Analytical Characterisation in support of Biologics Development

Transcription

1 Medlmmune Analytical Characterisation in support of Biologics Development Alistair Kippen PhD Director, Analytical Biotechnology Biomanufacturing Technology Summit (University of Maryland & NIST) Institute Bioscience and Biotechnology Research Rockville USA, 13 June 2014

2 Overview Characterisation of Biologics Analytical Tools &Requirements Structural Characterisation (LC-MS, HDX examples) ~ Summary Medlmmune

3 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

4 Process Control Strategy (From A-Mab case study) Prior Clinical Animal In-Vitro Knowledge Studies Studies Studies Product Quality Attributes 1 Safety and Efficacy Data Criticality Assessment High Criticality Attributes 1.Quality attributes to be considered antllor controlled by manufacturing process 2. Acceptable ranges for quality attributes to ensure drug safety and efficacy Process Targets for Quality Attributes Process Development and ~~ Design Space Characterization Input Material Controls Process Controls Procedural Controls Process Parameter Controls Testing In-Process Testing Specifications c 0 ~a U ~L a~ N N U 0 a 0 c ~ ċ0 a Attributes that tlo not need to be considered or controlled by manufacturing process Characterization & I Comparability Testing Stability Low Criticality Attributes ~ Process Monitoring ~ Product Understanding Process Understanding I~ I Medlmmune Link to document: &subarticlenbr=2864

5 Process Consistency Cell Bank Cell-line, Productivity Manufacture In-Process methods, scale-up &testing Purification Process impurities, aggregates, fragments ~ S' Drug Product Reference Material Lot Release Stability Medlmmune 5

6 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 product produced 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- and post-change product are identical, but that they are highly similar" I 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 Analytical comparability is the foundation ~ I Medlmmune

7 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 N-term sequencing Raman cief :.~ FFF FTIR HP-SEC Western blotting Mass Spectrometry Activity/binding assays SDS-PAGE RP-HPLC NMR Medlmmune Purity &stability Primary sequence Glycosylation Size heterogeneity Higher order structure

8 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) Functional &binding assays Post translation modifications Glycation Glycosylation Deamidation Oxidation 0

9 Sources of Structural Heterogeneity N-terminal leader processing/terminal clipping Incorrect N-terminal Glycosylation 2~., t ~` ) ' ` y. ~~~~ I~ Medlmmune Chemical r~ Oxidation Deamidation Charged isoforms C-terminal "ragged end" His tag Proteolytic cleavage

10 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

11 ~, D Define Parameters to Monitor: - e.g. Glycosylation Identify Product Attributes (Reference) Consider Analytical Strategy Tailored method development Determine Method Requirements 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)

12 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)...~.~ E... T , :.}.~.,...~.}... : ~ 101 ~ 1~ 1~ :... 9 ~t~: i ~ ~ ~ ~ ~ V : ~ :...f 7},y ps4 i ~,~ ~ n y~l :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 product 2-4, =Fragments 5-7 =Oxidation 8 =Elongation (impurity) ~ 9 = Deamidation Medlmmune 10 =Loss of N-terminal residue (clipping) 12

13 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 Medlmmune Growth Hormones Interferons Antibodies 13

14 Glycosylation: Complex N-Glycan Pathway Fucose Galactose Mannose N-Acetylglucosamine ~ Glucose Asn-X-Ser/Thr ER Mannosidase and Glucosidases Alpha Mannosidase 1A and 1B i i ~i i 2_6 ~ ~ ~ ~ High Mannose Structures Alpha Mannosidase II rlr_nac T1 ~+ 0+ ~ ~ ~ ~ ~ II U Hybrid Structures Medlmmune I~ Tetra-antennary ~+ ~+ ~ - ~- ~ n ~I r ~ ~~ ~ I n ~I r - ~ ~ n Tri-antennary Complex Structures ~+ ~+ r - - _ i r - ~ i ~--~i n ~ ~ ~ ~--!i n Bi-antennary i i 14

15 Glycosylation Comparability: Antibodies UPLC /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 ~~ Q F, 0 pl-a ~ ~~ t-6 ~ ~p ~ G1f P~ ~~ ~P'~'~ GO nt :Pt~. pt-~. ~", ~~P'_,~ G2f Medlmmune,.o,.o mo ~o ga re,ao,.o po.,o..o...,.

