Scientific and Regulatory Challenges in Development and...

Scientific and Regulatory Challenges in Development and Commercialization of BiosimilarsDr. Anurag S. RathoreProfessorDepartment of Chemical Engineering Indian Institute of Technology-DelhiNew Delhi, India

GBC 2017

96 experimental

conditions can be

evaluated in 3-4 hours

Outline

2

Challenges with biosimilars

Indian Guidelines on Similar Biologics

Comparison to other regulatory agencies

Major gaps in Indian regulatory system

Path forward

Anurag S. Rathore, BiotechCMZ.com

Challenges with Biosimilars

96 experimental

conditions can be


Characterization of Biosimilar Products

4Anurag S. Rathore, BiotechCMZ.com

Source : http://www.hospira.com/en/healthcare_trends/biologics

Huub Schellekens (2004), How similar do “biosimilars” need to be?, Nature Biotechnology, 22, 1357-1359

Biosimilars and reference biologics have the same amino acid sequences

Differences in clinically inactive components is generally quite minor

http://www.hospira.com/en/healthcare_trends/biologics

Limited ability to model safety and efficacy via non-clinical studies


A

B

C

Use of laboratory, non-clinical and

clinical studies for defining product

design space. A. Cotton Rat PK

Study Measuring Levels of

Motavizumab in BAL after Dosing.

B. Cotton Rat PK Study

Measuring Levels of Motavizumab

in Lung Homogenate after Dosing;

C. IM Prophylaxis in Cotton Rats

by Deamidated Motavizumab*

Will enable manufacturers to predict immunogenic behavior of products

Lessen reliance on clinical trials in human

Revolutionize approaches that are utilized today in drug discovery and molecular design.

•Schenerman, M.A. et al. (2009) Using a Risk Assessment Process to Determine Criticality of Product Quality Attributes. InQuality by Design for Biopharmaceuticals. Eds. Rathore AS, Mhatre R. Wiley Interscience, New York, 53-84.

Complexity of protein molecules


*M. Pathak, S. Dixit, S.

Muthukumar, and A. S.

Rathore, In-vitro

refolding of recombinant

human granulocyte

colony stimulating factor:

Mechanistic

understanding and its

application towards

process design, Journal of

Pharmaceutical and

Biomedical Analysis 126

(2016) 124-131.

Analytical characterization of complex moieties


• Most Ubiquitously used in conjunction with other separation techniques in analytical characterization workflows of biosimilars

• Provides Insights of primary structure, aggregation, Higher order structure of therapeutic proteins

Fragmentation

(CID/ETD)/Ion Mobility/HDX

MS

Chromatographic/

Electrophoretic

Separation

Mass

Spectrometry 2

Enzymatic

Treatment

(Papain/trypsin/

Chymotrypsin/IdeS

)

Peak Picking/

Database search

Biotherapeutic

Recombinant protein/

mAb

Biotherapeutic

fingerprint

Mass Spectrometry 1

Figure 1: Illustration of Mass Spectrometric workflow for protein Identification and characterization

LMVDCCETUX

Scope for

further

improvement

Illustration of Mass Spectrometric workflow for protein Identification and characterization

• Most Ubiquitously used in conjunction with other separation techniques in analytical characterization workflows of biosimilars

• Provides Insights of primary structure, aggregation, Higher order structure of therapeutic proteins

Fragmentation

(CID/ETD)/Ion Mobility/HDX

MS

Chromatographic/

Electrophoretic

Separation

Mass

Spectrometry 2

Enzymatic

Treatment

(Papain/trypsin/

Chymotrypsin/IdeS

)

Peak Picking/

Database search

Biotherapeutic

Recombinant protein/

mAb

Biotherapeutic

fingerprint

Mass Spectrometry 1

Figure 1: Illustration of Mass Spectrometric workflow for protein Identification and characterization

LMVDCCETUX

Scope for

further

improvement

Illustration of Mass Spectrometric workflow for protein Identification and characterization

Complexity of biotech processes

*A. S. Rathore, M. Pathak, S. K. Singh, E. K. Read, and K. Brorson, Fermentanomics: Relating Quality

Attributes of a Monoclonal Antibody to Cell Culture Process Variables and Raw Materials using

Multivariate Data Analysis, Biotechnology Progress, 31 (2015) 1586-1599.

