- Email: [email protected]
Outcome of the antibotabe mab development project
S40
Abstracts / Toxicon 123 (2016) S2eS90
Conclusions: Results suggest that incobotulinumtoxinA is an effective and well-tolerated therapeutic option for lower limb spasticity in young, lowweight children with CP and other motor disorders. Further studies in a larger population are needed to confirm safety and effectiveness in different muscles and after repeated injections. Keywords: Botulinum toxin A; Cerebral palsy; IncobotulinumtoxinA; Spasticity 102. AN EVALUATION OF THE cGMP MANUFACTURING PROCESS ECONOMICS AND HIGH-THROUGHPUT CHARACTERIZATION OF TARGETED SECRETION INHIBITORS Andy Dalby b, a Ipsen London,
Andrew Splevins a, Peter Hooker a, *, b Suzanne Farid . Bioinnovation, Abingdon, Oxfordshire, UK; London, UK
Horrocks a, b
Paul
University College
* Corresponding author: Ipsen Bioinnovation, 102 Park Drive, Abingdon, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
Introduction and objectives: Targeted secretion inhibitors (TSIs) are a novel class of recombinant biotherapeutics that use protein engineering to retarget botulinum neurotoxins for treatment of diseases with secretion disorders. SXN101959 has been successfully manufactured by Ipsen to Current Good Manufacturing Practice (cGMP) standards and is an example of an emerging recombinant TSI manufacturing platform. SXN101959 is a multi-domain, multi-functional recombinant protein expressed within Escherichia coli, composed of a light chain (LC/D) endopeptidase domain and a heavy chain (HN/D) comprising the translocation domain and the growth-hormone-releasing-hormone (GHRH) targeting peptide ligand. The Engineering and Physical Sciences Research Council (EPSRC) within the UK has established a Centre for Innovative Manufacturing in Emergent Macromolecular Therapies at University College London. The Centre provides an international lead in delivering biopharmaceutical manufacturing innovations for next-generation advanced therapies, which has included an evaluation of SXN101959. We present an assessment of SXN101959 using 2 research work streamsdWorkstream 1: an assessment of the process economics and manufacturability of SXN101959, including cost of goods; and Workstream 2: the development of rapid biophysical characterization decisional tools that assess aggregation propensity and facilitate formulation development. Methods: Workstream 1: Process and economic data from the cGMP manufacturing of SXN101959 were collated and combined into a spreadsheet-based mathematic model in order to determine the key cost factors and to allow future improvements that would reduce manufacturing costs. Workstream 2: The aggregation properties of SXN101959 were studied by size-exclusion high-performance liquid chromatography (SE-HPLC), static light scattering (SLS), intrinsic fluorescence, and monitoring binding of fluorescent dyes. These studies were performed in various buffers. Results and Conclusions: We present the key outcomes from the 2 SXN101959 research workstreams. Workstream 1: successful generation of a mathematic spreadsheet-based tool, able to identify key costs in the manufacturing process and to estimate effects of manufacturing scale on costs of goods; and Workstream 2: a suite of analytical tools able to provide data on the aggregation propensity of SXN101959 and so identify solvent conditions for minimizing aggregation. Funding: Ipsen and EPSRC Keywords: Botulinum neurotoxin (BoNT); Manufacture; Process development; Product characterization; SXN101959; Targeted secretion inhibitor (TSI) 103. RECOMBINANT BOTULINUM NEUROTOXIN SEROTYPE A1 (SXN102342): PROTEIN ENGINEERING AND PROCESS DEVELOPMENT
Introduction and objectives: Botulinum neurotoxin is effective in the treatment of several movement disorders (Simpson 2016). Native BoNT/A comprises a family of highly related neurotoxins produced by Clostridium botulinum bacteria, all neutralized by the same antisera. BoNT/A (subtype A1) is notable as the Hall strain BoNT/A used to produce abobotulinumtoxinA (Dysport), onabotulinumtoxinA (Botox), and incobotulinumtoxinA (Xeomin). An increased understanding of the structurefunction relationship of BoNT provides an opportunity to engineer recombinant (r) BoNTs with unique pharmacologic properties and therapeutic applications. Methods: Here we describe the construction, expression, purification, and characterization of rBoNT serotype A1 (rBoNT/A1). Results: This rBoNT comprises fully synthesized DNA, codon-optimized for heterologous, soluble expression in Escherichia coli. Purification is by hydrophobic interaction and ion exchange chromatography, coupled with an endoproteinase activation step that cleaves the expressed single-chain precursor protein and produces the fully active di-chain rBoNT/A1 product, functionally equivalent to the active agents present in commercial BoNT/ A1 products. Conclusions: The opportunity to successfully engineer and manufacture rBoNT/A1, as well as other genetically modified rBoNTs with differentiated pharmacologic properties, provides an alternative to current commercial BoNT products. Funding: Ipsen Reference Simpson DM, Hallett M, Ashman EJ, et al. Practice guideline update summary: botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity and headache: Report of the Guideline Development Subcommittee of the American Academy of Neurology. Neurology.2016;86(19):1818-1826. Keywords: Botulinum neurotoxin; (BoNT); Process development; Product characterization; Protein engineering; Serotype A1 104. OUTCOME OF THE ANTIBOTABE MAB DEVELOPMENT PROJECT Michael Hust. €t Braunschweig, Institut für Biochemie, Biotechnologie Technische Universita und Bioinformatik, Spielmannstrasse 7, 38106, Braunschweig, GermanyEmail address:[email protected]
