- Email: [email protected]
An innovative solution to the unique challenges in manufacturing recombinant botulinum neurotoxins
S56
Abstracts / Toxicon 123 (2016) S2eS90
Results: The patient was assessed at baseline (P0; pretreatment) and 6 weeks after the fourth treatment (P4) using Goal Attainment scaling (GAS) with a change score of 25.4. She reported significant improvements in performing ADLs, with gain of autonomy. There was a reduction in frequency and severity of dystonic postures from P0 to P4. Patient's follow-up showed maintenance of benefit at 9 months after the last treatment. Conclusions: We report a nonfunctional pseudodystonic hand, which improved after treatment with US-guided BT treatment, illustrating the potential usefulness of BT in the treatment of incapacitating dystonic postures secondary to central somatosensory deficits originating outside the basal ganglia. Keywords: Botulinum toxin; Dystonia; Pseudodystonia; Ultrasound guided 147. THE FIRST CASE OF BOTULISM TYPE F IN PORTUGAL Teresa Teixeira Lopes a, Margarida Saraiva a, *, Isabel Bastos Moura a, Rosa Andre Milit~ ao c, Adelaide Coelho d, Orta Santos Ribeiro b, d a a ~ Gomes , Conceiçao Costa Bonito , Claudia Pena , Isabel Campos nio Cunha a, Isabel Sousa a, Maria Manuel Toscano a, Elsa Soares d, Anto nia Calhau a. Tavares d, Maria Anto a Microbiology Laboratory, Reference Unit, Food and Nutrition Department, National Health Institute Doutor Ricardo Jorge, IP, Oporto, Portugal; b Intensive Care Service, St. Bernard Hospital, Centre Hospitalier de Setúbal, EPE, Setúbal, Portugal; c Neurology Service, St. Bernard Hospital, Centre Hospitalier de Setúbal, EPE, Setúbal, Portugal; d Department of Public Health, Regional Health Administration of Lisbon and Tagus Valley, IP, Lisbon, Portugal
reported cases of type F botulism, no source of exposure was identified in this patient.1 Keywords: Botulinum toxin type F; Clostridium botulinum; Neurotoxin; Portugal References 1. European Centre for Disease Prevention and Control (ECDC). Technical Report. Scientific Advice on Type F Botulism. Stockholm, Sweden: ECDC; 2013. http://www.ecdc.europa.eu/en/publications/Publications/botulismscientific-advice-type-F-botulism.pdf. 2. Centers for Disease Control and Prevention. Clostridium botulinum Monovalent and Polyvalent Antitoxins. Atlanta, Georgia: Centers for Disease Control and Prevention, US Dept of Health and Human Services;1987. 3. Austin JW, Sanders G. The Detection of Clostridium botulinum and Its toxins in Suspect Foods and Clinical Specimens. Compendium of Analytical Methods. vol. 2. 2009:1-9. 4. De Medici D, Anniballi F, Wyatt GM, et al. Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples. Appl Environ Microbiol. 2009; 75(20):6457-6461. 148. TRANSORAL ADMINISTRATION OF INCOBOTULINUMTOXINA FOR THE MANAGEMENT OF LARYNGEAL DYSTONIA Miguel Sebastia n Cortes b, Paúl Ricardo pez del Val a, *, Jose Javier Lo a b Vinueza Buitron , Yolanda Lois Ortega , Hector Valles Varea b, Elena pez García a, Elena Bellosta Diago b, Sonia Santos Lasaosa a. Lo a Neurology Service, Movement Disorders Unit, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain; b Otolaryngology Service, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain
* Corresponding author: National Health Institute Doutor Ricardo Jorge, Rua Alexandre Herculano, 321, 4000-055, IP, Porto, Portugal. E-mail address:margarida. [email protected].
* Corresponding author: Neurology Service, Movement Disorders Unit, Hospital Clínico Universitario Lozano Blesa, Avenida San Juan Bosco, 50009 Zaragoza, Spain. E-mail address:[email protected].
