1. Analytical Research & Development
May 2019

2. AR&D
Contents
Overview
Capabilities
Case Studies

3. AR&D
Overview

4. Highlights Diverse complexity profile of compounds 125+ projects
AR&D
Highlights
Overview
Diverse complexity profile of compounds
125+ projects
Have successfully developed suitable methods for Nucleosides, Amidites, Carbohydrates, Natural Products, Peptides and several non-chromophoric compounds, highly hygroscopic as well as volatile compounds
Over 125 projects delivered across globe for large pharma and biotech companies.
Phase-specific
approach
State-of-the-art Infrastructure
Expertise in developing clinical phase specific, yet robust and sensitive analytical methods using complementing techniques to determine each class of impurities.
Array of techniques, instruments and detectors for sensitive and specific analytical methods for impurity profiling, separations, structure elucidation and solid state characterization.

5. 90+ member team with diversified skill set and expertise
AR&D
90+ member team with diversified skill set and expertise
Overview
PhD 18%
10-22 years
20%
PhD
42%
MSc
58%
<5 years
44%
5-10 years
36%
MSc 82%
Education
Experience
TL/GL Education

6. Infrastructure Overview AR&D HPLC, UPLC Ion Chromatograph GC, GC-HS
ICP- MS
NMR
LC-MS, LC-MS/MS
GC - MS
DSC, TGA and PXRD
DVS, PSD
SFC and Prep HPLC
Flash chromatography
FTIR, UV visible
Digital Polarimeter
Auto Titrators and KF Coulometer
Strong network of external partners for other allied services.

7. Operating framework Conforming to latest global guidelines
AR&D
Operating framework
Overview
Conforming to latest global guidelines
GLP / GDP Compliant
ICH Q3A to Q3D (Impurities)
ICH Q2 R1 : (AMV)
USP 621 : (Chromatography)
USP 1225 : (Validation of compendial procedure)
FDA draft guidance
European guidelines
Internal SOPs
GLP (Good Laboratory Practice) as well as GDP (Good Documentation Practice) compliant

8. Phase-specific approach
AR&D
Phase-specific approach
Overview
Pre-Clinical
Ph-1 to Ph-2A
Ph-2B to Ph-3
& Commercial
Pre-Clinical/GLP Tox Development
“Fit for the purpose” methods
Focus on selectivity with all intermediates and known impurities.
Minimum method qualification, for sensitivity and stability in solution.
Early Phase Development
“Fit for the purpose” methods
Proven selectivity and specificity with all intermediates and known impurities.
Method qualification for sensitivity, linearity & range, recovery, precision (repeatability) and stability in solution.
Late Phase Development
Regulatory compliant
“Complete Method Development”.
Method validation as per the ICH guidelines (specificity, sensitivity, linearity & range, recovery, precision, robustness and stability in solution.
RRF established.

9. AR&D
Capabilities

10. Method development & validation GTI determination
AR&D
Capabilities
Method development & validation
GTI determination
Solid state characterization
Isolation & purification
Structure confirmation
Other specialized techniques

11. Method Development: LC method
AR&D
Method Development: LC method
Capabilities
Techniques
Methods
Reverse phase chromatography
Normal Phase chromatography
Chiral chromatography
Ion pair chromatography
Size exclusion chromatography
SIAM (Stability Indicating Assay Method)
Chromatographic Purity/ Related Substances
Assay determination
GTI determination
Enantiomeric Purity/Chiral Purity
Cleaning Method

12. Method Development: GC method
AR&D
Method Development: GC method
Capabilities
Techniques
Methods
Gas Chromatography with FID and Head Space Injection technology.
Gas Chromatography with Mass detector (CI and EI)
Gas chromatography with FID, ECD and TCD detectors.
Residual solvents and OVI methods
Chromatographic Purity/ Related Substances
Enantiomeric Purity/Chiral Purity
Assay determination
GTI determination

13. Method Validation and Transfer
AR&D
Method Validation and Transfer
Capabilities
Approach
Methods to Validate
Dedicated and separate team with experienced analysts for Analytical method Validation
As per and compliant to all the regulatory guidelines (ICH, USFDA, CDER, USP, EP)
Internal SOP (from protocol to reports)
Protocol based Analytical method transfer to Site QC
Identification Tests
Quantitative Tests for Impurities
Limit Tests for Control of Impurities
Quantitative tests for active moiety

