REGULATORY STUDIES

SUPPORT

Drug discovery services Regulatory Development

Selvita Services

• Target validation

• Hit identification

• Hit to lead

• Lead optimization

• Preclinical candidate nomination

• Broad target class and disease area expertise

Support for drug discovery in the areas of:

• Medicinal/Synthetic chemistry

• Assay development & screening

• In vitro pharmacology

• ADME/DMPK

• Structural biology

• CADD/ AI-driven drug discovery2

• Quality Control

• Release Analyses

• Impurity Studies

• Method Development

and Validation

Comparative studies of biosimilar products

• Pharmacodynamic studies

• Crystallography

• Proliferation studies in vitro

• Metabolic studies in vitro

• Process related impurities measurement

• Synthesis process optimization andparametrization

• Scale-up of processes & technology transfers

• Custom synthesis of NCEs, impurities/metabolites identification and synthesis

• Isotope labelling (2H, 13C, 15N)

• Computational chemistry

3

Methoddevelopment

Methodvalidation

Methodtransfer

Routinetesting

Regulatory aspects support

Development and

optimization of the

method based on

Quality by Design

approach

Validation of the

method developed

in house or provided

by the customer,

based on ICH

guidelines

Transfer of fully validated „ready to use” method to the customer or receiving the validated method from customer’s side for routine testing at Selvita

QC testing of finished products, full battery of physical, chemical, microbiological and biological tests performed in accordance with the specification

Support of CMC process on customer side, filling quality module of CTD, batch certification (FP, APIs), providing CoAs and CoCs confirmed by QP

1 2 3 4 5

Analytical and bioanalytical support of the drug developmentprocess

CERTIFICATES

EU-FDA mutual recognition agreement

5

• From February 7, 2019 Polish regulatory authority is recognized by FDA as equal to the US GMP inspection.

• This means that the FDA can now rely on a Polish GMP inspection to replace their own inspections.

• FDA inspection will be not needed for the following products:

• human chemical pharmaceuticals;

• human biologicals including immunologicals and biotherapeutics;

• active pharmaceutical ingredients;

• intermediate products and bulk pharmaceuticals.

https://www.fda.gov/internationalprograms/agreements/ucm598735.htm

GxP quality system – general overview

GMP GLP

FIRST CERTIFICATE 2009 2011

CERTIFICATE ISSUED BY The Chief Pharmaceutical Inspectorate The Bureau for Chemical Substances

CERTIFICATE SCOPE• physical and chemical testing• biological testing in quality control of medicines

• physical and chemical testing, mutagenicity studies

• chemical analyses and clinical chemistry testing• cytotoxicity tests of chemical substances• bioanalysis and pharmacokinetic studies

LAST INSPECTION/DEVIATIONS 4-5 Jun 2019 (critical 0, major 3, minor 8) 17-19 Sep 2019 (critical 0, major 2, minor 10)

NUMBER OF EXTERNAL/INTERNAL AUDITS Avg 10 external/4 internal audits per year

PERSONNEL IN GXP SYSTEM 85 in Quality Control, 5 in Quality Assurance (including 2 QPs)

DOCUMENTATION STRUCTURE Quality Manual, SMF, SOPs, Instructions and Analytical Methods, Laboratory Records, Raw Data

EQUIPMENT IN GXP SYSTEM 130 pieces of critical qualified equipment (OQ/PQ every year)

6

ANALYTICAL AND

REGULATORY STUDIES

Analytical Laboratory

8

Development of new drugs - ADME and PK studies

Development of bioanalytical methods

Analytical method development and validation

Stability and release testing

Impurity synthesis and identification

Support in CMC projects

Scope of services

CERTIFIED

Analytical services for the pharmaceutical industry

9

• Assay/identity (HPLC, GC, potentiometric titration)

• Identity (MS, NMR, classical methods)

• Impurity profiling and Identification

• Stress studies and degradation profiles

• Stability testing - shelf-life determination, storage and handling requirements

• Cleaning validation

• Residual solvents determination

• Dissolution profiles – pharmaceutical equivalence

• Metal analysis using AAS, ICP techniques

• Microbial purity testing

• Excipient determination - reverse engineering

• CMC projects – analytical method establishment and validation, analytical support of synthesis and formulation stability testing, release analysis

• ADME (solubility, liofilicity, metabolic and plasma stability, metabolite profiling, permeability, plasma protein binding, cytochrome P450 inhibition)

