal efficiency with Sartobind® S. Figure 3. (A) Purifica�on of engineered Cysteine-mAb-PBD ADC using Sartobind® Phenyl in stand-alone model. The Sartobind® S purified material (gram scale) was loaded to a 150 mL Sartobind® Phenyl membrane, then targeted and undesired ADC species were eluted sequen�ally. (B) DoE study to op�mize the aggregates removal with Sartobind® Phenyl purifica�on condi�ons. (C) HIC pr ofile of ADC prior/pos Sartobind® Phenyl purifica�on. B. C. A. 85 % recovery 98 % recovery HIC profile C Aggregates Free PBD-dimer D0 & D1 D2 Undesired species B. C. A. DAR 1.68 D1 D2 Undesired D0 DAR 1.94 80 % recovery HIC profile C D1 D2 Figure 4. (A) Purifica�on of engineered Cysteine-mAb-PBD ADC in tandem model. The quenched reac�on mixture (gram scale) was loaded to Sartobind® S which was tandemly connected to Sartobind® Phenyl. The conjuga�on species were separated through loading, washing, and mul�ple elu�on steps. (B) HIC pr ofile showing the crude reac�on mixture contains ADC variants with a range of DAR, residual PBD dimer, aggregates, and organic solvent. (C) HIC pr ofile of the tandem method purified ADC. The DAR of the ADC increased from 1.68 to 1.94. (D) SEC pr ofile showing the percentage of aggregate dropped from 5 % to less than 1 % a�er tandem model purifica�on. D. 5% > 1% Free PBD-dimer D0 & D1 D2 Undesired species Conclusions Acknowledgements We thank Sartorius for providing MODDE® so�ware for DoE analysis, and the membranes for screening and scale-up work. • Membrane based process consumed less buff er, shortened process and hold �mes (less GMP scien�st FTE days) which can reduce the cost and �me for a cGMP manufacturing campaign. • Membrane devices are scalable single-use, closed systems that improve manufacturing safety, elimina�ng the need of packing, qualifica�on, and cleaning valida�on studies associated with resin-based column chromatography. • Removal of free payload and undesired conjugate species by membrane chromatography presents a novel and efficient process that directly translates into improved efficiency both during process development and cGMP manufacturing. Intr oduc�on Sartobind® S stand-alone Sartobind® Phenyl stand-alone Tandem Model Contact: [email protected] and [email protected] www.abzena.com World ADC 2019 Cambridge, UK - San Diego, CA - Bristol, PA Sartobind® S DBC mAb 32 mg/ml MMAE-ADC 37 mg/ml PBD-ADC 34 mg/ml Sartobind® Phenyl DBC mAb 13 mg/ml MMAE-ADC 14 mg/ml PBD-ADC 14.9 mg/ml Before A�er
Transcript of Development of an ADC Process with Single-Use Membrane ...€¦ · World ADC 2019 Cambridge, UK -...
Development of an ADC Process with Single-Use Membrane TechnologyJuan Carlos Cordova1, Sheng Sun1, Jieyu Zhou1, Jesse Novak1, Jeffrey Bos1, Srinath Thirumalairajan1, Ian Schwartz2, Sanjeevani Ghone1
1. Abzena, 360 George Pa�erson Blvd. Bristol, PA 19007, United States2. Sartorius North America, 565 Johnson Avenue. Bohemia, NY 11716, USA
Membrane chromatography is rou�nely used to remove host cell proteins,viral par�cles, and aggregates during an�body downstream processing. Theapplica�on of membrane chromatography to the field of an�body-drugconjugates (ADCs) has been applied in a limited capacity and in onlyspecialized scenarios.Here, we u�lized the characteris�cs of the membrane adsorbers, Sartobind® Sand Phenyl, for aggregate and payload clearance whilst polishing the ADC in asingle chromatographic run. The Sartobind® S membrane was used in theremoval of excess payload while the Sartobind® Phenyl was used to polish theADC by clearance of unwanted DAR species and aggregates. The Sartobind® Smembrane reproducibly achieved log-fold clearance of free payload with a tenmembrane volume wash. Applica�on of the Sartobind® Phenyl decreasedaggregates and higher DAR species while increasing DAR homogeneity. TheSartobind® S and Phenyl membranes were placed in tandem to simplify theprocess in a single chromatographic run. With the op�mized binding, washingand elu�on condi�ons, the tandem membrane approach was performed in ashorter �mescale with minimum solvent consump�on with high yield. Theapplica�on of the tandem membrane chromatography system presents anovel and efficient purifica�on scheme that can be realized during ADCmanufacturing.