Botulinum neurotoxins (BoNTs) are among the most toxic substances known and are listed as “dirty dozen” agents and possible bioweapons. The results of the European Union AntiBotABE project are presented. In brief, antibodies were generated against botulinum neurotoxin (BoNT) serotypes A, B, and E using macaque immune phage display libraries. To test their neutralization capacities, anti-BoNT antibody fragments (single-chain variable fragment [scFv] or scFv-Fc format) directed against the BoNT HC fragments were tested in ex vivo (mouse phrenic nerve-hemidiaphragm) assays, and the antibodies directed against the light chains were tested in vitro and ex vivo. The anti-HC and antielight chain antibodies with the best neutralizing profiles were selected and tested in nonlethal in vivo protection (mouse flaccid paralysis) assays alone and in combination. Subsequently, these antibodies were germline-humanized to reduce potential immunogenicity and produced as human immunoglobulin G (IgG). The in vivo efficacy of these antibodies was verified in nonlethal and lethal mouse experiments. Combining the anti-HC antibody with the corresponding antielight chain antibody showed a strong synergistic effect. Interestingly, a single antibody against the BoNT/E light chain was protective in vivo. These five antibodies against three botulinum toxin serotypes are intended for further clinical development as an oligoclonal drug cocktail. Keywords: Antibody engineering; Botulinum toxins; IgG; In vivo neutralization; In vivo protection; Phage display; scFv; scFv-Fc
Andy Hooker*, Shilpa Palan, Matthew Beard. Ipsen Bioinnovation, Abingdon, Oxfordshire, UK
105. MYSTICOL: A CONTROLLED STUDY OF MYOBLOC IN THE TREATMENT OF SIALORRHEA IN PARKINSON'S DISEASE (PD) AND OTHER NEUROLOGICAL CONDITIONS
* Corresponding author: Ipsen Bioinnovation Ltd., 102 Park Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
a
Stuart Isaacson a, Lawrence Severt b, Thomas Clinch c, *. Parkinson’s Disease and Movement Disorder Center, Boca Raton, FL, USA;
Abstracts / Toxicon 123 (2016) S2eS90
Conclusions: Results suggest that incobotulinumtoxinA is an effective and well-tolerated therapeutic option for lower limb spasticity in young, lowweight children with CP and other motor disorders. Further studies in a larger population are needed to confirm safety and effectiveness in different muscles and after repeated injections. Keywords: Botulinum toxin A; Cerebral palsy; IncobotulinumtoxinA; Spasticity 102. AN EVALUATION OF THE cGMP MANUFACTURING PROCESS ECONOMICS AND HIGH-THROUGHPUT CHARACTERIZATION OF TARGETED SECRETION INHIBITORS Andy Dalby b, a Ipsen London,
Andrew Splevins a, Peter Hooker a, *, b Suzanne Farid . Bioinnovation, Abingdon, Oxfordshire, UK; London, UK
Horrocks a, b
Paul
University College
* Corresponding author: Ipsen Bioinnovation, 102 Park Drive, Abingdon, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
Introduction and objectives: Targeted secretion inhibitors (TSIs) are a novel class of recombinant biotherapeutics that use protein engineering to retarget botulinum neurotoxins for treatment of diseases with secretion disorders. SXN101959 has been successfully manufactured by Ipsen to Current Good Manufacturing Practice (cGMP) standards and is an example of an emerging recombinant TSI manufacturing platform. SXN101959 is a multi-domain, multi-functional recombinant protein expressed within Escherichia coli, composed of a light chain (LC/D) endopeptidase domain and a heavy chain (HN/D) comprising the translocation domain and the growth-hormone-releasing-hormone (GHRH) targeting peptide ligand. The Engineering and Physical Sciences Research Council (EPSRC) within the UK has established a Centre for Innovative Manufacturing in Emergent Macromolecular Therapies at University College London. The Centre provides an international lead in delivering biopharmaceutical manufacturing innovations for next-generation advanced therapies, which has included an evaluation of SXN101959. We present an assessment of SXN101959 using 2 research work streamsdWorkstream 1: an assessment of the process economics and manufacturability of SXN101959, including cost of goods; and Workstream 2: the development of rapid biophysical characterization decisional tools that assess aggregation propensity and facilitate formulation development. Methods: Workstream 1: Process and economic data from the cGMP manufacturing of SXN101959 were collated and combined into a spreadsheet-based mathematic model in order to determine the key cost factors and to allow future improvements that would reduce manufacturing