• Study of physicochemical properties

(KF, osmolality, logD, pKa, DSC, melting/freezing point, IR, NMR, PSD, SEM-EDS, XRPD)

• Genotoxicity testing of impurities (performed by the Cell & Molecular Laboratory)

Analytical Services for the Pharmaceutical Industry

10

• Impurity identification

• In-process manufacturing/formulation support

• Analytical method validation/verification

• Characterization and qualification of reference standard

• Stability testing (for demo batches and GMP batches)

• GMP lot release testing or

• Analytical method transfer

ANALYTICAL SUPPORT FOR CHEMISTRY, MANUFACTURING AND CONTROLS (CMC) PURPOSE

Analytical method development and pre-validation:

assay, identity, purity, stress tests, residual solvents,

techniques: H(U)PLC-DAD/UV/CAD/ELSD/MS, GC(HS)-FID/MS

Analytical Services for the Pharmaceutical Industry

11

• Long-term stability testing

(including refrigerator, freezer and below -20⁰C)

• Intermediate stability testing

• Accelerated stability testing

• Forced degradation study

• Photo stability testing

• Other (freeze/thaw, in-use stability, open-pot stability)

STABILITY TESTING

STORAGE AND ANALYSES, FOR FORMULATIONS, DRUG SUBSTANCE AND DRUG PRODUCT

Analytical Services for the Pharmaceutical Industry

12

• Appearance

• Identification by HPLC-DAD, UV, HPLC-CAD,

GC-FID-HS, LC/MS , IR

• Uniformity of mass

• Loss on drying

• Hardness

• Disintegration time

• Dissolution

• Assay by HPLC-DAD, UV, HPLC-CAD, GC-FID-HS,

LC/MS

RELEASE AND SHELF-LIFE EVALUATION OF RAW MATERIALS, DRUG SUBSTANCES

AND DRUG PRODUCTS FOR SPECIFICATION COMPLIANCE

• Related Substances: identified and unidentified

impurities by HPLC-DAD, UV, HPLC-CAD,

GC-FID-HS, LC/MS

• Microbiology (microbial purity limits, bacteria, fungi,

E.coli)

• Analysis by ICP-OES, ICP-MS - catalyst residues

• Analysis by AAS - metal content

Analytical Methods

13

• For small and big (biosimilar) molecules

• According to the requirements of the respective

guidance

• ICH Topic Q2 (R1) Validation of Analytical Procedures:

Text and Methodology, CPMP/ICH/381/95

• U.S. Department of Health and Human Services,

Food and Drug Administration: Guidance for Industry,

Analytical Procedures and Methods Validation for Drugs

and Biologics, July 2015

• Committee for Medicinal Products for Human Use (CHMP):

Guideline on bioanalytical method validation, 21 July 2011,

EMEA/CHMP/EWP/192217/2009

• U.S. Department of Health and Human Services,

Food and Drug Administration: Guidance for Industry,

Bioanalytical Method Validation, May 2018

METHOD DEVELOPMENT, VERIFICATION, VALIDATION AND TRANSFER

• According to the general pharmacopoeial requirements (methods

and monographs); customer’s methods and specifications;

internal procedures

• Verification of analytical methods

• Optimization and development of analytical methods

• Pre-validation

• Validation

• Transfer

Analytical Documentation

14

• Report on analytical method development

• Report on pharmacopoeial analysis

• Report on analyses for specification compliance

(stability, release)

• Certificate of Analysis

• Parts of the Common Technical Document

• Analytical method – Standard Operating Procedure

• Validation protocol and report

• Transfer protocol and report

• Raw/integrated/calculated data

Equipment (GLP/GMP)

15

• GC(HS)/FID (Agilent)

• GC(HS)/FID (Shimadzu)

• GC/FID/MS (Agilent) - ESI/quadrupole

• GC(HS)/MS (Shimadzu) - ESI/quadrupole

• UHPLC/HPLC/MS (Bruker) - ESI/APCI/ion trap

• UHPLC/MS (Thermo Fisher Scientific) - ESI/APCI/TSQ

• UHPLC/MS (Thermo Fisher Scientific) - H-ESI/APCI Q/Orbitrap

• UHPLC/MS (Shimadzu) - ESI/APCI/QQQ

• UHPLC/MS (Agilent) - ESI/APCI/QQQ

• HPLC-VWD (Dionex/Thermo Scientific)

