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Full IND Program

Deliverables: API (non-GMP and GMP) and FIH Drug Product (GMP)

The Challenge

  • Only previously synthesized on milligram scale by a medicinal chemistry route
  • 8-step synthesis with overall yield of 5-10%
  • 5 chromatography steps with non-crystalline IMs
  • No analytical methodologies

Compound 1 Introduction

  • Avista was contracted to perform IND-enabling and follow-up campaign support on Compound 1
    • Process research and development to improve throughput and robustness of the medicinal chemistry route
    • Analytical development of API, intermediate, and starting material methods along with stability protocols
    • Pharmaceutics research and development towards powder-in-capsule, powder- in-bottle, and suspension/solution formulations
    • API manufacturing to deliver non-GMP and GMP batches of Compound 1
    • Drug Product manufacturing to deliver Phase I clinical supply and resupply

Medical Chemistry Route

  • 8 Steps with over all yield of 5-10%
  • 5 steps involved chromatography
  • N-Boc protected intermediates not crystalline (oil/foam)
  • Yields for the alkyne reduction and final step were low; penultimate step yield varied greatly
  • Final step volume >250mL/g

Step 1 – SnAr Reaction

  • Resolution/crystallization developed for cGMP starting material [2]
    • Improved chiral and achiral purity to >99%
    • Intermediate 2-tartrate salt is crystalline (freebase was an oil)
  • EtOH as reaction solvent instead of MeCN
  • Aqueous workup and chromatographic purification replaced by EtOH/water precipitation of compound [4]
    • Throughput dramatically improved
      • Current Vmax (EtOH/water) = 9 mL/g compared to >100 mL/g original route
  • Yield increased from 78% to 95%
  • Repeated 5 times on 7.0 – 7.5 kg scale
    • 96% yield, 99.9 area% purity

Step 2 – Demethylation / Triflate Formation

  • Medicinal chemistry reagents were retained for this step
    • Reduced HCl/dioxane stoichiometry
  • Intermediate phenol not crystalline
    • Telescoped into triflate formation after aqueous workup
    • Original procedure called for concentration of phenol intermediate to dryness
  • Chromatographic purification of [5] was replaced by MeOH/water precipitation
    • Previous Vmax (including chromatography) > 100 mL/g
    • Current Vmax (Aqueous workup for phenol) = 17 mL/g
  • Repeated 3 times on 5.0 kg scale, average yield 78% with 99.3% purity  

Step 3 – Sonogashira/Reduction/Hydrolysis

  • Reordering of Steps
    • N-Boc deprotection moved up in the sequence
      • Chromatography replaced with acid-base extractions for purging of catalysts, ligands, and non-basic impurities
      • Solutions of non-crystalline, basic amine intermediates, telescoped into the next steps
      • All three chromatographic purifications were removed
  • Transfer hydrogenation used for alkyne reduction instead of H2 pressure – no specialized equipment required
  • No decarboxylated impurity detected with intermediate 8, unlike the Med Chem route (which required reverse phase chromatography to remove)
    • Current hydrolysis conditions: 1eq. NaOH, neutralized with 1 eq. HCL at reaction completion
    • Compound 8 is precipitated from iPrOH/heptane along with NaCl (no effect on subsequent step)
  • Number of transformations from intermediate 5 to 8 remained the same; however, number of 
isolations reduced from 4 to 1
  • Yield increase from 30% to 55% over the 4 steps. 5 runs at 3 kg average scale afforded 55% yield with 86% purity

Step 4 – Macrolactamization

  • EDCI retained as coupling agent; HOBt additive replaced with DMAP
    • Less potential hazards than HOBt or HOAt
    • Easier to remove in the workup and purification
  • DCM was utilized as solvent due to poor starting material and API solubility in suitable class 3 solvents
  • Amino acid 8 is dosed into EDCI/DMAP solution over 5 – 6 h
    • Reaction volume dramatically reduced from > 250 mL/g to 20 mL/g
  • Isolated yield was improved from 40% to 60%
    • Chromatography was removed
    • Charcoal/silica treatment of organic layer following aqueous workup is necessary
    • API polish filtration takes place after charcoal/silica, in DCM
    • Solvent-swap to iPAc is performed to precipitate the API
    • Run several times >2 kg scale with average yield of 60% and >98% purity

Step 5 – API Isolation

  • MEK trituration was developed
    • >100 solvents and solvent combinations were screened
    • Optimized to purge dimer by-product
      • Dimer is reduced from 3-4 area% in crude reaction to <0.3 area% after MEK trituration
    • Reduces residual solvents to within ICH limits
      • GC in-process control utilized to determine completion
    • MEK trituration generates desired polymorph and has been used for batch combination into a single lot
  • Run at 3.0 kg scale
    • 84% yield
    • 5 area% purity (0.15 area% dimer impurity)
    • >99.9 area% chiral purity
    • 99.7% potency
    • <0.1% ROI
    • All solvents and metals were below ICH limits