16 Glycosylation Comparability: Complex Glycoprotein

17 Glycosylation Comparability: Complex Glycoprotein 2AB LC-MS characterisation of glycans obo611 MEDI-524_PIC_+P 349 (7.384) Cm (344:362) _ 1 27$ I aa~~ ~ ~,tv:, " Molecular ion GOF / 19~G GOF-GN Fragment ions ~i~ ~ ~8 138D.2087 X380. M2F+GN ss 5 276D Bz M 3 F 6~ i 48 M2F ~ as Na adducts _ ~ K adducts D D D D i

18 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] ~ «1 25,7 i 90 ~ G2F+S ~' so,ssz.s ~~~~ 26Q6.4 G2F 78 G 1 F ' ' GOF 2 ao.' 50, ~.s G3F+S 1 T q~ ao ~.._ ' G2F+S2 1 G 3 F ~ fi iss~.a as.2 9F94S l G3F+S2 san.l G3F+S 1141 I I1 29.8'! ~ Mass ~mlz} JOb: 223D9, N-Qly, , Op MC D: fi N-GFy.dat Acquired: 14:18:00, March 30, 2011

19 Complex Glycoprotein analysis MALDI-MS vs Q-ToF-MS.~... r.,n Q-ToF Synapt G2 Q-TOF-MS spectra of Glycoprotein X The right tool! Compound A, Calibration using cluster of Protein A. Matrix Sinapinic Acid. ~~R q xt04~ '~ ~'4 S 1.2 m [M+H]' i 1.0 '' I D.8 [M+2H]~ ~ D.6 ~ t4 i 1~ ~ h 0.2,` y~j~, g ~,i~ m/z Bruker Ultraflextreme MALDI-TOF MS spectra of Glycoprotein X Medimmune

20 Complex Glycoprotein analysis Sequence integrity confirmation From MALDI-TOF (single broad peak) use ISD for site-specific sequence information z+2 ~ tpi c 12 c 15 c 18 c11 c14 c17 c70 c13 c16 19 c Y1 20 c 24 c31 c21 z+22b ~~ a31 c23 c26 ~ I ' c 30 c 25 c 28 c 27 ~~c29 c Match Firors ~ MS,'MS Fagmerrts I MSMS r'~nalysis ~ RIASError: 0.03 Da Imear regresswn~. f(x1 = ~ X Coneletlon quotient: Y~ c33 c35 32 c 3q ~ z+2 38 ~~~~ ~ ~ 4000 ~~r~~ ~ o o o o ~ ~ c 44 y 47 z+2 49 c a3 c as z;2 so mlz ~i i " i r i r i ~ i i i i i i i i i i i i i D ( ( a ISD fragmentation (top down sequencing) Site-specific sequence modifications/comparability Medlmmune

21 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, extrinsic factors (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

22 Product Characterisation: HDX-MS Example Originator (Reference) Monoclonal antibody M a rketted 1.4 nm Kd ~.sr~4 5k~ Arg29 '`. I.~ Biosuperior Same linear epitope Only 13 residues difference Different affini 35 pm K ~10-fold ore potent...how was this achieved? ~ys~~ V-: Courtesy of P. Kwong lab, NIH Medlmmune

23 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

24 HDX: vhcdr1 Conformational Dynamics Originator 10 s 30 s 1 min 2 min 10 min 30 min 60 min 120 min Deuterium Labelling time ~ Biosuperior E~ 'X Deuterium incorporation - Biosuperior in CDR1 (s~gle site mutation) is more flexible and/or less structured Medlmmune ~~

25 H DX: vhcdr2 Conformational Dynamics Originator 10 s 30 s 1 min 2 min 10 min 30 min 60 min 120 min Deuterium Labelling time ~ ~,~~ j~ ~. Biosuperior o iii o 0 0 Deuterium incorporation o - Biosuperior in CDR2 (two site mutations) is identical to originator (reference) Medlmmune 7J

26 3D Visualisation of HDX-MS Data Highlighting 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 min p

27 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

28 Acknowledgments ~~~~~~~~Lindo Emilie Solier ~~~~~~~~~~Phillips ~~~~~~~~Higazi David Lowe Mark Schenerman Medlmmune ~~