Medlmmune

Analytical Characterisationin support of BiologicsDevelopment

Alistair Kippen PhDDirector, Analytical Biotechnology

Biomanufacturing Technology Summit(University of Maryland & NIST)Institute Bioscience and Biotechnology ResearchRockville USA, 13 June 2014

Overview

Characterisation of Biologics

Analytical Tools &Requirements

Structural Characterisation (LC-MS, HDX examples)

~ Summary

Medlmmune

Characterisation of Biologics

♦ Biologics (produced in biological/cell-based systems) exhibit inherent variability— Changes in manufacturing processes/scale-up can lead to changes in Quality Attributes— Structural changes (e.g. from process change) leading to functional changes that relates to

demonstration of consistency (or impact) to efficacy &safety

♦ Comparability (ICH Q5E) forms the basis of characterisation (ie scientific principles)— Analytical, Non-Clinical (Tox/PK) and Clinical requirements

♦ Analytical comparability is the foundation— Reliance on detailed methodologies that are sensitive to differences in Quality Attributes— Providing comprehensive analytical comparison to reference— Including stability data under intended, accelerated &stressed conditions

♦ Biologics development driven through the Target Product Profile— Reference product range established from multiple lots (target specifications)— The more critical Quality Attributes, the more comparability required— Demonstrating Biologic as ̀safe &efficacious' as reference (with tailored non-clin/clin)

Control Strategy for all biologics is a combination of:

Criticality of Quality Attributes, Process control &Analytical testing strategy

Medlmmune 3

Process Control Strategy(From A-Mab case study)

Prior Clinical Animal In-VitroKnowledge Studies Studies Studies

1 High CriticalityAttributes

Product QualityAttributes

Safety andEfficacy Data 1.Quality attributes to be

considered antllor controlledby manufacturing process

CriticalityAssessment

2. Acceptable ranges forquality attributes to ensuredrug safety and efficacy

Attributes that tlo not need tobe considered or controlledby manufacturing process

Low CriticalityAttributes

Product Understanding

Process Targets Processfor Quality Development and ~~Design

Attributes Characterization Space

Input Material Controls

Process Controls

Procedural Controls

Process ParameterControls

Testing

In-Process Testing

Specifications

Characterization & IComparability Testing

Stability

~ Process Monitoring ~

Process Understanding

c0~aU

~La~NN

U0a

0c~.c0a

I~ I Medlmmune Link to document: http://www.casss.orq/displaycommon.cfm?an=1 &subarticlenbr=2864

Process Consistency

Cell Bank

Manufacture

Purification

Drug Product~ S'

Medlmmune

Cell-line, Productivity

In-Process methods, scale-up &testing

Process impurities, aggregates, fragments

ReferenceMaterial

Lot Release Stability

5

Comparability

♦ Changes through manufacturing/process development lifecycle— Cell line, suppliers (eg raw materials, media), purification methods, formulation, scale/site

♦ Impact assessed by comparability studies (ICH Q5E) to be tightly controlled— Basis of 18 biosimilars approved (EU), Ph. Eur & EMA guidelines introduced

— "The goal of the comparability exercise is to ensure quality, safety and efficacy of the drug productproduced by a changed [manufacturing] process"

— "Determination of comparability can be based on a combination of analytical testing, and in some cases,non-clinical and clinical data"

— "Demonstration of comparability does not necessarily mean that the quality attributes of the pre- andpost-change product are identical, but that they are highly similar"

♦ Revised comparability guideline is the basis for biosimilars (Legal &Regulatory)

♦ FDA Draft Guidance's on Biosimilarity (April/May 2014)

♦ EMA Guideline on Similar Biological Medicinal Products (Draft 2013)— Stepwise approach with comprehensive physicochemical &biological characterisation (that defines

nature of non-clinical &clinical studies to be performed)— Not expected that all quality attributes will be identical and minor differences may be acceptable— Comparable does not mean Identical. Extent of change defines development program, from release

analytics (e.g. small process change) to Clinical studies

I • Analytical comparability is the foundation~ I Medlmmune

Analytical testing The Endless toolbox!