Stable Polymorph Screen


  • To ensure that the most thermodynamically stable polymorph was consistently isolated, a stable form screen was conducted
    • Crystallization techniques included slow cooling, cooling, slow evaporation, etc.
  • Two anhydrous polymorphs were discovered
  • Competitive slurries indicated Form A was more stable which was fully characterized in-house by XRPD, DSC, TGA, PLM, DVS, NMR, and FT-IR


Compound 1 GMP Route and Deliveries

cGMP 200 L Pfaudler

  • Bond connections and number of transformations are the same as medicinal chemistry route
  • Number of isolations were reduced from 8 to 5
  • All chromatographic purifications were removed
  • Throughput was improved for all steps
  • Reaction robustness and reproducibility was increased
  • Reproducible yield of 20% to API that meets desired purity, potency, and physical form specifications
  • Time from start of Process R&D through first GMP delivery, including a 50 g demonstration batch and GLP delivery, was 11 months
  • Delivery amounts have included:
    • Two non-GMP deliveries (550 g and 1 kg)
    • 3 GMP deliveries (215 g up to 2.5 kg) and another (3 kg) in progress

Analytical Development and Manufacturing Support

  • Method Development and Qualification/Validation
    • Impurities method, including forced degradation
    • Residual solvents, assay and chiral purity methods
      • Chiral method resolved multiple diastereomers and reporting tied in with achiral impurities method
    • Elemental impurities by ICP-MS
    • Scientifically sound method for XRD analysis
    • IPC, starting material and intermediate methods
      • Challenging IPC method for alkyne reduction developed
  • Methods developed are suitable for release testing of DS and neat powder in capsule drug product
  • Reference standard qualification
  • Multiple pilot (non-GMP) stability studies performed (DS, PIC, Ora-Plus® and Ora-Sweet ® formulations)

Released >10 batches of DS and DP including clinical trial material and set 9 ICH stability studies in-house

Pediatric Formulation Development

Develop a pediatric formulation for an immediate dosing need

  • Initial formulation was developed using a mixture of Ora- Plus® and Ora-Sweet ®
    • Ora-Plus® and Ora-Sweet ® are pre-manufactured mixtures of excipients specifically designed for oral suspensions
    • Excipients are known, but levels are proprietary

Avista responded quickly to study dose uniformity, time to achieve initial suspendability, and in-use stability of drug substance in formulation

  • Pros:
    • Suspendability of drug substance was maintained 
for 4 hours
    • Drug substance was stable for up to 6 months
    • Formulation could be prepared in the clinical setting
  • Cons:
    • Level of excipients are pre-defined and cannot be 
    • Use of propylparaben as a preservative in the EU is discouraged

Develop a commercial pediatric formulation suitable for filling

  • Reverse engineer Ora-Plus® and Ora-Sweet ®
    • Determine which excipients are necessary to 
suspend drug substance
    • Design of Experiments surrounding the level of excipients
    • Replace propylparaben with ethylparaben or other anti-microbial excipients
  • DOE performed
    • Four excipients were found to be critical to provide suspendability
    • 24 factorial design with 4 center point experiments
    • Objective: find a design space where the formulation maintains suspendability of the API after 1 and 2 hours of initial shaking
      • Large design space after suspension at rest for 1-hr targeting a label claim (LC) of 90-110%
      • Design space narrows for same LC after 2-hrs at rest


DP Manufacturing

  • Xcelodose Powder in Capsule
    • Four batches at multiple strengths
    • Batch sizes ranged from 4k–30k capsules
    • Fill completed within one month
  • Bottle Packaging and Labeling
    • Multiple bottle counts
    • Packaging and labeling completed within one week
  • Pediatric Powder in Bottle
    • Kylix syrup bottles for ease of clinical dispensing – Multiple weights ranged from 0.5g-5g
  • Clinical Shipments
    • Shipments to multiple sites
    • Domestic and international (supported by QP audit)

The Outcome

  • 8-step API synthesis optimized and scaled-up
    • Chromatography eliminated
    • Throughput, reproducibility and yields improved
  • Multiple non-GMP and GMP kilo-scale API deliveries
  • Analytical Methods developed and qualified (SM, IM, IPC, API and DP)
  • API fully characterized with final stabile form selected
  • FIH drug product developed, manufactured, bottled, labeled and delivered to clinics
  • On-demand Pediatric formulation developed and delivered for in-clinic use
  • On-going support for future campaigns (API/DP) and stability storage & testing

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