Comparability Assessment of Biotherapeutics Using Analytical Strategies

1Arnaud Delobel, PhD – 4th edition Bioproduction of Immunotherapies – Romainville, France – September 24th 2019
Advances in analytical
strategies for comparability
assessment
Arnaud Delobel, PhD
Director, R&D
4th edition Bioproduction of Immunotherapies – Romainville, France – September 24th 2019

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Comparability studies: what, why, when?
• Process changes occur throughout the lifecycle of the product, from
early through late development stages, but also after marketing approval.
• The goal of the comparability study is to demonstrate that the products
pre and post-change are comparable
• The results can validate the use of previous safety and efficacy data to
support the next phases of development
• First comparability exercise is between nonclinical material used for IND and
Phase 1 clinical material

Regulatory framework
• FDA Guidance concerning demonstration of comparability of human biological products, including therapeutic
biotechnology-derived products (1996)
https://www.fda.gov/drugs/guidancecomplianceregulatoryinformation/guidances/ucm122879.htm
• FDA Points to consider in the manufacturer and testing of monoclonal antibody products for human use (1997)
https://www.fda.gov/downloads/biologicsbloodvaccines/guidancecomplianceregulatoryinformation/otherrecommendationsformanufacturers/ucm153182.
pdf
• ICH Q6B Specifications: Test Procedures and acceptance criteria for biotechnological/biological products. ICH
1999.
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
• ICH Q5E Comparability of biotechnological/biological products subject to changes in their manufacturing process
(2004)
http://www.ich.org/products/guidelines/quality/article/quality-guidelines.html
• EMA - Guideline on the requirements for quality documentation concerning biological investigational medicinal
products in clinical trials (2016)
http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2016/07/WC500209618.pdf

Phase appropriate comparability
Phase of development Scope of comparability Acceptance criteria
Nonclinical and Phase I
clinical study
• Release assays
• Characterisation assays
No predefined acceptance
criteria required
Between Phases 1, 2 and 3
• Release assays
• Characterisation assays
• Extended characterisation (incl. peak isolation
and characterisation)
• In-process assays and controls
• Stability studies, if appropriate
• Forced degradation studies, if appropriate
(selected conditions)
Pre-defined acceptance criteria
based on limited experience, and
limited statistical analysis
After pivotal study
• Release assays
• Extended characterisation assays (incl. peak
isolation and characterisation)
• In-process assays and controls
• Stability studies
• Forced degradation studies
Pre-defined acceptance criteria
based on statistical analysis

Which analytical techniques?

Higher-Order Structure comparison by
HDX/MS

What is HDX?
H
Addition of D2O
J. R. Engen (2009), Analytical Chemistry
10.1021/ac901154s
Most accessible H
exchanges faster
Least accessible H
exchanges more slowly
H vs. D
1 Da 2 Da
We measure H-D exchange
as the mass increase of
a protein or peptide

Hydrogen-Deuterium Exchange
Important factors that affect deuterium exchange
rates:
The amount of deuteration
on the protein backbone can be directly related to protein
structure, conformational change, and protein-protein
interaction.
Amide hydrogen in protein backbone
 The rate of exchange in solution phase is in the range that LC-MS
can measure from seconds to hours
 One NH in every AA except proline
Hydrogen to carbon
 No exchange
Side chain Hydrogen
 Exchanges too fast
 Washes off in LC-MS
D2O
added
 Solvent accessibility
 Hydrogen bonds
 Temperature
 pH

HDX-MS workflows at protein and peptide level
LC separationESI-QTOF
(Xevo G2-XS)

• Humira® (adalimumab 10 mg/mL)
▪ Heat-stressed at 60°C for 24 hours
▪ Buffer-exchanged in Na Phosphate 10 mM, 100 mM NaCl, pH 6.8
▪ Final concentration: 30 µM
• HDX
▪ 15 s, 1 min, 10 min, 1 h, 2 h in 90% D2O (same buffer)
• Quench/denaturation/reduction
▪ Na Phosphate 100 mM, pH 2.3 + 400 mM TCEP + 4 M guanidine
▪ 1:1 mixture at 1.0°C for 2 min
Experimental design

• Global folding conserved
• Almost no peptide with difference > 1 Da for single incubation times
• Some peptides with large summed differences
▪ Which amino-acids are most impacted by heat stress?
▪ For each aa: determination of mean of summed uptake across all overlapped peptides, normalized by their amount
of exchangeable H
Butterfly plot
∆𝑈𝑝,𝐻 =
∆𝑈𝑝𝑡
𝐻𝑝
∆𝑈𝑝(𝑡) = 𝑈𝑝,1(𝑡) − 𝑈𝑝,2(𝑡)
𝐻𝑝 = 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑎𝑎 − 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑃𝑟𝑜 − 1
N-glycosylation

