Relevance of Stability Studies on Demonstrating Biosimilarity...Aim: To adjust the stress condition...

Relevance of Stability Studies on Demonstrating Biosimilarity

Laura Giribaldi, PhD

Amega Biotech, Argentina

CASSS, CMC Strategy Forum Latin America 2019March 13, Brasilia

Development, Production and Commercialization of Development, Production and Commercialization of BIOSIMILARSBIOSIMILARS

4414 years producing Biosimilars14 years producing Biosimilars

442 Production Plants2 Production Plants

442 R&D Labs2 R&D Labs

441 Preclinical site1 Preclinical site

AMEGA BIOTECH: AMEGA BIOTECH:

CONFIDENTIAL

441 Preclinical site1 Preclinical site

44R&D scientific staff 50 peopleR&D scientific staff 50 people

4415 Biosimilars developed15 Biosimilars developed

44Exports to over 40 countriesExports to over 40 countries

TECHNOLOGICAL PLATFORMS

BACTERIAL EXPRESSION (E. Coli, Inclusion bodies)BACTERIA (E. coli, soluble)

PEPTIDES (E. coli)CHO, PerfusionCHO, fed-batch

PEGYLATIONPEGYLATION

AMEGA BIOTECH R&D SITES

Gemabiotech R&D Lab

Staff : Total 22

(4 PhD, 7 MS, 11 Tech)

R&D platforms

- Molecular Biology

- Cell lines & Bioprocesses

Zelltek R&D Lab

Staff : Total 20


R&D platforms

- Bioprocess

- Purification

Gemabiotech preclinical Site

Staff : Total 10


-Medical Direction

-Histopathology laboratory- Cell lines & Bioprocesses

- Purification

- Analytical dev

(MS: ESI-MS/MS, MALDI-TOF MS)

- Cell Banks QC

- Purification

- Pilot Upstream Facility

- Analytical dev

- Hybridoma development

- Post-transfer process validation

-Animal facilities

-Preclinical studies

-In vivo biological activity

Assays

DP

Protein content

pH

Osmolarity

ANALYTICAL CAPABILITIES

Glycosylation

N-glycans, charged

N-glycans, structure

N-glycan occupation

O-glycans

Monosaccharides

Sialic acidIdentity and structure

Aminoacid sequence (MS/MS)

Biologic activity

Binding assays

Cell proliferation assays

Viral Intereference assays

TNF Interference assay

In vivo EPO assay

In vivo FSH assay

Enzymatic assays

Preclinical tests (rats)Toxicity Local toleranceImmunogenicity PK

Impurities

SEC Aggregation

Aggregation DLS

Hidrophobic variants

Nonreducing SDS-PAGE

Charged variants

Oxidation and deamination (MS)

Aminoacid sequence (MS/MS)

Intact mass (MS)

Immunoidentification

Isoelectric point

Peptide mapping

Disulfide bonds (MS)

Free cystein

Thermodynamic structural stability

Fluorescence spectroscopy

Enzymatic assays

ADCC

CDC

– To establish degradation pathways.

– To differentiate and elucidate the structure of degradation products.

– To determine the intrinsic stability of a drug substance informulation.

– To reveal the degradation mechanisms.

Forced degradation studies

– To reveal the degradation mechanisms.

– To understand the chemical properties of drug molecules.

– To generate more stable formulations.

– To produce a degradation profile similar to that of what would beobserved in a formal natural stability study.

– To solve stability-related problems.

–To be included in comparability exercise –Biosimilars and Manufacturing process

Forced degradation studies

Biosimilars and Manufacturing processchanges

Forced Degradation Studies for Biosimilar Development

Howde, Berkowitz. BIOPHYSICAL CHARACTERIZATION OF PROTEINS IN DEVELOPING BIOPHARMACEUTICALS, 2015

“A stress is applied in a well-controlled repeatable way to two biopharmaceuticalsamples that only slightly differ in the native state, but the difference is too small todetect. On applying and then removing the stress, the resulting states may now moreeasily reveal the subtle underlining difference that initially existed”

Aim: To adjust the stress condition to achieve a measurable degradation

Temperature (60°C) hours 2 8 24 1 3 7

Acid hydrolisis (pH 3) hours 2 8 24 1 3 7

Selected expositionCondition Exposition

Sample: DP Product A – 40,000 IU/ml

1- Exploratory studies

Exposure Exposure

Acid hydrolisis (pH 3) hours 2 8 24 1 3 7

Basic hydrolisis (pH 8) hours 2 8 24 1 3 7

Oxidation (H2O2 0.2% v/v) hours 6 16 24 48 66 6 16 24

Oxidation (H2O2 2% v/v) hours 6 16 24

Agitataion (200 rpm) days 1 3 7 30 45

Freeze/Thawing cycles 16 30 60 60

After studying several exposition times, experimental conditions were selectedin order to achieve the adequate degradation (not too harsh that most of drugproduct has degraded and not too light that degradation level is betweenmethod variability)