Botulinum toxin type F (BoNT/F) from the stool of a patient with symptoms of botulism was detected in Portugal in July 2016. A strain that exhibited the phenotypic characteristics of Clostridium botulinum serotype F was also isolated. All known human cases have been caused by toxin types A, B, E and, more rarely, type F. In the vast majority of cases, the toxin Feproducing organism was C botulinum and sometimes C baratii.1 Ingestion of contaminated food or, more rarely, infection of an exposed wound or growth in an immunologically immature intestinal tract (infant botulism) or adult intestinal colonization can produce the clinical syndrome of botulism.1 A 53-year-old man was admitted to the Emergency Department of St. Bernard Hospital reporting symptoms of botulism, including blurred vision, diplopia, ptosis, dysarthria, dysphagia, dry mouth, and bilateral nonreactive mydriasis. He reported nausea, abdominal distress, and vomiting after dinner on the previous day. The patient was hospitalized syndrome. with a possible diagnosis of botulism vs atypical Guillain-Barre He sought treatment in the intensive care unit with urinary retention, flaccid paraparesis, progressive respiratory distress, and needing respiratory assistance. A serum sample and a stool sample resulting from a microenema were collected and sent to the laboratory. Epidemiological and environmental investigations were carried out by local public health teams, supported by the regional health authority. Interviews with family members were conducted to identify suspected foods. A selection of canned foods of the same brand as that ingested by the patient was sent to the laboratory. The serum and stool samples were tested with the standard mouse bioassay2, and the stool sample was also tested for the presence of BoNTproducing Clostridia (with multiplex-PCR and culture).3,4 BoNT/F was detected in the stools. In the serum sample the assay was not conclusive. The food samples were analyzed for the presence of toxin (mouse bioassay)2 and for BoNT-producing Clostridia (with multiplex-PCR and culture).3,4 BoNT and BoNT-producing Clostridia were not detected. Some of the few cases of BoNT/F in adults reported were due to adult colonization and others to food-borne botulism.1 As in the majority of
Introduction and objectives: Laryngeal dystonia is a movement disorder of the muscles within the larynx, which most commonly manifests as spasmodic dysphonia (SD). The use of botulinum toxin (BTX) in the treatment of SD has a major clinical impact with a marked improvement of symptoms. One of the injection routes of administration of BTX is the transoral approach. The objective of this study is to describe our experience using the transoral technique for administration of incobotulinumtoxinA in patients with SD. Methods: We have treated 15 patients (11 female; 4 male) diagnosed of adductor or abductor SD during 134 therapeutic administrations of incobotulinumtoxinA via the transoral route with direct injection into the vocal cord. A dose of 5 international units (IU) per vocal cord was administered. Ten patients (66.67%) had adductor SD, 3 (20%) had abductor SD; in 1 patient (6.67%), the laryngeal disorder was integrated in a process of generalized dystonia, and in 1 patient (6.67%), SD was accompanied by cervical dystonia. The procedure was carried out under local anesthesia with aerosolized lidocaine diluted according to the anatomical area, and the injection was performed using a Chiba needle with the help of a flexible fibroendoscope. In order to assess the degree of therapeutic response, a patient-reported self-assessment scale (1: no improvement; 5: very much improved) was developed. The site of injection was the same regardless of the type of laryngeal dystonia. Results: Patients received a mean dose of 5 IU per vocal cord (minimum 2.5 IU and maximum 7.5 IU). Average duration of the therapeutic beneficial effect was 5.3 months (95% CI: 4.9 -5.7 months). In our group of patients, 29.3% rated the improvement as marked-moderate and 64.7% rated it as very much improved. Patients reported adverse effects in 11.9% of treatment sessions, most commonly transient and mild dysphagia, dysphonia, and choking. Conclusions: In our experience, the transoral injection technique of botulinum toxin for the treatment of SD (both adductor and abductor forms) is an effective, easy to perform, and well-tolerated treatment provided good anesthesia and relaxation of treated patients is achieved. Keywords: IncobotulinumtoxinA; Laryngeal dystonia
Abstracts / Toxicon 123 (2016) S2eS90
149. LIGHT-CHAIN-MODIFIED RBONT/A1 MUTANTS WITH IMPROVED STABILITY pez de la Paz, Daniel Scheps, Fred Hofmann, Marcel Manuela Lo Jurk, Jürgen Frevert*. Merz Pharmaceuticals GmbH, Potsdam, Germany * Corresponding author: Merz Pharmaceuticals GmbH Potsdam, Hermannswerder Haus 15, 14473 Potsdam, Germany. E-mail address:[email protected].
