- Email: [email protected]
An evaluation of the cGMP manufacturing process economics and high-throughput characterization of targeted secretion inhibitors
Abstracts / Toxicon 123 (2016) S2eS90
Harald Hefter*, Alexander Jansen, Marek Moll, Marius Ringelstein, Philipp Albrecht. Department of Neurology, Heinrich-Heine-University Düsseldorf, Germany
S39
Doris Hexsel. Hexsel Dermatologic Clinics; Brazilian Center for Studies in Dermatology, Dom Pedro II 1592, Porto Alegre, RS 90550-141, Brazil. E-mail address:doris@ hexsel.com.br
Table Incidence and prevalence of NABs, applied doses, and treatment duration. Parameter
Hemifacial Blepharospasm Cervical Other spasm dystonia focal dystonia
Spasticity
Number of patients Prevalence of NABs Mean incidence of NABs 10-year prevalence of NABs Mean single dose Doses, 95% CI Duration of treatment (yrs) Duration, 95% CI
47
51
343
29
0
3 (6%)
65 (16%) 9 (17%)
5 (15%)
0
1.071 %
2.848 %
5.917 %
4.66 %
0
10.71 %
28.48 %
59.17 %
46.6 %
58 42e74 5.27
94 73e115 5.6
602 323 536e667 287-359 5.62 2.87
4.60e5.93 4.89e6.32
5.15 e6.09
43
834 786e884 3.22
2.43-3.32 2.72 e3.71
* Corresponding author: Rehabilitation Unit, Hospital General Universitario, Calle Hermanos Falco 37, 02006, Albacete, Spain. E-mail address:[email protected].
Introduction and objectives: Repetitive intramuscular application of botulinum neurotoxin type A (BoNT/A) is a vaccination with a large molecule (150 kDa). Complete avoidance of neutralizing antibodies (NABs) is nearly impossible. We were interested in estimating the incidence and determining the prevalence of NABs for different indications. Methods: A monocentric cross-sectional study was performed at the outpatient clinic of the University of Düsseldorf for 5 indications (hemifacial spasm, blepharospasm, cervical dystonia, other focal dystonia, and spasticity of the upper/lower extremity). Blood samples were taken from patients who presented regularly to be injected every 3 months and had given written informed consent. Each sample was screened by enzymelinked immunosorbent assay (ELISA) to determine whether antibodies against BoNT/A were present. ELISA-positive samples were further analyzed by means of mouse hemidiaphragm assay for NABs. For each patient, demographic and treatment-related data were extracted from the ACCESS database: age, sex, diagnosis, BoNT/A preparation, duration of treatment, single dose of each injection, and time point at which blood sample was taken. Doses of ona-BoNT/A and inco-BoNT/A were converted into unified doses by a factor of 4; doses of abo-BoNT/A stayed unchanged. Results: The table below shows the number of patients; prevalence and mean incidence of NABs; estimated prevalence for 10-year treatment; mean single dose (in abo-BoNT/A doses) and 95% confidence interval (CI); mean treatment duration and 95% CI. There was a strong correlation between the mean single dose and the prevalence of NABs (y ¼ 0.0052x + 0.0722; r2 ¼ 0.9534). Conclusions: The main influence on NAB induction was mean single dose when injections were given regularly every 3 months. In patients with hemifacial spasm and blepharospasm, mean single dose and NAB induction were low; they were much higher in cervical and other dystonias. In spasticity, high doses were used, leading to a higher prevalence of NABs than in cervical dystonia. Keywords: Botulinum toxin; Dystonia; Neutralizing antibodies; Spasticity 100. EVIDENCE-BASED REVIEW OF BOTULINUM TOXIN INJECTIONS FOR AESTHETIC USE
Over the last 25 years, botulinum toxin type A (BoNT-A) has been used in clinical and cosmetic dermatology, and has become one of the most requested procedures for facial rejuvenation. It is considered the best treatment for dynamic wrinkles, as a simple, safe, and highly effective therapeutic modality. BoNT-A is the only serotype approved for aesthetic use, but BoNT-B has also been used successfully by a few authors. The most frequent aesthetic use of BoNT-A is for the reduction of expression lines in the upper, middle, and lower face. BoNT-A use results in changes in the shape and position of important facial structures, such as the eyebrows and lips, and treats gummy and asymmetric smiles, as well as other asymmetries. In addition to thousands of publications regarding its cosmetic use, updated consensus guidelines in the aesthetic field were recently published in the literature, providing physicians with important information on technique and guidelines for BoNT-A use. Recent trends in the aesthetic use of BoNT-A focus on a more natural look, improving rhytides and skin quality, and preserving patients’ expressiveness. The injection of microdoses of BoNT-A for cosmetic purposes delivers multiple intradermal or subdermal injections of low doses of BoNT-A into the overlying skin and superficial layer of the facial and neck muscles. Reports of emerging aesthetic uses of BoNT-A, such as for rosacea and the improvement of oily skin and associated conditions, have also been published in the scientific literature. A careful clinical evaluation of every patient together with the proper knowledge of facial muscles and techniques, pre and post procedure recommendations, correct handling of the products, and use of standard doses are very important to obtain the best results. 101. EFFECTIVENESS AND SAFETY ASSESSMENT OF INCOBOTULINUMTOXINA (XEOMIN) INJECTIONS IN THE LOWER-LIMB MUSCLES OF YOUNG, LOW-WEIGHT CHILDREN WITH CEREBRAL PALSY Soraya Hijazi a, *, Martín Arcas b. Rehabilitation Unit, Hospital General, Albacete, Spain; General, Albacete, Spain
a
b
Pain Unit, Hospital
* Corresponding author: Rehabilitation Unit, Hospital General Universitario, Calle Hermanos Falco 37, 02006, Albacete, Spain. E-mail address:[email protected].