♦ Effective comparability reliant on appropriate analytical methods

♦ Improvement in method detectability and sensitivity

♦ Notably mass spectrometry methods, allowing detailed characterisation of Biologics

♦ Providing improved definition and control of product attributes

CD

AUC

IEC

DLS

:.~FTIR

Mass Spectrometry

Raman FFF cIEF

Western blotting HP-SEC

RP-HPLC SDS-PAGE

N-term sequencing

Medlmmune

NMR

• Purity &stability• Primary sequence• Glycosylation• Size heterogeneity• Higher order structure

Activity/binding assays

Consideration of Quality Attributes(rather than analytical test)

♦ QbD of Biologics development: QTPP considerations described in ICH Q8 (R2)

♦ Consider intended use (clinical setting, dose, delivery, etc)♦ Based on characteristics of reference product (range from multiple lots)

♦ Target specifications (ICH Q6B), including stability (shelf-life)

♦ Criticality assessment of Quality Attributes (cQA's) —defines degree of comparability

Size heterogeneity

Fragmentation/elongation

Aggregation

Particles

Higher order structure

SS bridges

Mutimeric state

Charge• heterogeneity

Medlmmune

Primary sequence

Amino acid modification

Integrity (deletions/insertions)

Post translation modifications

Glycation

Glycosylation

Deamidation

Oxidation

Functional &binding assays

0

Sources of Structural Heterogeneity

N-terminal leader processing/Terminal clipping

Incorrect N-terminal

Chemical r~• Oxidation• Deamidation• Charged isoforms

I~ Medlmmune

Glycosylation

., •2~

t

• ~` •) '