Introduction and objectives: As a proteolytic enzyme, the light chain (LC) of botulinum neurotoxins (BoNTs) can be inactivated and denatured by heat and/or reactive oxygen species present in the cell and/or during isolation, purification, and storage processes. Stabilization of the LC of BoNT/A (LC/A) by reducing vulnerability toward oxygen, enhancing thermostability, and/or increasing resistance against proteolytic attack would thus be advantageous for the development of BoNT-based therapeutics. We tackled this task by rational design of mutations at critical sites for the stability of the LC. Methods: Stabilization of the structure of LC/A was addressed by decreasing the flexibility of loop regions and by mutation of amino-acid residues susceptible to oxidation. Based on amino-acid-relative solventaccessible surface area in the LC structure (molecular modeling software WHAT IF) and on LC/A loop flexibility as explored by means of molecular dynamics code simulations in explicit water using Desmond, residues were deleted and loop-truncated mutants generated. Substitutions of oxidation-vulnerable LC amino acid residues were identified by rational design using the BioLuminate software package of € dinger (BioLuminate, Version 1.1, Schro € dinger, New York, NY, USA). Schro Stability against proteolytic attack of the mutants was assessed by incubation with trypsin and pronase as model proteases. The in vitro activity and in vivo effect of mutants was analyzed in the endopeptidase assay and mouse running assay (MRA), respectively. Results: Incubation with hydrogen peroxide demonstrated that the stability of BoNT/A1-LC mutants with up to 8 substituted amino-acid residues was markedly increased with respect to wild-type (WT) LC. Mutants with suitably truncated loops showed a higher resistance against trypsin or pronase without substantial loss of enzymatic activity and WT-like in vivo activity according to MRA. Conclusions: The LC of BoNT/A can be stabilized by mutating oxidationprone amino-acid residues or by rigidification of flexible loops. A BoNT/A holotoxin with a suitably modified LC would provide a therapeutic agent with a higher stability. Keywords: Light chain; Oxidation; Oxidative stability; Recombinant BoNT; Surface loop; Thermostability 150. AN INNOVATIVE SOLUTION TO THE UNIQUE CHALLENGES IN MANUFACTURING RECOMBINANT BOTULINUM NEUROTOXINS Laura Lovelock a, Daniel Kwan a, Kevin Moore a, Andrew Splevins a, Phillip Marks b, Andy Hooker a, Peter Horrocks a, *. a Ipsen Bioinnovation Limited, Oxford, UK; b Ipsen Biopharm Limited, Wrexham, UK * Corresponding author: Ipsen Bioinnovation Limited, 102 Park Drive, Milton Park, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
Introduction and objectives: When manufacturing recombinant botulinum neurotoxins (rBoNTs), the full-length toxin is expressed as a single polypeptide chain. However, for rBoNTs to achieve full biological activity, the light chain must be able to dissociate from the heavy chain, to allow effective diffusion into the cytoplasm following translocation from the endosome. In the native toxin, there are two chains, heavy and light, that are joined by a disulfide bridge. To replicate this in recombinant manufacturing, where the molecule is expressed as a single polypeptide chain, the protein sequence linking the heavy and light chains (the
S57
activation loop) is cleaved enzymatically during the downstream manufacturing process. Ensuring full but specific cleavage of this activation loop is a known issue. Separation and removal of single chain toxin from the cleaved dichain toxin by traditional protein manufacturing methodologies is difficult due to the high degree of sequence and structural homology. Methods: We have developed a manufacturing approach that forces the enzymatic cleavage reaction beyond completion and then removes unwanted truncated products by protein chromatography. Proteolysis screens were employed to monitor the activation reaction progress, and generation of fully cleaved rBoNT. Chromatography resins are assessed for their ability to purify full-length di-chain product away from unwanted single chain or truncated impurities, which is monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Conditions for isolating the full-length di-chain product from the single chain precursor were identified. Separation of the truncated impurity from the desired full-length di-chain product was successfully achieved, and a consistent manufacturing process, that produces material that is 100% activated and over 95% pure has been developed. Conclusion: A process for manufacturing full-length, di-chain recombinant BoNT has been developed that utilizes a novel proteolytic activation approach. Keywords: Activation; Botulinum neurotoxin (BoNT); Chromatography; Manufacture; Proteolysis; Recombinant 151. ENGINEERING NEUROTOXINS