• UHPLC-DAD (Agilent)

• HPLC-DAD (Dionex/Thermo Scientific)

• HPLC/VWD/Corona (Dionex/Thermo Scientific) Detector (CAD)

• HPLC/PDA (Dionex/Thermo Scientific)

• HPLC/DAD/ELSD (Dionex/Thermo Scientific, Agilent)

• HPLC/VWD/RI (Dionex/Thermo Scientific)

• HPLC/UV/FLD (Dionex/Thermo Scientific)

• HPLC/DAD (Dionex/Thermo Scientific) - non-aqueous conditions

• IC (Dionex/Thermo Scientific)

• Capillary electrophoresis CESI 8000 Plus (Sciex)

• Isoelectric focusing electrophoresis (IEF), iCE3 (bio-techne)

• Spectrophotometer UV-VIS (Shimadzu, Agilent)

• Dissolution analysis SR8+ (Hanson Research)

• ICP-MS (Agilent), ICP-OES (Shimadzu), AAS (Thermo Fisher Scientific)

• FTIR (Thermo Fisher Scientific), NIR (Thermo Fisher Scientific)

• KF (Metrohm, Crison)

• Osmometer

• Particle analyser

• Disintegration DISI (Hanson Research), hardness (ERWEKA), friability (ERWEKA)

• Stability chambers (Memmert)

Biopharmaceuticals and Biosimilars Services

16

All studies are designed, optimized and performed based

on the EMA and ICH guidelines, as well as European

and US Pharmacopoeias.

Selvita has established an extensive set of in house tests

suggested by the ICH Q6B guideline to perform during

biopharmaceuticals’ characterization.

STRUCTURAL CHARACTERIZATION

PHYSICOCHEMICAL PROPERTIES

IN VITRO PHARMACODYNAMIC

STUDIES

BATCH-TO-BATCH CONSISTENCY

STUDIES

IN VITRO PROLIFERATION

STUDIES

PROCESS-AND-PRODUCT RELATED

IMPURITIES

17

Biopharmaceuticals Analysis at Selvita

Types of biopharmaceuticals Assays perfomed at CMBD

Bioassay

Cell proliferation and cytotoxicity assays, Neutralization assays, Apotosis assay,cAMP assay

Binding assay

ELISA and SPR based

Identification assays

• SDS-PAGE with Silver Staining/Coomassie Brilliant Blue Staining underreducing and non-reducing conditions

• SDS-PAGE with Iodine Staining (detection of protein PEGylation)

• Western Blot analysis

• Iso Electrical Focusing with Comassie Brilliant Blue Staining

Purity assays

SDS-PAGE with Silver Staining/Coomassie Brilliant Blue Staining under reducing

and non-reducing conditions

Iso Electrical Focusing with Comassie Brilliant Blue Staining

Growth factors and hormones:• GM-CSF (Molgramostim)

(Human Granulocyte Macrophage Colony-StimulatingFactor)

• G-CSF (Filgrastim) and PEG-G-CSF (Pegfilgrastim)

(Human Granulocyte Colony-Stimulating Factor)

• PTH (Teriparatide)

(Human Parathyroid Hormone)

• Insulin and insulin analogs

Monoclonal antibodies:▪ Anti hVEGF (Anti Human Vascular Endothelial Growth Factor)

▪ Bevacizumab

▪ Ranibizumab

▪ Anti hTNF-α (Anti HumanTumor Necrosis Factor α)

▪ Etanercept (Fusion protein)

▪ Adalimumab

▪ Infliximab

▪ Anti hHER2 (Anti Human Epidermal Growth Factor Receptor 2)

▪ Trastuzumab

Bioassay development and validation

18

Selvita approach to in vitro bioassay development & validation (GMP)

In-vitro biossay development in accordance with USP <1032> ‘Design and development

of biological assays’.

Validation of non-compendial methods conducted according to the requirements of

ICH Topic Q2 (R1) Validation of Analytical Procedures: Text and Methodology,

CPMP/ICH/381/95, London, 1995.

Analysis of results is performed using PLA 3.0 Bioassay Software (Stegmann Systems

GmbH).