Introduction and objectives: Long-term botulinum neurotoxin (BoNT) treatment in children with cerebral palsy (CP) requires avoidance of a secondary nonresponse and formation of anti-BoNT antibodies. In this retrospective case series, we assessed the safety and efficacy of incobotulinumtoxinA (Xeomin), a BoNT formulation free from complexing proteins, for lower-limb spasticity in young, low-weight children with CP or other motor disorders. Methods: Nine children (ages 2.5 to 6 years) with CP (n¼8) or West syndrome (n¼1), with low weight (mean, 17.7; standard deviation [SD], 4.8 kg) and spastic hemiplegia or diplegia were treated with incobotulinumtoxinA. All injections were performed by the same practitioner with ultrasound guidance, and under general anesthesia in 43% of cases. Assessments performed before and 3 months after treatment included the Modified Ashworth Scale (MAS) and subjective global impressions of change rated by the physician and parent/caregiver. Parents/caregivers were asked 3 months after treatment about the occurrence of adverse effects. Results: The mean (SD) dose of incobotulinumtoxinA administered was 212.9 (62.4) U, corresponding to a mean dose of 12 U/kg body weight (maximum total dose, 300 U). Muscles injected were the gastrocnemius (87.5% of patients), tibialis posterior (75.0%), adductors (75.0%), psoas (75.0%), soleus (37.5%), and rectus femoris (37.5%). Three months after treatment, a positive response was reported for 77% of children, with objective ( 1 point improvement in MAS score) and subjective (global impression of change rated by the physician and parent/caregiver) improvements. No adverse effects were reported.
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;
Harald Hefter*, Alexander Jansen, Marek Moll, Marius Ringelstein, Philipp Albrecht. Department of Neurology, Heinrich-Heine-University Düsseldorf, Germany
S39
Doris Hexsel. Hexsel Dermatologic Clinics; Brazilian Center for Studies in Dermatology, Dom Pedro II 1592, Porto Alegre, RS 90550-141, Brazil. E-mail address:doris@ hexsel.com.br
Table Incidence and prevalence of NABs, applied doses, and treatment duration. Parameter
Hemifacial Blepharospasm Cervical Other spasm dystonia focal dystonia
Spasticity
Number of patients Prevalence of NABs Mean incidence of NABs 10-year prevalence of NABs Mean single dose Doses, 95% CI Duration of treatment (yrs) Duration, 95% CI
47
51
343
29
0
3 (6%)
65 (16%) 9 (17%)
5 (15%)
0
1.071 %
2.848 %
5.917 %
4.66 %
0
10.71 %
28.48 %
59.17 %
46.6 %
58 42e74 5.27
94 73e115 5.6
602 323 536e667 287-359 5.62 2.87
4.60e5.93 4.89e6.32
5.15 e6.09
43
834 786e884 3.22
2.43-3.32 2.72 e3.71
* Corresponding author: Rehabilitation Unit, Hospital General Universitario, Calle Hermanos Falco 37, 02006, Albacete, Spain. E-mail address:[email protected].