DESTRUCTION
SITES
INTO
BOTULINUM
Laura Lovelock, Patrick Stancombe, Amelie Laurendon, Verity Cadd, Gavin Hackett, Keith Foster, John Chaddock, Dina Anderson*. Ipsen Bioinnovation, Milton Park, Abingdon, UK * Corresponding author: Ipsen Bioinnovation Ltd, 102 Park Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
Introduction and Objectives: Botulinum neurotoxins (BoNTs) are highly effective therapeutics for the treatment of neuromuscular disorders (Chen 2013). However, for disorders afflicting large muscle groups, the efficacy of BoNT therapeutics can be limited by the need for higher doses and associated risks such as systemic spread (Simpson 2008). In a drive to develop BoNT therapeutics that can be administered at higher doses, we have taken steps to engineer modified BoNTs that possess protease-labile destruction sites that are cleaved by plasma proteases to deactivate the toxin in the blood. Methods and Results: We identified unstructured solvent-exposed loops by sequence analysis and introduced sequences that can be cleaved by plasma-resident proteases. Modified BoNT/A molecules were expressed in Escherichia coli, purified, and evaluated for their susceptibility to exogenous protease. Molecules were assessed in embryonic spinal cord neuron (eSCN) synaptosome associated protein (SNAP)-25 potency to measure BoNT destruction by the exogenous protease, plasma kallikrein (KLKB1). As well as sites that were not cleavable by KLKB1, positions were successfully identified in the light chain and heavy chain of BoNT/A1 that rendered the molecule susceptible to cleavage by exogenous protease, resulting in a significant (10-fold) decrease in toxin activity in a rat eSCN assay. Conclusions: The results that we present here identify 1 region within the light chain ([LC]; T323-V333) and 2 regions within the N-terminal domain of the heavy chain ([HN]; Q557-I566 and N642-K671) that are suitable for the insertion of plasma-protease-labile destruction sites. We also map the first steps in developing a BoNT therapeutic with a decreased risk of systemic adverse effects. The results show that introducing a protease cleavage site to the HN domain of BoNT/A1 results in a toxin that is susceptible to KLKB1. To engineer a BoNT/A1 with even greater sensitivity to
Abstracts / Toxicon 123 (2016) S2eS90
Results: The patient was assessed at baseline (P0; pretreatment) and 6 weeks after the fourth treatment (P4) using Goal Attainment scaling (GAS) with a change score of 25.4. She reported significant improvements in performing ADLs, with gain of autonomy. There was a reduction in frequency and severity of dystonic postures from P0 to P4. Patient's follow-up showed maintenance of benefit at 9 months after the last treatment. Conclusions: We report a nonfunctional pseudodystonic hand, which improved after treatment with US-guided BT treatment, illustrating the potential usefulness of BT in the treatment of incapacitating dystonic postures secondary to central somatosensory deficits originating outside the basal ganglia. Keywords: Botulinum toxin; Dystonia; Pseudodystonia; Ultrasound guided 147. THE FIRST CASE OF BOTULISM TYPE F IN PORTUGAL Teresa Teixeira Lopes a, Margarida Saraiva a, *, Isabel Bastos Moura a, Rosa Andre Milit~ ao c, Adelaide Coelho d, Orta Santos Ribeiro b, d a a ~ Gomes , Conceiçao Costa Bonito , Claudia Pena , Isabel Campos nio Cunha a, Isabel Sousa a, Maria Manuel Toscano a, Elsa Soares d, Anto nia Calhau a. Tavares d, Maria Anto a Microbiology Laboratory, Reference Unit, Food and Nutrition Department, National Health Institute Doutor Ricardo Jorge, IP, Oporto, Portugal; b Intensive Care Service, St. Bernard Hospital, Centre Hospitalier de Setúbal, EPE, Setúbal, Portugal; c Neurology Service, St. Bernard Hospital, Centre Hospitalier de Setúbal, EPE, Setúbal, Portugal; d Department of Public Health, Regional Health Administration of Lisbon and Tagus Valley, IP, Lisbon, Portugal