Batch Mode Fed- Batch Mode Fed- Batch with microaeration

Everchanging landscape of characterization tools


Manual Picking

Automatic Picking

TEM Micrograph of mAbs

mAbs frozen in thin

Vitreous Ice

Schematic

Representation of a

Novel Method for

Aggregate

Characterization

Mathematical

Algorithm

Size exclusion chromatography

Charge variants impact cell proliferation

10

A

C

B

Variant% Variant in the

product pool (A)

Sensitivity: Change in proliferative

activity per unit change in % variant (B)Net effect of the variant : A*B

A1 0 0 0

A2 1.7 1.11 1.887

A3 2.5 -0.88 -2.2

A4 4.3 48.28 207.604

A5 6.5 -1.2 -7.8

A6 4.9 2.02 9.898

A7 2.6 33.05 85.93

M 47.5 -5.3 -251.75

B1 23.7 0.13 3.081

B2 5.1 1.9 9.69

B3 0.82 -2.5 -2.05

B4 0 -0.17 0

B5 0 0.13 0

B6 0 0.01 0

B7 0 0 0

155.64Cell Proliferation


Figure : Summary of the regression model correlating charge heterogeneity with

cell proliferation

*S. K. Singh, G. Narula, and A. S. Rathore, Should charge variants of monoclonal

antibody therapeutics be considered critical quality attributes?, Electrophoresis, 37 (2016)

2338-2346.

Guidelines on Similar Biologics

96 experimental

conditions can be


6/8/2017

12

Product Name Active Substance Therapeutic Areas Company

Alzumab Itolizumab Psorasis Biocon

Basalog Insulin glargine Diabetes Biocon

Biomab EGFR Nimotuzumab Head and Neck

Cancer

Biocon

Canmab Trastuzumab Breast Cancer Biocon

Grafeel Filgrasim Cancer Dr. Reddy’s Lab

Insugen Human Insulin Diabetes Biocon

Mabtas Rituximab Lymphoma Intas Pharma

Nufil Filgrastim Cancer Biocon

Filgrastim Filgrastim Cancer Lupin

Peg-filgrastim Pegfilgrastim Cancer Lupin

Peg grafeel Pegfilgrastim Cancer Dr. Reddy’s Lab

Reditux Rituximab Lymphoma Dr. Reddy’s Lab

Relibeta Interferon beta Multiple Sclerosis Reliance Life

Sciences

Religrast Filgrastim Cancer Reliance Life

Sciences

Rituximab Rituximab Non-Hodgkin’s

lymphoma

Zenotech

Laboteries

Biosimilars in India

India is one of the leading contributors in the world Biosimilar market

In 2000, the first “similar biologics” was approved and marketed in India for hepatitis B vaccine

In 2012, “similar biologics” guideline was first released and implemented in India

Over 50 biopharmaceuticals have been approved for marketing in India, with more than half of them being “similar biologics”

Comparison to Other Regulatory Agencies

96 experimental

conditions can be


S.NO PARAMETERS EU US SOUTH KOREA INDIA

1. Laws and

Regulation

Directive 2001/83/EC—

implemented by the

European Medical

Agency (EMA)

Committee for

Medicinal Products for

Human Use

Biologics Price

Competition and

Innovation (BPCI) Act

of 2009

Guideline on

evaluation of

biosimilar products,

2009

Guidelines on similar

biologics: regulatory

requirements for

marketing

authorisation in India

2. Regulatory

Authority

European Medical

Agency (EMA)

Food and Drug

Administration (FDA)

Ministry of Food and

Drug Safety (MFDS)

Central Drugs

Standard Control

Organization (CDSCO)

and Department of

Biotechnology (DBT)3. Definition of

biosimilars

Biological products

which demonstrated its

equivalence to an

already approved

reference product with

regard to quality, safety,

and efficacy

A product highly

similar to the reference

product without

clinically meaningful

differences in safety,

purity and potency

A biological product

shown to be

comparable, in terms

of quality, safety, and

efficacy, to a

reference drug

A biological product or

drug produced by

genetic engineering

techniques and

claimed to be similar in

terms of safety,

efficacy, and quality to

a reference biological4. Pre-litigation

Procedure

None Present. Absent Absent

• In Pre-litigation procedure, bio-

similar sponsors may challenge an

unexpired patent in an attempt to

enter the market before the original

patent expires.