` y.•

~~~~• •

C-terminal "ragged end"• His tag• Proteolytic cleavage

Choosing the right engine(Quality Attribute to testing strategy)

♦ Appropriate subset of analytical toolbox applied to product characterisation♦ Use of sensitive & orthagonal methods (head-head with reference)

— Comparison of physicochemical/structural/biological results, identify variants, Consistency

♦ LC-MS/MS: Identity, detailed structure and purity assessment— Primary sequencing, identification of impurities, degradation pathway (Stability)— Structural MS (HDX-MS, native MS, ion mobility, high mass MALDI, ETD)— Peptide Mapping: structural sequence modifications (eg deamidation, oxidation)

♦ Alongside routine tests:— SDS-PAGE/BioAnalyser: Molecular weight, purity— RP-HPLC: High resolution determination of purity, structure &fragmentation— SEC (UV/MACS) & AUC: Molecular weight distribution, aggregation— DSC (Differential Scanning Calorimetry): Transition temperatures, structural stabi— DLS (Differential Light Scattering): Size, distribution— CE (Capillary Electrophoresis): Purity, charged-state analysis— CD (Circular Dichroism): Secondary &tertiary structural analysis

I •~ Medlmmune 10

~,

Identify ProductAttributes

(Reference)

DefineParameters to

Monitor:- e.g. Glycosylation

Determine MethodRequirements

D

ConsiderAnalyticalStrategy

Tailored methoddevelopment

♦ Systematic process engineering to meet product requirements♦ Early identification (define target) &monitoring of Quality Attributes:

- Timely development of the right tools (novel analytical screen)- Cell line and process development (e.g. glycosylation as a key input)

Product Characterisation: Degradation (LC-MS)

♦ Mass spectrometry methods, allowing detailed characterisation of Biologics

♦ RP-HPLC separation of product-related sequences

♦ LC-MS/MS structural characterisation by mass (sequence/modifications)

..... 9 ~t~:

.~ • .............~ E..... T ............................................................................................................................................................................4......,

~ 101 ~ 1~ 1~: .}.~.,.....................................................................................~.}.... : :i ~ ~ ~ ~ ~ V : 1 .................. . ................................ ~... .......... .....................................................................................................:......f

7},yi~ ~ n y~lps4 ,~

:a E ~dt f~~ 'p ~~ ~~~ i5 ~ a{ '~ '~r'~f~ ~ ~~ )i

_ ̀a,~~'~Fryr"F`~~,~,'`h,~~ iAl 4~ ~ ~~w,.P,4~,,:~'~ y~ .,,, ~ : y~ i~u •11~ X9:4 ~a~a , Y'}~ .} ,_.." '~•r~e,~t.y+'1fyrY_=r=~~.•, ~,,x;~,,,q^aye.. ~, 3.,~1'+~*'~'f~-r'

a ~~-~. isq', .(~l ~.'d' ~~ ~~ A$I ?~ 1<:~~ is '~j SLtl]j iJ x~ ° '!,~ Mi iii !i ce L5~ !1W 3J~

1=Native product2-4, 11-13 =Fragments5-7 =Oxidation8 =Elongation (impurity)

~ 9 = DeamidationMedlmmune 10 =Loss of N-terminal residue (clipping) 12

Product Characterisation: Glycosylation

♦ One of the most widely occurring and functionally important PTMs

♦ Can be significant cause of batch variability &non-comparability

♦ Glycosylation variants can modulate:

— Protein Folding, Stability, Immunogenicity, Biological activity, PK

♦ Complex heterogeneous structures

— Mature glycans formed by trimming (in ER and Golgi) and elongation (Golgi)

♦ Potential critical attribute for biologics

Growth HormonesInterferons

Medlmmune Antibodies13

Glycosylation: Complex N-Glycan Pathway

Fucose• Galactose• Mannose■ N-Acetylglucosamine~ Glucose

Alpha Mannosidase 1A and 1B

•

Medlmmune

i i~i i2_6 ~ ~

~— — — — — — ~High Mannose

Structures

I~

Alpha Mannosidase II

Tetra-antennary

Asn-X-Ser/Thr

ER Mannosidase and Glucosidases

rlr_NAc T1

~+ 0+•~—————~

~ ~~ — —II

U

Hybrid Structures

~+ ~+~ - ~-

~— — n ~I

r ~ ~~ ~I— — n ~Ir - ~ ~

— — — — — —n Tri-antennary

Complex Structures

~+ ~+r - - _ _ _ i

r - ~ i

~--~i in~ — ~ ~

~--!i in — — — — — —

Bi-antennary14

Glycosylation Comparability: AntibodiesUPLC /Mass Spectrometry characterisation

♦ Display predictable Fc glycans in CHO cell lines

— Complex bianntennary, low sialylation, low galactosylation

♦ Good platform testing strategies for ID: 2AB -labelling UPLC

— Glycan released form protein with PNGaseF

— Labelled with 2-aminobenzamide (2AB)

— Analysed by UPLC (HILIC)

♦ Comparison to standard required for structural assignment

•~-~~~~.

pt:• t~

~~!t°l~~P~-~~ Mans

~p~ :~ GOf

■P~ ~~

~P'~'~ GO

F,__ 0Qpl-a ~ ~~ t-6

~ ~p ~ G1f

nt:Pt~. pt-~.

~",~~P'_,~ G2f

Medlmmune,.o ,.o mo ~o ga re ,ao ,.o po .,o ..o ....,.

Glycosylation Comparability: Complex Glycoprotein

Glycosylation Comparability: Complex Glycoprotein2AB LC-MS characterisation of glycans

obo611MEDI-524_PIC_+P 349 (7.384) Cm (344:362)

100 811.1216_

1 27$ I aa~~~ ~,tv:,.

1.61222

1583.272025"

GOF /

19~G

1584.3418182

1584.3711150

GOF-GN

12.1078 ~i~803.1291 111

103~ 1380.249 585.3092

93 ~8

138D.2087 X380.

M2F+GN ss 5276D

1218.2611 Bz

830.0984 M 3 F 6~792.1467

4846 i M2F 19.2383~ 586.3358

as 1382.2753 3729 +Na adducts

746.3959 530.5759 1015.1942 1 1.9136 1220.2156 _1605.282618 25 19 ~ 16 2313832886

16.2269 14 +K adducts11

D 800 900 1000 1100 1200 1300 140D 1500 160D 1700 1

Molecular ion

Fragment ions

19D0 2000 2100 i

Glycosylation Comparability:Permethylation MALDI-TOF-MS characterisation

• Remove glycans & methylate for detection by MALDI

Mans ~ ,.R~ ~yager Spec #1=>Sh -' - ivBC(32,0.5,0.1)[BP = 573.3, 4356]

1001579.8 ~ «

1 25,7 i

90 ■ ■

~ G2F+S

so ,ssz.s~'

■ ~~~~ 26Q6.4

G2F78 G 1 F '

'-. q~

64 2245.2

GOF 2° ao.'

50 , ~.s G3F+S 1 T■ ■ 1835.9 __.~ .._ •' •

aoG2F+S2 1

G 3 F ~ 3055.630

9F94S l G3F+S2iss~.a

22as.21886.0 2071.1

1362.fi 7 7.8 1880.

1141 I I1 29.8'! ~2.9 9.1 57.

1109.0

JOb: 223D9, 95346 N-Qly, 300311, Op MCD:1...1300311 9534fi N-GFy.datAcquired: 14:18:00, March 30, 2011

1828.4

san.l G3F+S

3778.87

2547.8 3267.2 3986.6Mass ~mlz}

Complex Glycoprotein analysisMALDI-MS vs Q-ToF-MS

.~....r.,n

Q-ToF Synapt G2

Q-TOF-MS spectra of Glycoprotein X

Bruker Ultraflextreme

Medimmune

The right tool !

Compound A, Calibration using cluster of Protein A. Matrix Sinapinic Acid. ~~R

'~ q xt04~

~'4 m [M+H]'

S i

1.2

'' I1.0

D.8

[M+2H]~• ~

D.6 ~

t4 i

1~ ~ h

0.2 ,` y~J~, g~,i~

50000 75000 100000 125000 150000 775000 200000 225000 250000m/z

MALDI-TOF MS spectra of Glycoprotein X

Complex Glycoprotein analysisSequence integrity confirmation

• From MALDI-TOF (single broad peak) use ISD for site-specific sequence information

z+2 ~ tPi

13

12

10

25

c 12 c 15 c 18

c11 c14 c17

c70 c13 c16

19 c Y1

20 c 24c31

c21 z+22b ~~ a31

c23 c26' c 30

c 25 c 28

c 27

~ I ~~c29 c

1000 1600 2000 2500 3000 3500

Match Firors ~ MS,'MS Fagmerrts I MSMS r'~nalysis

~ RIASError: 0.03 Da Imear regresswn~. f(X1 = -0.054170 ~ 0.000016 X Coneletlon quotient: 0.528494

0.10

0.00

Y~

c33 c35

32 c 3q

~z+2 38