14. Isolation and Purification
AR&D
Isolation and Purification
Capabilities
Instrumentation
Outcomes
Super critical Fluid Chromatography (Thar SFC )
Preparative HPLCs
Novasep column (600 mm x 80 mm)
Flash Chromatography from Combiflash
Quick enrichment and Isolation of impurities for structure confirmation
Purification of products

15. Structure Confirmation
AR&D
Structure Confirmation
Capabilities
Instruments & Techniques
Outcomes
GC MS
LC MS / LC-MS –MS
NMR (1D- NMR (1H, 13C, 19F, 31P, DEPT 90 &135, 1D NoE with Variable Temperature)
2D NMR (COSY, TOCSY, HSQC, HMBC, NOESY, ROESY, EXSY AND DOSY)
FTIR
Structure confirmation of impurities and products using complementing techniques
Absolute configuration confirmation
Complete structure confirmation report

16. GTI determination Complementing Techniques Methods Capabilities AR&D
Liquid Chromatography (HPLC/UPLC with UV, CAD detectors)
Gas chromatography with FID, ECD and TCD detectors
Mass spectroscopy (GC-MS)
Mass Spectroscopy (LC-MS, LC-MS/MS with SIM and MRM modes)
Sensitive Limit test method
Quantitative method

17. Solid State Characterization
AR&D
Solid State Characterization
Capabilities
Techniques
Outcomes
XRPD
DSC
PSD
Microscopy
TGA
DVS
Polymorph screening, Identification and Quantification
Polymorphic transitions determination
Pseudo polymorphism determination
Particle size determination
Crystal structure orientation and determination
Hygroscopicity studies
Sorption and desorption rate
Surface and bonding water studies

18. Other specialized techniques
AR&D
Other specialized techniques
Capabilities
Technique features
Methods
ICP-MS
UV-Vis Spectrometer
FT-IR Spectrometer
KF-Meter
Columetric KF instrument
Potentiometric Titrator
Trace metals analysis
Cleaning methods
Functional groups identification
Assay determination
Moisture determination

19. AR&D
Case studies

20. Related substances by HPLC
AR&D
Related substances by HPLC
Case studies
Specific, precise, robust, sensitive, linear and accurate method was developed and validated for the thirteen specified impurities along with other related substances by HPLC for Phase 3 NCE compound. Targeted Reporting Threshold is 0.03%

21. Thionyl Chloride content in Tac-Cl
AR&D
Thionyl Chloride content in Tac-Cl
Case studies
Tac-Cl is the reagent used for phase 3 intermediate.
Thionyl chloride is used as a reagent for the preparation of Tac-Cl. Tac-Cl contain the trace level of Thionyl chloride, we have observed about 15 to 20% of dimer impurity formation due to the presence of 0.1% level of Thionyl chloride in Tac-Cl. Determining Thionyl chloride in Tac-Cl is challenge, this was overcome by GC analysis using below derivatizing technique. Based on this investigation instead of Thionyl chloride, acetyl chloride reagent was used for chlorination of Tac-acid.

22. Cyanide content estimation by UV-Vis
AR&D
Cyanide content estimation by UV-Vis
Case studies
The toxicity of Cyanide is well known.
Generally cyanide content was estimated in Ion Exchange Chromatography (IC), however it is expensive, sample interference challenge and time consuming technique.
Developed a high sensitive (Detection up to 1 ppm) derivatization method, simple, fast and less expensive technique for estimating the cyanide content by using UV-Visible Spectroscopy.
A colored complex (pink) will be formed after derivatization and the intensity of complex is proportional to cyanide content in the compound.
UV-Visible method (Derivatization): Advantages
Simple and fast
Less expensive
Highly sensitive (Can be detected up to 1ppm)
User friendly (Easy to Operate)
Universal (Can be adopted to other products).
Validated method.