costs. Workstream 2: The aggregation properties of SXN101959 were studied by size-exclusion high-performance liquid chromatography (SE-HPLC), static light scattering (SLS), intrinsic fluorescence, and monitoring binding of fluorescent dyes. These studies were performed in various buffers. Results and Conclusions: We present the key outcomes from the 2 SXN101959 research workstreams. Workstream 1: successful generation of a mathematic spreadsheet-based tool, able to identify key costs in the manufacturing process and to estimate effects of manufacturing scale on costs of goods; and Workstream 2: a suite of analytical tools able to provide data on the aggregation propensity of SXN101959 and so identify solvent conditions for minimizing aggregation. Funding: Ipsen and EPSRC Keywords: Botulinum neurotoxin (BoNT); Manufacture; Process development; Product characterization; SXN101959; Targeted secretion inhibitor (TSI) 103. RECOMBINANT BOTULINUM NEUROTOXIN SEROTYPE A1 (SXN102342): PROTEIN ENGINEERING AND PROCESS DEVELOPMENT
Introduction and objectives: Botulinum neurotoxin is effective in the treatment of several movement disorders (Simpson 2016). Native BoNT/A comprises a family of highly related neurotoxins produced by Clostridium botulinum bacteria, all neutralized by the same antisera. BoNT/A (subtype A1) is notable as the Hall strain BoNT/A used to produce abobotulinumtoxinA (Dysport), onabotulinumtoxinA (Botox), and incobotulinumtoxinA (Xeomin). An increased understanding of the structurefunction relationship of BoNT provides an opportunity to engineer recombinant (r) BoNTs with unique pharmacologic properties and therapeutic applications. Methods: Here we describe the construction, expression, purification, and characterization of rBoNT serotype A1 (rBoNT/A1). Results: This rBoNT comprises fully synthesized DNA, codon-optimized for heterologous, soluble expression in Escherichia coli. Purification is by hydrophobic interaction and ion exchange chromatography, coupled with an endoproteinase activation step that cleaves the expressed single-chain precursor protein and produces the fully active di-chain rBoNT/A1 product, functionally equivalent to the active agents present in commercial BoNT/ A1 products. Conclusions: The opportunity to successfully engineer and manufacture rBoNT/A1, as well as other genetically modified rBoNTs with differentiated pharmacologic properties, provides an alternative to current commercial BoNT products. Funding: Ipsen Reference Simpson DM, Hallett M, Ashman EJ, et al. Practice guideline update summary: botulinum neurotoxin for the treatment of blepharospasm, cervical dystonia, adult spasticity and headache: Report of the Guideline Development Subcommittee of the American Academy of Neurology. Neurology.2016;86(19):1818-1826. Keywords: Botulinum neurotoxin; (BoNT); Process development; Product characterization; Protein engineering; Serotype A1 104. OUTCOME OF THE ANTIBOTABE MAB DEVELOPMENT PROJECT Michael Hust. €t Braunschweig, Institut für Biochemie, Biotechnologie Technische Universita und Bioinformatik, Spielmannstrasse 7, 38106, Braunschweig, GermanyEmail address:[email protected]
Botulinum neurotoxins (BoNTs) are among the most toxic substances known and are listed as “dirty dozen” agents and possible bioweapons. The results of the European Union AntiBotABE project are presented. In brief, antibodies were generated against botulinum neurotoxin (BoNT) serotypes A, B, and E using macaque immune phage display libraries. To test their neutralization capacities, anti-BoNT antibody fragments (single-chain variable fragment [scFv] or scFv-Fc format) directed against the BoNT HC fragments were tested in ex vivo (mouse phrenic nerve-hemidiaphragm) assays, and the antibodies directed against the light chains were tested in vitro and ex vivo. The anti-HC and antielight chain antibodies with the best neutralizing profiles were selected and tested in nonlethal in vivo protection (mouse flaccid paralysis) assays alone and in combination. Subsequently, these antibodies were germline-humanized to reduce potential immunogenicity and produced as human immunoglobulin G (IgG). The in vivo efficacy of these antibodies was verified in nonlethal and lethal mouse experiments. Combining the anti-HC antibody with the corresponding antielight chain antibody showed a strong synergistic effect. Interestingly, a single antibody against the BoNT/E light chain was protective in vivo. These five antibodies against three botulinum toxin serotypes are intended for further clinical development as an oligoclonal drug cocktail. Keywords: Antibody engineering; Botulinum toxins; IgG; In vivo neutralization; In vivo protection; Phage display; scFv; scFv-Fc
Andy Hooker*, Shilpa Palan, Matthew Beard. Ipsen Bioinnovation, Abingdon, Oxfordshire, UK
105. MYSTICOL: A CONTROLLED STUDY OF MYOBLOC IN THE TREATMENT OF SIALORRHEA IN PARKINSON'S DISEASE (PD) AND OTHER NEUROLOGICAL CONDITIONS
* Corresponding author: Ipsen Bioinnovation Ltd., 102 Park Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
a
Stuart Isaacson a, Lawrence Severt b, Thomas Clinch c, *. Parkinson’s Disease and Movement Disorder Center, Boca Raton, FL, USA;