5. Reference Product On the basis of a

complete dossier,

reference product has

to be authorised in EU

Section 351(a) of the

Public Health Service

(PHS) Act enshrines

reference product

characteristics and in

the light of this

section reference

product has to be

licensed with a full

biologicals license

application

Only a biological

product that was

licensed on the

basis of a full

registration dossier

can serve as a

reference biological

The reference drug is

used in

demonstrating the

comparability of a

biosimilar product

through quality, non-

clinical, and clinical

studies

6. Jurisdiction Bolar Exemption under

article 10 (6) of

Directive 2004/27/Ec

empowers to conduct

necessary trials or

studies for biosimilar

approval

Follow-on Biologics

(FOB) is not

constructive

infringement

Exemption is

accorded from

infringement under

35 U.S.C 271 (e) (1)

for preclinical and

clinical investigation

FOB is constructive

infringement

sufficient for federal

district court

jurisdiction under

section 35 U.S.C.

271(e)(2)(c)

Not defined Not defined


7. Data Requirement Purity,

Physiochemical

properties,

Biological activity,

Clinical studies,

Preclinical, and

Immunogenicity

studies

Analytical data that show

similar to the reference ,

animal studies, Clinical

studies, identity of

mechanism of action

Extensive side- by -side

characterization,

physiochemical

properties, Biological

activity, immunochemical

properties and

immunogenicity studies

Biological activity,

Clinical studies,

Preclinical, and

Immunogenicity

studies

8. Substitution Substitution is

determined at the

member-state level,

and therefore this

topic is not directly

addressed in EMA

guidance

Interchangeable—the

applicant has to show

that the biological

product can be expected

to produce the same

clinical result as the

reference product in any

patient. Biosimilars that

are deemed

interchangeable could be

substituted for reference

products without

physicians’ orders

Not addressed Not addressed

9. Formulation Same strength and

route of

administration,

otherwise further

studies required

Same strength and route

of administration

Dosage form and

strength must be the

same

Same strength

and route of

administration


10. Exclusivity period for

reference product

“8+2+1” rule .

11 Years, comprising 10

years for new biologics

(8-year data exclusivity

and 2-year market

exclusivity) and a 1-year

extension for a new

indication.

12 Years, A section 42

USC 262 (k) , application

may not be filed until 4

years after reference

product approval.

Not specified Not specified

11. Stability

Requirement

Accelerated and under

stress condition.

Accelerated and Long

Term studies.

Accelerated and under

stress condition.

Accelerated and based

on real-time stability

study.

12. Market exclusively

for biosimilar

products

No exclusivity 6 months to 1year

exclusivity for first

biosimilar.

No exclusivity No exclusivity

13. Post-marketing

Surveillance

Mandatory with updated

risk management plan.

Mandatory with

submission of reports in

FDA Adverse Event

Reporting System

(FAERS).

Pharmacovigilance

plan must be

submitted.

Pharmacovigilance plan

must be submitted.

14. Clinical Trials Mandatory but extent

negotiable.

Mandatory but extent

negotiable.

Phase I studies

mandatory, Phase III

studies may be

abbreviated in some

situations

Only preclinical studies

and Phase III trials are

mandatory.

• Comparative clinical trials required .

• Clinical study design- Preferred for

equivalence trials with pre-specified and

justified margins.

• Safety data from patients and study

duration should be provided.

• Critical and Key Quality Attributes (CQA,

KQAs should be included

• Phase3 CT-single arm study with at-least

100 evaluable subjects.

• Confirmatory clinical safety and efficacy

study waived.

In EU & US ,

• Phase 1: pharmacokinetics (PK) and

pharmacodynamics (PD) study

• Phase 3 study : safety and efficacy study

• Immunogenicity assessment :- Both in

Phase 1 and 3


15. Naming and

labelling

Commercial name,

appearance, and

packaging should differ

and INN should be the

same for related

biosimilars.

Not addressed Not addressed Not addressed

16. Non-clinical data Non-clinical studies

should be comparative

and designed to detect

differences in responses

between the biosimilar

and the reference

product.

Generally, an application

should include data

derived from animal

studies to help to show

that the product is

biosimilar to a reference

product.

Non-clinical studies

could be done with

methods such as

assays in vivo, PK/PD,

and studies of toxic

effects, to establish the

comparability of the

biological or PD

activity between the

reference and

biosimilar product.

In-vitro cell-based

bioassays or animal

studies should be used

to establish

comparability of the

biosimilar and the

reference product.

17. Extrapolation Extrapolation to other

diseases and settings for

which reference products

have EMA approval is on

a case-by-case basis.

Indication extrapolation

is possible on a case-by-

case basis.

In some circumstances,

a biosimilar product

might receive

extrapolated

authorisation for other

indications of the

reference product.

A similar biological can

be extrapolated to

other clinical

applications in some

circumstances, such as

when similarity in

quality or preclinical

assessment has been

established between

the biosimilar and the

reference product.