Agitation (

C4-HPLC

D e

g r

a d

a t

i o

n

p r

o f

i l e

s

SEC-HPLC In vitro biological activity

2-To establish stability indicating nature of a developed method

D e

g r

a d

a t

i o

n

p r

o f

i l e

s

2- To establish stability indicating nature of a developed method

D e

g r

a d

a t

i o

n

p r

o f

i l e

s

C4-HPLC SEC-HPLC In vitro biological activity

D e

g r

a d

a t

i o

n

p r

o f

i l e

s

Stability indicating methods analysis

Analytical method

SEC

C4 HPLC

In vitro BA

ELISA

Sialic acid

Western blot

SDS-PAGE

Freeze

Thawing1 3 7 1 3 7 1 3 7

Stress conditionTemperature (h) Acid hydrolisis (h) Basic hydrolisis (h) Oxidation (h)

Agitation6 16 24

Stability indicating methods and the conditions that promote degradation for this product were identified.

SDS-PAGE

N-glycans WAX

CZE

Fluorescence spec

Thermal Shift

CD

RMN

No degradation detected

Degradation detected

3- Comparability of the type and extent of degradation profile between biosimilar and reference product

Temperature

3 h 7 h 3 h 7h

Temperature

3 h 7 h 3 h 7h

1- Non stressed Product A2, 3- AmegaBiotech DP4, 5- Reference product

Similar degradation profile was obtained for AmegaBiotech DP and Reference product bothin SDS-PAGE and Western blot

Comparability analysis

30000

40000

50000

60000

70000

80000

In v

itro

Bio

logi

cal A

ctiv

ity

(IU

/ml)

Biological Activity

30

40

50

60

70

80

90

100

Agg

rega

tes

and

dim

mer

s co

nte

nt

(%)

HMW Impurities

Similar behavior was observed for two batches of AmegaBiotech Product A (blue) and two batches of reference product (red)

0

10000

20000

Control 60°C pH 3 + 60°C

In v

itro

Bio

logi

cal A

ctiv

ity

(IU

/ml)

0

10

20

30

Control 60°C pH 3 + 60°C

Agg

rega

tes

and

dim

mer

s co

nte

nt

(%)

Etanercept: FDS and comparability

Conditions Duration Analysis performed

25°C ± 3 °C 8 and 15 days SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay

Sample: DP Product B – 50 mg/ml

50°C ± 3 °C 8 and 15 days SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay

Low pH

(pH 3- 25 ± 3 °C) 8 and 15 days

SEC-HPLC HIC-HPLC TNF-α neutralization cell based assay

High pH

(pH 9, 25 ± 3 °C) 8 and 15 days


Oxidation

(0.5% v/v H2O2) 3 days


HMWs variants

Slope ratios were used to compare degradation rates

Truncated variants

Degradation kinetics were similar for AmegaBiotech Product B (green) and Reference product (blue)

Size exclusion chromatography - High performance liquid chromatography (SEC-HPLC)Stress condition: Oxidation

Refe

rence P

roduct

The degradation profile of all samples was highly similar since they all show an increase of Product B fragments (LMWs), denoted by peaks with less retention time than the peak of monomeric form

Am

egaB

iote

chP

roduct

Potency (TNF-a neutralization) In vitro cell based assay

No significant differences were found between degradation rates of both products

CD near UV- Tertiary structuree

llip

tic

ity

(de

g/m

ol)

Ref product pH 3 15 days




AB Product B pH 3 15 days


AB Product B control

Control Condition

Mo

lar

ell

ipti

cit

y

Wavelength (nm)




CD near UV- Tertiary structure

Acid Hydrolisis- 8 days

ell

ipti

cit

y(d

eg

/mo

l)

days

days








Unlike erythropoietin, CD is able to detect changes in etanerceptstructure.Similar spectra are obtained for AmegaBiotech and Reference products after acid hydrolisis (7 and 15 days)

Mo

lar

ell

ipti

cit

y

Wavelength (nm)

days

days




CD near UV- Tertiary structure

Acid Hydrolisis- 15 days

ell

ipti

cit

y(d

eg

/mo

l)

days

days








Unlike Product A, CD is able to detect changes in Product B structure.Similar spectra are obtained for AmegaBiotech and Reference products after acid hydrolisis (8 and 15 days)

Mo

lar

ell

ipti

cit

y

Wavelength (nm)

days

days




Conclusions

• Both for Product A and Product B, forced degradation conditions wereselected, which allowed sufficient degradation to occur over a reasonabletime period

• Stability indicating methods were product-specific

• Degradation was studied not only at the final state but also at intermediatestates (kinetics)states (kinetics)

• FDS showed similarity between compared samples

• FDS allowed to increase product knowledge

• Forced degradation studies constitute a powerful tool for comparabilitystudies in biosimilar development, since they leverage opportunities toidentify structural differences between them

Thank you!Thank you!

Relevance of Stability Studies on Demonstrating Biosimilarity...Aim: To adjust the stress condition...

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