reported cases of type F botulism, no source of exposure was identified in this patient.1 Keywords: Botulinum toxin type F; Clostridium botulinum; Neurotoxin; Portugal References 1. European Centre for Disease Prevention and Control (ECDC). Technical Report. Scientific Advice on Type F Botulism. Stockholm, Sweden: ECDC; 2013. http://www.ecdc.europa.eu/en/publications/Publications/botulismscientific-advice-type-F-botulism.pdf. 2. Centers for Disease Control and Prevention. Clostridium botulinum Monovalent and Polyvalent Antitoxins. Atlanta, Georgia: Centers for Disease Control and Prevention, US Dept of Health and Human Services;1987. 3. Austin JW, Sanders G. The Detection of Clostridium botulinum and Its toxins in Suspect Foods and Clinical Specimens. Compendium of Analytical Methods. vol. 2. 2009:1-9. 4. De Medici D, Anniballi F, Wyatt GM, et al. Multiplex PCR for detection of botulinum neurotoxin-producing clostridia in clinical, food, and environmental samples. Appl Environ Microbiol. 2009; 75(20):6457-6461. 148. TRANSORAL ADMINISTRATION OF INCOBOTULINUMTOXINA FOR THE MANAGEMENT OF LARYNGEAL DYSTONIA Miguel Sebastia n Cortes b, Paúl Ricardo pez del Val a, *, Jose Javier Lo a b Vinueza Buitron , Yolanda Lois Ortega , Hector Valles Varea b, Elena pez García a, Elena Bellosta Diago b, Sonia Santos Lasaosa a. Lo a Neurology Service, Movement Disorders Unit, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain; b Otolaryngology Service, Hospital Clínico Universitario Lozano Blesa, Zaragoza, Spain
* Corresponding author: National Health Institute Doutor Ricardo Jorge, Rua Alexandre Herculano, 321, 4000-055, IP, Porto, Portugal. E-mail address:margarida. [email protected].
* Corresponding author: Neurology Service, Movement Disorders Unit, Hospital Clínico Universitario Lozano Blesa, Avenida San Juan Bosco, 50009 Zaragoza, Spain. E-mail address:[email protected].
Botulinum toxin type F (BoNT/F) from the stool of a patient with symptoms of botulism was detected in Portugal in July 2016. A strain that exhibited the phenotypic characteristics of Clostridium botulinum serotype F was also isolated. All known human cases have been caused by toxin types A, B, E and, more rarely, type F. In the vast majority of cases, the toxin Feproducing organism was C botulinum and sometimes C baratii.1 Ingestion of contaminated food or, more rarely, infection of an exposed wound or growth in an immunologically immature intestinal tract (infant botulism) or adult intestinal colonization can produce the clinical syndrome of botulism.1 A 53-year-old man was admitted to the Emergency Department of St. Bernard Hospital reporting symptoms of botulism, including blurred vision, diplopia, ptosis, dysarthria, dysphagia, dry mouth, and bilateral nonreactive mydriasis. He reported nausea, abdominal distress, and vomiting after dinner on the previous day. The patient was hospitalized syndrome. with a possible diagnosis of botulism vs atypical Guillain-Barre He sought treatment in the intensive care unit with urinary retention, flaccid paraparesis, progressive respiratory distress, and needing respiratory assistance. A serum sample and a stool sample resulting from a microenema were collected and sent to the laboratory. Epidemiological and environmental investigations were carried out by local public health teams, supported by the regional health authority. Interviews with family members were conducted to identify suspected foods. A selection of canned foods of the same brand as that ingested by the patient was sent to the laboratory. The serum and stool samples were tested with the standard mouse bioassay2, and the stool sample was also tested for the presence of BoNTproducing Clostridia (with multiplex-PCR and culture).3,4 BoNT/F was detected in the stools. In the serum sample the assay was not conclusive. The food samples were analyzed for the presence of toxin (mouse bioassay)2 and for BoNT-producing Clostridia (with multiplex-PCR and culture).3,4 BoNT and BoNT-producing Clostridia were not detected. Some of the few cases of BoNT/F in adults reported were due to adult colonization and others to food-borne botulism.1 As in the majority of
Introduction and objectives: Laryngeal dystonia is a movement disorder of the muscles within the larynx, which most commonly manifests as spasmodic dysphonia (SD). The use of botulinum toxin (BTX) in the treatment of SD has a major clinical impact with a marked improvement of symptoms. One of the injection routes of administration of BTX is the transoral approach. The objective of this study is to describe our experience using the transoral technique for administration of incobotulinumtoxinA in patients with SD. Methods: We have treated 15 patients (11 female; 4 male) diagnosed of adductor or abductor SD during 134 therapeutic administrations of incobotulinumtoxinA via the transoral route with direct