Analytical Comparability of Filgrastim Biosimilars

96 experimental

conditions can be

evaluated in 3-4 hoursN. Nupur, S. K. Singh, G. Narula, and A. S. Rathore, Assessing

analytical comparability of biosimilars: GCSF as a case study, J.

Chromatogr. B, doi:10.1016/j.jchromb.2016.05.027

Analytical Comparability of Filgrastim


Molecular attributes Method/Technique used Filgrastim

Primary structure and

Molecular identity

LC-ESI-MS, MALDI-TOF-MS, Peptide

mapping, N-terminal sequence analysis

Purity and Molecular

integrity

HP-SEC, SDS-PAGE, CE-SDS analysis

Charged isoforms HP-IEC, cIEF

Secondary and higher

order structure analysis

CD spectroscopy, Fluorescence

spectroscopy

Glycan analysis UHPLC

Binding Affinity SPR, Octet

Biological Activity Cell based assays



Non reduced SDS PAGE Reduced SDS PAGE

25 KDa

15 KDa

25 KDa

15 KDa

Neup Lupf Emgr Cols Neuk Graf Neup Lupf Emgr Cols Neuk Graf

SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine

SampleName: neupogen

AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

SE-HPLCRP-HPLC

-30

-20

-10

0

10

20

30

40

50

195 215 235 255C

D (

md

eg)

Wavelength (nm)

NeupogenLupifilEmgrastColstimNeukine

Analytical Comparability : Intact Mass by MS


SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes


SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes


SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

SampleName: grafeel

SampleName: lupfil

SampleName: emgrast

SampleName: colstim

SampleName: neukine


AU

0.00

0.02

0.04

0.06

Minutes

10.00 15.00 20.00 25.00 30.00

18798.93

18798.70

18798.66

18798.75

18798.88

18799.14

Alkylation

(+57.02 Da)

at A37/A43



CQAs Primary structurePotency

(%)

Higher order

structure

Aggregates and degradation isoforms

Charge heterogeneity

Intact Mass

Sequence coverage

High-molecular

weight variants

Truncated variants

Oxidized/ reduced variants

Method MSPeptide

mappingIn vitro CD

SEC (RT of main peak)

SDS PAGERPC (RT of main

peak)Neupogen® 18798.74

All biosimilars

show similar number and abundance

of peaks against

Neupogen®

100

All biosimilarscorrespond

withNeupogen®

indicated by two dips at 208 and

222 nm

14.82 All biosimilars showed a principal

band around 18.8 kDa

with similar position and

intensity against

Neupogen®

28.13Biosimilar

118798.87 96.4 14.88 27.94

Biosimilar 2

18798.59 89.5 14.99 27.97

Biosimilar 3

18798.74 95.3 14.93 28.06

Biosimilar 4

18798.76 90.1 14.89 28.14

Biosimilar 5

18798.87 94.9 14.98 28.03

Analytical Comparability of Rituximab Biosimilars

96 experimental

conditions can be


Analytical Comparability : Functional Characterization


0

20

40

60

80

100

120

140

160

Me

an F

luo

resc

en

t in

ten

sity

(F

LU)

2 hr 4 hr 8 hr

0

0.5

1

1.5

2

2.5

3

3.5

0 0.2 0.4 0.6 0.8 1 1.2A

bso

rban

ce a

t 4

90

nm

mAb Concentration (mg/ml)

Ristova

Biosimilar 1

Biosimilar 2

Biosimilar 3

Biosimilar 4

Biosimilar 5

0

0.5

1

1.5

2

2.5

3

3.5

0 0.2 0.4 0.6 0.8 1 1.2

LDH

re

leas

e

mAb concentration (mg/ml)

Ristova

Biosimilar 1

Biosimilar 2

Biosimilar 3

Biosimilar 4

Biosimilar 5

A- Anti-CD20 antibody mediated ADCC measured by lactate dehydrogenase(LDH) release, B- CDC measured by MTS assay of Rituximab biosimilars withrespect to Ristova

Mean fluorescent intensityshowing binding affinity ofRituximab biosimilars towardsRaji cells expressing CD20receptor with respect to Ristova

Cell based binding assay to CD20 (FACS)

ADCC and CDC assay



Glycoformmasses (Da)