The development of ca�on-exchange and hydrophobic interac�on membranechromatography for site-specific ADC process is described using an engineeredcysteine-mAb with Pyrrolobenzodiazepine(PBD)-dimer as a model conjuga�onsystem. Key process parameters such as product yield, efficiency of free PBD-dimer and aggregate removal were evaluated.
Abstract
Process Diagrams
Figure 1. Process diagram. (A) Bind/Elute using Sartobind® S to remove residual payload,and (B) Bind/Elute using Sartobind® Phenyl to remove aggregate in stand-alone model; (C)Simplified process to purify target ADC with Sartobind® S and Phenyl in tandem model.
A. B.
C.
Results
C/C0 = 0.19%, 24.13 ng/mLC/C0 = 0.15%, 19.63 ng/mL
B. C. D.
A.
Figure 2. (A) Removal of residual PBD using Sartobind® S in stand-alone model. Thequenched reac�on mixture (gram scale) was loaded to a 150 mL Sartobind® S membrane,then washed with 10 mv binding buffer prior to elute. (B) HIC and (C) SEC profiles showingthe removal of free PBD dimer. (D) LC-MS method showing PBD dimer removal efficiencywith Sartobind® S.
Figure 3. (A) Purifica�on of engineered Cysteine-mAb-PBD ADC using Sartobind® Phenyl instand-alone model. The Sartobind® S purified material (gram scale) was loaded to a 150mL Sartobind® Phenyl membrane, then targeted and undesired ADC species were elutedsequen�ally. (B) DoE study to op�mize the aggregates removal with Sartobind® Phenylpurifica�on condi�ons. (C) HIC profile of ADC prior/pos Sartobind® Phenyl purifica�on.
B. C.
A.
85 % recovery
98 % recovery
HIC profile C
Aggregates
Free PBD-dimer
D0 & D1
D2
Undesired species
B. C.
A.
DAR 1.68
D1
D2
Undesired D0
DAR 1.9480 % recovery
HIC profile C
D1
D2
Figure 4. (A) Purifica�on of engineered Cysteine-mAb-PBD ADC in tandem model. Thequenched reac�on mixture (gram scale) was loaded to Sartobind® S which was tandemlyconnected to Sartobind® Phenyl. The conjuga�on species were separated throughloading, washing, and mul�ple elu�on steps. (B) HIC profile showing the crude reac�onmixture contains ADC variants with a range of DAR, residual PBD dimer, aggregates, andorganic solvent. (C) HIC profile of the tandem method purified ADC. The DAR of the ADCincreased from 1.68 to 1.94. (D) SEC profile showing the percentage of aggregate droppedfrom 5 % to less than 1 % a�er tandem model purifica�on.
D.
5%> 1%
Free PBD-dimerD0 & D1D2Undesired species
Conclusions
Acknowledgements
We thank Sartorius for providing MODDE® so�ware for DoE analysis, and themembranes for screening and scale-up work.
• Membrane based process consumed less buffer, shortened process andhold �mes (less GMP scien�st FTE days) which can reduce the cost and�me for a cGMP manufacturing campaign.
• Membrane devices are scalable single-use, closed systems that improvemanufacturing safety, elimina�ng the need of packing, qualifica�on, andcleaning valida�on studies associated with resin-based columnchromatography.
• Removal of free payload and undesired conjugate species by membranechromatography presents a novel and efficient process that directlytranslates into improved efficiency both during process developmentand cGMP manufacturing.
Introduc�on
Sartobind® S stand-alone
Sartobind® Phenyl stand-alone
Tandem Model
Contact: [email protected] and [email protected]
www.abzena.comWorld ADC 2019 Cambridge, UK - San Diego, CA - Bristol, PA
Sartobind® S DBC
mAb 32 mg/mlMMAE-ADC 37 mg/ml
PBD-ADC 34 mg/ml
Sartobind® Phenyl DBC
mAb 13 mg/mlMMAE-ADC 14 mg/ml
PBD-ADC 14.9 mg/ml
Before
A�er
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