Introduction and objectives: Repetitive intramuscular application of botulinum neurotoxin type A (BoNT/A) is a vaccination with a large molecule (150 kDa). Complete avoidance of neutralizing antibodies (NABs) is nearly impossible. We were interested in estimating the incidence and determining the prevalence of NABs for different indications. Methods: A monocentric cross-sectional study was performed at the outpatient clinic of the University of Düsseldorf for 5 indications (hemifacial spasm, blepharospasm, cervical dystonia, other focal dystonia, and spasticity of the upper/lower extremity). Blood samples were taken from patients who presented regularly to be injected every 3 months and had given written informed consent. Each sample was screened by enzymelinked immunosorbent assay (ELISA) to determine whether antibodies against BoNT/A were present. ELISA-positive samples were further analyzed by means of mouse hemidiaphragm assay for NABs. For each patient, demographic and treatment-related data were extracted from the ACCESS database: age, sex, diagnosis, BoNT/A preparation, duration of treatment, single dose of each injection, and time point at which blood sample was taken. Doses of ona-BoNT/A and inco-BoNT/A were converted into unified doses by a factor of 4; doses of abo-BoNT/A stayed unchanged. Results: The table below shows the number of patients; prevalence and mean incidence of NABs; estimated prevalence for 10-year treatment; mean single dose (in abo-BoNT/A doses) and 95% confidence interval (CI); mean treatment duration and 95% CI. There was a strong correlation between the mean single dose and the prevalence of NABs (y ¼ 0.0052x + 0.0722; r2 ¼ 0.9534). Conclusions: The main influence on NAB induction was mean single dose when injections were given regularly every 3 months. In patients with hemifacial spasm and blepharospasm, mean single dose and NAB induction were low; they were much higher in cervical and other dystonias. In spasticity, high doses were used, leading to a higher prevalence of NABs than in cervical dystonia. Keywords: Botulinum toxin; Dystonia; Neutralizing antibodies; Spasticity 100. EVIDENCE-BASED REVIEW OF BOTULINUM TOXIN INJECTIONS FOR AESTHETIC USE
Over the last 25 years, botulinum toxin type A (BoNT-A) has been used in clinical and cosmetic dermatology, and has become one of the most requested procedures for facial rejuvenation. It is considered the best treatment for dynamic wrinkles, as a simple, safe, and highly effective therapeutic modality. BoNT-A is the only serotype approved for aesthetic use, but BoNT-B has also been used successfully by a few authors. The most frequent aesthetic use of BoNT-A is for the reduction of expression lines in the upper, middle, and lower face. BoNT-A use results in changes in the shape and position of important facial structures, such as the eyebrows and lips, and treats gummy and asymmetric smiles, as well as other asymmetries. In addition to thousands of publications regarding its cosmetic use, updated consensus guidelines in the aesthetic field were recently published in the literature, providing physicians with important information on technique and guidelines for BoNT-A use. Recent trends in the aesthetic use of BoNT-A focus on a more natural look, improving rhytides and skin quality, and preserving patients’ expressiveness. The injection of microdoses of BoNT-A for cosmetic purposes delivers multiple intradermal or subdermal injections of low doses of BoNT-A into the overlying skin and superficial layer of the facial and neck muscles. Reports of emerging aesthetic uses of BoNT-A, such as for rosacea and the improvement of oily skin and associated conditions, have also been published in the scientific literature. A careful clinical evaluation of every patient together with the proper knowledge of facial muscles and techniques, pre and post procedure recommendations, correct handling of the products, and use of standard doses are very important to obtain the best results. 101. EFFECTIVENESS AND SAFETY ASSESSMENT OF INCOBOTULINUMTOXINA (XEOMIN) INJECTIONS IN THE LOWER-LIMB MUSCLES OF YOUNG, LOW-WEIGHT CHILDREN WITH CEREBRAL PALSY Soraya Hijazi a, *, Martín Arcas b. Rehabilitation Unit, Hospital General, Albacete, Spain; General, Albacete, Spain
a
b
Pain Unit, Hospital
* Corresponding author: Rehabilitation Unit, Hospital General Universitario, Calle Hermanos Falco 37, 02006, Albacete, Spain. E-mail address:[email protected].
Introduction and objectives: Long-term botulinum neurotoxin (BoNT) treatment in children with cerebral palsy (CP) requires avoidance of a secondary nonresponse and formation of anti-BoNT antibodies. In this retrospective case series, we assessed the safety and efficacy of incobotulinumtoxinA (Xeomin), a BoNT formulation free from complexing proteins, for lower-limb spasticity in young, low-weight children with CP or other motor disorders. Methods: Nine children (ages 2.5 to 6 years) with CP (n¼8) or West syndrome (n¼1), with low weight (mean, 17.7; standard deviation [SD], 4.8 kg) and spastic hemiplegia or diplegia were treated with incobotulinumtoxinA. All injections were performed by the same practitioner with ultrasound guidance, and under general anesthesia in 43% of cases. Assessments performed before and 3 months after treatment included the Modified Ashworth Scale (MAS) and subjective global impressions of change rated by the physician and parent/caregiver. Parents/caregivers were asked 3 months after treatment about the occurrence of adverse effects. Results: The mean (SD) dose of incobotulinumtoxinA administered was 212.9 (62.4) U, corresponding to a mean dose of 12 U/kg body weight (maximum total dose, 300 U). Muscles injected were the gastrocnemius (87.5% of patients), tibialis posterior (75.0%), adductors (75.0%), psoas (75.0%), soleus (37.5%), and rectus femoris (37.5%). Three months after treatment, a positive response was reported for 77% of children, with objective ( 1 point improvement in MAS score) and subjective (global impression of change rated by the physician and parent/caregiver) improvements. No adverse effects were reported.
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;