Heatmap
E V Q L V E S G G G L V Q P G R S L R L S C A A S G F T F D D Y A M H W V R Q A P G K G L E W V S A
I T W N S G H I D Y A D S V E G R F T I S R D N A K N S L Y L Q M N S L R A E D T A V Y Y C A K V S
Y L S T A S S L D Y W G Q G T L V T V S S A S T K G P S V F P L A P S S K S T S G G T A A L G C L V
K D Y F P E P V T V S W N S G A L T S G V H T F P A V L Q S S G L Y S L S S V V T V P S S S L G T Q
T Y I C N V N H K P S N T K V D K K V E P K S C D K T H T C P P C P A P E L L G G P S V F L F P P K
P K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P R E E Q Y
N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A L P A P I E K T I S K A K G Q P R E P
Q V Y T L P P S R D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P
V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H Y T Q K S L S L S P G K
135 140 145 150
155 160 165 170 175
105 110 115 120 125 130
205 210 215 220 225
180 185 190 195 200
255 260 265 270 275
230 235 240 245 250
305 310 315 320 325
280 285 290 295 300
355 360 365 370 375
330 335 340 345 350
405 410 415 420 425
380 385 390 395 400
55 60 65 70 75
430 435 440 445 450
1 5 10 15 20 50
80 85 90 95 100
25 30 35 40 45
D I Q M T Q S P S S L S A S V G D R V T I T C R A S Q G I R N Y L A W Y Q Q K P G K A P K L L I Y A
A S T L Q S G V P S R F S G S G S G T D F T L T I S S L Q P E D V A T Y Y C Q R Y N R A P Y T F G Q
G T K V E I K R T V A A P S V F I F P P S D E Q L K S G T A S V V C L L N N F Y P R E A K V Q W K V
D N A L Q S G N S Q E S V T E Q D S K D S T Y S L S S T L T L S K A D Y E K H K V Y A C E V T H Q G
L S S P V T K S F N R G E C
251 5 10 15 20
55 60 65 70 75
30 35 40 45 50
80 85 90 95 100
135 140 145 150
155 160 165 170 175
105 110 115 120 125
205 210
130
180 185 190 195 200
214
min max∆𝑼 𝒂𝒂
CDR

Most and least impacted amino acids
C-terminus
N-glycosylation
site
CDRHC
N-terminus

Comparison of FcRn binding
by Surface Plasmon
Resonance (SPR, Biacore)

• Adalimumab (Humira®) : Anti-TNFα antibody
• 2 strategies
Binding to the target
CM5 sensor chip Protein A sensor chip
- Human antibody capture kit (10 000 RU)
- Capture Humira (~50 RU)
- Regeneration : 3M MgCl2
- 300 sec (association) – 600 sec (dissociation)
- 1:1 model
- Capture Humira (~50 RU)
- Regeneration : 3M MgCl2
- 300 sec (association) – 600 sec (dissociation)
- 1:1 model

• CM5 sensor chip
• Histidine capture kit
• Immobilisation : 8000 RU
• Regeneration : Glycine pH 1.5
Binding to Fcγ receptors
FcγR1a FcγR3a
1:1 model Steady-state affinity model

• CM5 sensor chip
• Histidine capture kit
• Immobilisation : 8000 RU
• Regeneration : Glycine pH 1.5
Binding to FcRn
Bivalent model

• 3 runs, 3 Preps/run, 2 analysts
Validation : precision

Sensorgrams similarity
• Interesting during comparability studies, stability studies or for batch-to-batch
consistency
• Comparison of kinetics/affinity is sometime difficult :
▪ Incorrect fitting model due to complex interaction
▪ Complex fitting model with several ka, kd and KD
▪ Fitting model is sometime different between normal samples and stressed samples
• Comparison of kinetics/affinity based on sensorgrams shape and not only on report
point or kinetics/affinity constants.

Sensorgrams similarity : principle

Sensorgrams similarity score calculation

Glycosylation analysis by
LC/MS

Use of RapiFluor-MS for
quick sample prep and high
sensitivity in both FLR and
MS
N-glycans release and derivatisation
Adapted from Waters poster « Rapid preparation of released N-glycans
for HILIC analysis using a novel fluorescence and MS-active labeling reagent » (2015)

Dextran ladder injection
RT > GU
Sample injection
RT-based candidate identification
MS-based identification confirmation
database
Identification principle for RFMS-derivatised glycans

Analysis of released N-glycans in HILIC mode
Acquity UPLC BEH Glycan (Waters)
150 x 2.1 mm, 1.7µm
Monoclonal antibody standard
RapiFluor-MS labeling
3 independent sample preparations

Gain in sensitivity using RapiFluor-MS vs 2-AB
FLR
Injection of 10x less material
Similar FLR signal (i.e. 10x sensitivity gain)
10x increase in MS (i.e. 100x sensitivity gain)
Low energy MSE
High energy MSE
0.6% relative intensity
RFMS
z = 3
2-AB
z = 2

Conclusion
• Comparability studies are critical in the
development of NBEs
• Reliable analytical tools are key
• New technologies can be useful to get
more information, more quickly

Acknowledgements
• Claire BUTRE
• Estelle LARA
• Fabian VANDERMEERS

arnaud.delobel@quality-assistance.be
+32 71 53 47 81
www.quality-assistance.com
Technoparc de Thudinie, 2
B-6536 Donstiennes (Belgium)
Thank you for
your attention
Any question?

Comparability Assessment of Biotherapeutics Using Analytical Strategies

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