injection into the vocal cord. A dose of 5 international units (IU) per vocal cord was administered. Ten patients (66.67%) had adductor SD, 3 (20%) had abductor SD; in 1 patient (6.67%), the laryngeal disorder was integrated in a process of generalized dystonia, and in 1 patient (6.67%), SD was accompanied by cervical dystonia. The procedure was carried out under local anesthesia with aerosolized lidocaine diluted according to the anatomical area, and the injection was performed using a Chiba needle with the help of a flexible fibroendoscope. In order to assess the degree of therapeutic response, a patient-reported self-assessment scale (1: no improvement; 5: very much improved) was developed. The site of injection was the same regardless of the type of laryngeal dystonia. Results: Patients received a mean dose of 5 IU per vocal cord (minimum 2.5 IU and maximum 7.5 IU). Average duration of the therapeutic beneficial effect was 5.3 months (95% CI: 4.9 -5.7 months). In our group of patients, 29.3% rated the improvement as marked-moderate and 64.7% rated it as very much improved. Patients reported adverse effects in 11.9% of treatment sessions, most commonly transient and mild dysphagia, dysphonia, and choking. Conclusions: In our experience, the transoral injection technique of botulinum toxin for the treatment of SD (both adductor and abductor forms) is an effective, easy to perform, and well-tolerated treatment provided good anesthesia and relaxation of treated patients is achieved. Keywords: IncobotulinumtoxinA; Laryngeal dystonia
Abstracts / Toxicon 123 (2016) S2eS90
149. LIGHT-CHAIN-MODIFIED RBONT/A1 MUTANTS WITH IMPROVED STABILITY pez de la Paz, Daniel Scheps, Fred Hofmann, Marcel Manuela Lo Jurk, Jürgen Frevert*. Merz Pharmaceuticals GmbH, Potsdam, Germany * Corresponding author: Merz Pharmaceuticals GmbH Potsdam, Hermannswerder Haus 15, 14473 Potsdam, Germany. E-mail address:[email protected].
Introduction and objectives: As a proteolytic enzyme, the light chain (LC) of botulinum neurotoxins (BoNTs) can be inactivated and denatured by heat and/or reactive oxygen species present in the cell and/or during isolation, purification, and storage processes. Stabilization of the LC of BoNT/A (LC/A) by reducing vulnerability toward oxygen, enhancing thermostability, and/or increasing resistance against proteolytic attack would thus be advantageous for the development of BoNT-based therapeutics. We tackled this task by rational design of mutations at critical sites for the stability of the LC. Methods: Stabilization of the structure of LC/A was addressed by decreasing the flexibility of loop regions and by mutation of amino-acid residues susceptible to oxidation. Based on amino-acid-relative solventaccessible surface area in the LC structure (molecular modeling software WHAT IF) and on LC/A loop flexibility as explored by means of molecular dynamics code simulations in explicit water using Desmond, residues were deleted and loop-truncated mutants generated. Substitutions of oxidation-vulnerable LC amino acid residues were identified by rational design using the BioLuminate software package of € dinger (BioLuminate, Version 1.1, Schro € dinger, New York, NY, USA). Schro Stability against proteolytic attack of the mutants was assessed by incubation with trypsin and pronase as model proteases. The in vitro activity and in vivo effect of mutants was analyzed in the endopeptidase assay and mouse running assay (MRA), respectively. Results: Incubation with hydrogen peroxide demonstrated that the stability of BoNT/A1-LC mutants with up to 8 substituted amino-acid residues was markedly increased with respect to wild-type (WT) LC. Mutants with suitably truncated loops showed a higher resistance against trypsin or pronase without substantial loss of enzymatic activity and WT-like in vivo activity according to MRA. Conclusions: The LC of BoNT/A can be stabilized by mutating oxidationprone amino-acid residues or by rigidification of flexible loops. A BoNT/A holotoxin with a suitably modified LC would provide a therapeutic agent with a higher stability. Keywords: Light chain; Oxidation; Oxidative stability; Recombinant BoNT; Surface loop; Thermostability 150. AN INNOVATIVE SOLUTION TO THE UNIQUE CHALLENGES IN MANUFACTURING RECOMBINANT BOTULINUM NEUROTOXINS Laura Lovelock a, Daniel Kwan a, Kevin Moore a, Andrew Splevins a, Phillip Marks b, Andy Hooker a, Peter Horrocks a, *. a Ipsen Bioinnovation Limited, Oxford, UK; b Ipsen Biopharm Limited, Wrexham, UK * Corresponding author: Ipsen Bioinnovation Limited, 102 Park Drive, Milton Park, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