Biosimilar5 Biosimilar4 Biosimilar3 Biosimilar2 Biosimilar1 Ristova

G0F/G0F 147116.4 147115.2 147112.6 147107.9 147110.1 147104.6

G0F/G1F 147275.3 147275.0 147270.7 147269.3 147263.2 147266.2

G0F/G2F 147433.2 147432.4 147428.6 147430.3 147412.3 147427.4

G1F/G2F 147582.0 147579.3 147577.1 147588.3 147554.4 147585.1

G2F/G2F 147734.2 147734.7 147722.1 147744.9 147699.9 147741.3

Figure 1: A1- ESI-TOF-MS spectra, A2- Deconvoluted spectra for most abundantglycoforms, B- Total compound chromatogram (TCC) mirror plot for peptidemapping for reduced trypsin digest of Rituximab biosimilars and Ristova

Ristova

Ristova

Ristova

Ristova

Ristova

Biosimilar1

Biosimilar2

Biosimilar3

Biosimilar4

Biosimilar5

Ab

un

dan

ce

Mass to charge ratio

Analytical Comparability : Process Variants


-50

0

50

100

150

200

250

2 4 6 8

Re

spo

nse

(m

AU

)

Time (min)

CEX without CpB digestion

Ristova

Biosimilar 1

Biosimilar 2

Biosimilar 3

Biosimilar 4

Biosimilar 5

-50

0

50

100

150

200

250

2 4 6 8

Re

spo

nse

(m

AU

)

Time (min)

CEX after CpB digestion

Ristova

Biosimilar 1

Biosimilar 2

Biosimilar 3

Biosimilar 4

Biosimilar 5

A- CEX-chromatogram without CpB, B- CEX-chromatogram with CpB digestion ofRituximab biosimilars with respect to Ristova for assessing charge variants

CEX for

charge

variants

-50

0

50

100

150

200

10 15 20 25

Re

spo

nse

(m

AU

)

Time (min)

SEC for aggregates

Ristova

Biosimilar 1

Biosimilar 2

Biosimilar 3

Biosimilar 4

Biosimilar 5

Non reduced SDS PAGE

A- Non reduced SDS PAGE for assessing LMWs, B- SE-chromatogram forassessing HMWs of Rituximab biosimilars with respect to Ristova

A B

SDS PAGE

and SEC

for high

and low

molecular

weight

variants



CQAs Primary structureHigher order structure

Intact MassSequence coverage

Method MSPeptide

mappingFar UV CD Near UV CD Fluorescence

Ristova®Five abundant

glycoformsobtained after deconvolution

of the mass spectrum

All biosimilarsshow similar number and

abundance of peaks against

Ristova®

All biosimilarscorresponds with Ristova® indicated by

dip at 218 nm

All biosimilarscorresponds with Ristova® indicated by dips for Phe,

Try, Tyr

All biosimilarscorresponds with Ristova® indicated

by λmax at 344 nm

Biosimilar 1

Biosimilar 2

Biosimilar 3

Biosimilar 4

Biosimilar 5



CQAs Aggregates and degradation isoforms Charge heterogeneity

High and low molecular weight variantsAcidic

%Main

%Basic

%Acidic

%Main

%Basic

%

MethodSEC

(main %)DLS (nm)

SDS PAGE CEX without CpB CEX after CpB digestion

Ristova® 99.4 11

All biosimilars show principal band around 148 kDa with

similar position and intensity against Ristova®

15.22 71.91 12.86 12.66 85.51 1.82

99.6 11 8.87 56.44 34.69 10.02 81.73 8.25Biosimilar 198.1 10 9.56 55.91 34.53 9.79 82.99 7.22

Biosimilar 299.6 11 13.71 45.89 40.41 13.17 78.84 7.99

Biosimilar 399.4 11 12.87 70.01 17.12 11.42 84.72 3.86

Biosimilar 499.4 11 9.51 32.65 57.84 13.81 78.81 7.38Biosimilar 5



CQAs Functional characterization

Cell based assay Affinity (KD)

Method FACS ADCC CDC SPR

Ristova®

All biosimilars showed dose response curve, EC50 values within the range

with respect to Ristova®

4.7E-07Biosimilar 1 1.3E-07

Biosimilar 2 3.3E-07




Summary

96 experimental

conditions can be


Global Regulations for Biosimilars

Differences exist amongst various jurisdictions:US is the most conservative (interchangeability)

Differences with respect to legal accountability of the regulatory bodies

Substantial differences in clinical requirements:Extent of clinical program

Number of patients

Indian experience:99% of the patients cannot afford biotherapeutics

While regulations are similar to global regulations, there are issues with interpretation for approval

While the products on the market seem to be of satisfactory to the most part, some are not

A more rigorous approach needs to be put in place

34 Anurag S. Rathore, BiotechCMZ.com

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Scientific and Regulatory Challenges in Development and...

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