~~~~4000 ~~r~~

z+2 49

c 44 y 47

c a3 c as z;2 so

5000 5500 mlz

a0 0 0 ~ ~ ° ° °

~ ❑ ❑ o o ° oo ~ ~~i i " i r i r i ~ i i i i i i i i i i i i i1200.0 1600.0 2000.0 2400.D 2800.0 3200.0 3600.0 40(10.0 4400.0 5800 0 5200.(

• ISD fragmentation (top down sequencing)• Site-specific sequence modifications/comparability

Medlmmune

Product Characterisation: HDX-MS

♦ Hydrogen-Deuterium exchange mass spectrometry

♦ Valuable for evaluation of comparability— Exquisitely sensitive to the molecule (sequence), folding (subtle changes to structure /

conformational variants through eg. production, cell line evaluation), formulation, extrinsicfactors (temperature, pH, etc)

— Can define comparability /equivalence at site-specific level— Need to reduce experimental ̀ noise' (very sensitive)

♦ Use of HDX-MS to compare a marketed product with a ̀next generation'Biosuperior

Medlmmune

Product Characterisation: HDX-MS Example

Originator (Reference)

♦ Monoclonal antibody

♦ M a rketted

♦ 1.4 nM Kd

Biosuperior

♦ Same linear epitope

♦ Only 13 residues difference

♦ Different affini 35 pM K

♦ ~10-fold ore potent

♦ ...How was this achieved?

Medlmmune

~.sr~4 5k~Arg29 '` .

I .~

~ys~~ V-:

Courtesy of P. Kwong lab, NIH

Biosuperior Mutations (Crystal Structures)

- Originator (Left: heavy chain -dark green; light chain -bright green)- Biosuperior (Right: heavy chain -pale blue; light chain -cyan)- Mutations represented in yellow- Improved fit to hydrophobic pocket in epitope

I ~ I Medimmune 23

HDX: vhCDR1 Conformational Dynamics

Originator

10 s 30 s 1 min 2 min 10 min 30 min 60 min 120 minDeuterium Labelling time ~

BiosuperiorE~ 'X

Deuterium incorporation

- Biosuperior in CDR1 (s~gle site mutation) is more flexible and/or less structured

Medlmmune ~~

H DX: vhCDR2 Conformational Dynamics

Originator

10 s 30 s 1 min 2 min 10 min 30 min 60 min 120 minDeuterium Labelling time ~

Biosuperior

~ ,~~ j~ ~.

o iii o0 0Deuterium incorporation o

- Biosuperior in CDR2 (two site mutations) is identical to originator (reference)

Medlmmune 7J

3D Visualisation of HDX-MS DataHighlighting Dynamic Differences Between mAbs

Biosuperior vh (CDR1 ) Originator vh (CDR1)

- Full HDX timecourse within single image (interior T=O, exterior T=120mins)- CDR1 of Biosuperior demonstrates higher D-incorporation (more flexible)- Characterises changes (mutations) to site-localised specific information

100% 0%Centre: T= 0 min

I~I Medlmmune D incor oration Outer: T = 120 minp

Summary

♦ Characterisation of Biologics— Biologics are complex, inherent variability, process-dependent changes— Comparability forms the basis— Analytical comparability is the foundation

♦ Appropriate Analytical tools (methods)— Well-characterised, comprehensive analytical comparison to reference— Physicochemical, structural &biological properties— Sensitive to differences in defined Quality Attributes (attribute-based testing)

♦ Structural Characterisation— Recent advances in mass spectrometry, a powerful tool— Provides sensitive data for several quality attributes— Need to adapt testing strategy for demands of complex products— HDX-MS can rationalise structure-function relationships

I~ I Medlmmune Thank you !27

Acknowledgments

~~~~~~~~Lindo

♦ Emilie Solier

~~~~~~~~~~Phillips

~~~~~~~~Higazi

♦ David Lowe

♦ Mark Schenerman

Medlmmune ~~