Introduction and objectives: When manufacturing recombinant botulinum neurotoxins (rBoNTs), the full-length toxin is expressed as a single polypeptide chain. However, for rBoNTs to achieve full biological activity, the light chain must be able to dissociate from the heavy chain, to allow effective diffusion into the cytoplasm following translocation from the endosome. In the native toxin, there are two chains, heavy and light, that are joined by a disulfide bridge. To replicate this in recombinant manufacturing, where the molecule is expressed as a single polypeptide chain, the protein sequence linking the heavy and light chains (the
S57
activation loop) is cleaved enzymatically during the downstream manufacturing process. Ensuring full but specific cleavage of this activation loop is a known issue. Separation and removal of single chain toxin from the cleaved dichain toxin by traditional protein manufacturing methodologies is difficult due to the high degree of sequence and structural homology. Methods: We have developed a manufacturing approach that forces the enzymatic cleavage reaction beyond completion and then removes unwanted truncated products by protein chromatography. Proteolysis screens were employed to monitor the activation reaction progress, and generation of fully cleaved rBoNT. Chromatography resins are assessed for their ability to purify full-length di-chain product away from unwanted single chain or truncated impurities, which is monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Results: Conditions for isolating the full-length di-chain product from the single chain precursor were identified. Separation of the truncated impurity from the desired full-length di-chain product was successfully achieved, and a consistent manufacturing process, that produces material that is 100% activated and over 95% pure has been developed. Conclusion: A process for manufacturing full-length, di-chain recombinant BoNT has been developed that utilizes a novel proteolytic activation approach. Keywords: Activation; Botulinum neurotoxin (BoNT); Chromatography; Manufacture; Proteolysis; Recombinant 151. ENGINEERING NEUROTOXINS
DESTRUCTION
SITES
INTO
BOTULINUM
Laura Lovelock, Patrick Stancombe, Amelie Laurendon, Verity Cadd, Gavin Hackett, Keith Foster, John Chaddock, Dina Anderson*. Ipsen Bioinnovation, Milton Park, Abingdon, UK * Corresponding author: Ipsen Bioinnovation Ltd, 102 Park Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RY, UK. E-mail address:[email protected].
Introduction and Objectives: Botulinum neurotoxins (BoNTs) are highly effective therapeutics for the treatment of neuromuscular disorders (Chen 2013). However, for disorders afflicting large muscle groups, the efficacy of BoNT therapeutics can be limited by the need for higher doses and associated risks such as systemic spread (Simpson 2008). In a drive to develop BoNT therapeutics that can be administered at higher doses, we have taken steps to engineer modified BoNTs that possess protease-labile destruction sites that are cleaved by plasma proteases to deactivate the toxin in the blood. Methods and Results: We identified unstructured solvent-exposed loops by sequence analysis and introduced sequences that can be cleaved by plasma-resident proteases. Modified BoNT/A molecules were expressed in Escherichia coli, purified, and evaluated for their susceptibility to exogenous protease. Molecules were assessed in embryonic spinal cord neuron (eSCN) synaptosome associated protein (SNAP)-25 potency to measure BoNT destruction by the exogenous protease, plasma kallikrein (KLKB1). As well as sites that were not cleavable by KLKB1, positions were successfully identified in the light chain and heavy chain of BoNT/A1 that rendered the molecule susceptible to cleavage by exogenous protease, resulting in a significant (10-fold) decrease in toxin activity in a rat eSCN assay. Conclusions: The results that we present here identify 1 region within the light chain ([LC]; T323-V333) and 2 regions within the N-terminal domain of the heavy chain ([HN]; Q557-I566 and N642-K671) that are suitable for the insertion of plasma-protease-labile destruction sites. We also map the first steps in developing a BoNT therapeutic with a decreased risk of systemic adverse effects. The results show that introducing a protease cleavage site to the HN domain of BoNT/A1 results in a toxin that is susceptible to KLKB1. To engineer a BoNT/A1 with even greater sensitivity to