23. Azide content estimation by GC-MS
AR&D
Azide content estimation by GC-MS
Case studies
In conventional HPLC method, estimation of Azide content is not achieved due to low sensitivity, developed an accurate and robust analytical method by using GC-MS. Since it is a genotoxic impurity acceptable specification limit is not more than 3 ppm for a phase 2 NCE.
Genotoxic impurities estimation is always challenge to meet Regulatory requirements as specification limits are very stringent.
Validated method.

24. GTI content estimation by LC-MS
AR&D
GTI content estimation by LC-MS
Case studies
LC-MS method was developed for the estimation of Genotoxic impurity in NCE Product in phase 3.
In conventional HPLC method estimation of this impurity is not achieved due to blank interference and low sensitivity of the method. Developed a robust analytical method for quantifying this impurity by using LC-MS with ESI source in Positive ionization mode.
Validated method.
Highlights:
LOQ of GTI is 2.5 ppm
LOD of GTI is 0.75 ppm

25. Amidites purity by 31P-NMR method
AR&D
Amidites purity by 31P-NMR method
Case studies
Amidites are class of Nucleotides, Phosphoramidites are key building blocks for the automated, solid supported syntheses of oligonucleotide-based drugs, They have been used as primers and probes for the polymerase chain reaction (PCR) for DNA sequencing and implications, and for the synthesis of artificial genes.
Analysis of Amidites & identification of impurities are very difficult in regular HPLC. The impurities can be classified into three types P(III), P(V)-Oxidised & P(V)-Hydrolysed impurities.
By analysing the 31P-NMR spectrum, we can determine the content and type of impurity either it is P(III) or P(V).
This method is simple and accurate compare to HPLC.
Advantages
Simple and fast
Less expensive
Highly sensitive (Can be detected up to 0.05%)
Eco friendly (Less solvents consumption)
Analysis time is about 10 min.
Detection of non phosphorus impurities are not possible.
Does not required internal standard for quantification.

26. Amidites purity by 31P-NMR method
AR&D
Amidites purity by 31P-NMR method
Case studies
ppm---- UV-Active P(III)
ppm----UV-Inactive P(III) imp’s
30-40 ppm---- Oxidised P(V) imp’s
0-20 ppm---- Hydrolysed P(V) imp’s

27. Purification and Isolation
AR&D
Purification and Isolation
Case studies
Purification & Isolation of E4 by Flash Chromatography
E4 crude was purified by Flash chromatography
Input : 10 Kg of E4 crude with 60% purity
Flash Method Conditions:
Column : Grace C18-330g (250x50mm) 40um
Flow : 80 mL/min
Mobile phase :Water :Acetonitrile (05:95)
Runtime : 30 min
Loading :10 g/inj
Solubility : Acetonitrile
After purification E4 was obtained, 4.5k g with 94.0% purity.

28. Purification and Isolation
AR&D
Purification and Isolation
Case studies
Purification & Isolation of Racemic compound by Prep HPLC
Chiral separation of (50: 50) racemic mixture was purified Prep HPLC using Novasep DAC system.
Input Quantity : 4.0 Kg of Racemic mixture
Prep LC Method Conditions:
Column : Chiral Pak IG(250x80mm) 20um
Flow :300 mL/min
Mobile Phase: Methanol : DCM (50:50)
Runtime(Stack time) : 20 min
Loading :6.5 g/inj
Diluent : Methanol : DCM (50:50)
After purification the Isomer-I was obtained 1.8 Kg with 99.9% purity and Isomer-II was obtained 1.75 Kg with 99.9% purity.

29. Structural characterization
AR&D
Structural characterization
Case studies
Structural elucidation study was carried out using NMR and MS data
Annotated 1D NMR and 2D NMR data(COSY, HSQC, HMBC and NOESY)
Based on spectral data structure was unequivocally determined

30. Structural characterization
AR&D
Structural characterization
Case studies
Spectral Data:
Mass
1H-NMR

31. Structural characterization
AR&D
Structural characterization
Case studies
Spectral Data:
13C NMR
COSY

32. Structural characterization
AR&D
Structural characterization
Case studies
Spectral Data:
HMBC
HSQC

33. Sai Life Sciences
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