Exemplary scope of in vitro bioassay validation

• system suitability

• specificity

• repeatability (6 assays performed on the same day by single Scientist)

• accuracy (six potency levels of the sample: 175%, 150%, 130%, 100%,

70% and 50%, assay performed by 3 scientists on 3 different days)

• linearity and range

• intermediate precision (3 scientists)

• robustness

• mAbs binding to target

antigen

(e.g. TNFα, VEGF)

including on-off kinetics

using SPR method

• Binding to representative

isoforms of the relevant

three Fc gamma receptors

(FcγRI, FcγRII and FcγRIII),

neonatal Fc receptor (FcRn)

and complement (C1q) using

SPR method

• Neutralization assays for

anti-TNFα mAbs:

− Inhibition of hTNFα-induced

cytotoxicity/apoptosis

− Inhibition of hTNFα-induced

expression of adhesion

molecules

• Neutralization assays for

anti-VEGF mAbs:

− Cell growth inhibition assay

• Antibody-dependent cell-

mediated cytotoxicity

(ADCC) assay

• Jurkat cells stably expressing

FcƴRIIIa and luciferase

reporter under NFAT control

• LDH-based method using

mTNF-CHO cells and CD16

(v/f)-NK-92 cells

• Complement-dependent

cytotoxicity (CDC) assay

In vitro Comparability Studies of Biosimilar mAbs

Binding to target antigen(s)

Binding to FcγRs and complement

Fab-associatedfunctions

Fc-associatedfunctions

Comparability in vitro Studies of Biosimilar Insulin and Insulin Analogues

• IR-A and IR-B binding assay including on-off kinetics using SPRmethod

• IR binding assay using MST and FP methods

IR binding assays

• Radiometric glucose uptake assay in 3T3-L1 adipocytes

• Radiometric lipogenesis assay in 3T3-L1 cells

• Inhibition of stimulated lipolysis assay in 3T3-L1 adipocytes (FFA evaluation)

Metabolic assays

• Cell-based assay measuring phosphorylation/ dephosphorylation of IR-A, IR-B (stable cell lines) and IGF-1R(HepG2 and MCF7 cells) (AlphaLISA, ELISA)

• Cell-based assay measuring activation of IRS-1 protein, Akt1 and MAP kinase in HepG2 and MCF7 cells (ELISA)

Intrinsic activity

• Analysis of the mitogenic activity of insulin analogs in primary and established cell lines, e.g. SaOS-2, MCF7, MCF10A and HMEC

• Evaluation of cell viability using MTS, CellTiter-Glo® Luminescent Cell Viability Assay and BrdU assay

Mitogenic activity

• Identification of process-related impurities:

• Host Cell Proteins (HCPs)

• DNA Content

Impurities

Standard curve HCPcode: #41599

Log conc. Total HCP eq.[ng/ml]

OD

[40

5n

m]-

OD

[49

2n

m]

0.0 0.5 1.0 1.5 2.0

0.0

0.5

1.0

1.5

2.0

2.5

log(agonist) vs. response -- Variable slope (four parameters)

Best-fit values

Bottom

Top

LogEC50

HillSlope

EC50

Span

Std. Error

Top

LogEC50

HillSlope

95% Confidence Intervals

Top

LogEC50

HillSlope

EC50

Goodness of Fit

Degrees of Freedom

R square

Absolute Sum of Squares

Sy.x

Constraints

Bottom

Number of points

Analyzed

OD[405nm]-OD[492nm]

= 0.2640

4.766

2.080

0.9621

120.2

= 4.502

1.398

0.2427

0.09645

1.690 to 7.843

1.546 to 2.614

0.7498 to 1.174

35.15 to 411.4

11

0.9954

0.02160

0.04432

Bottom = 0.2640

14

Equipment for Biosimilar Studies

21

MicroscaleThermophoresis

(Monolith® NT.115, NanoTemperTechnologies)

GE Healthcare Biacore™ label-freeinteraction analysiswith Biacore T200

instrument

MicroBeta®2 (PerkinElmer) Microplate

ScintillationAnd Luminescence

Counter

Multi-mode Microplate Readers:• Synergy™2 (Biotek)• Synergy™ Neo2 (Biotek)• Spark (Tecan)

CBS Isothermal(Panasonic) Cell Lines Long Term

Storage System in Nitrogen Vapour

Phase

/company/selvita/

SelvitaKrakow

/Biotechnology-Company/Selvita-SA

THANK YOU FOR ATTENTION!

/company/selvita/

SelvitaKrakow

/Biotechnology-Company/Selvita-SA

Get in touch with us: sales@selvita.com