This presentation looks at the different technologies available for detection of particles generated during the drug development lifecycle and their control using a formulation approach for particles generated as a result of agitation and freeze/thaw, events commonly observed during sample shipment and temperature excursions.
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Particles in the Biotech Product Life Cycle: Analysis, Identification and Control
1. PARTICLES IN THE BIOTECH
PRODUCT LIFE CYCLE: ANALYSIS,
IDENTIFICATION AND CONTROL
Dr Tara Sanderson, Formulation Services Manager, SGS M-Scan
2. 2
KEY MESSAGES
Why is it important to characterise and control particles in
the product?
What different types of particles are often seen in the
product?
Summary of mechanisms of proteinaceous particle
generation
Overview of instrumentation useful for particle analysis
Higher risk areas of particle generation in a drug
development program and routes of control
Case study: Reformulation of a mAb showing significant
aggregation following shipment and temperature
excursions – useful HTS techniques to incorporate
3. 3
WHY DO WE NEED TO CONTROL PARTICLE
LEVELS?
Potential to cause immunogenic responses
Regulators require demonstrable limitation, control and
identification of product-related impurities
Can impact product stability and shelf life
4. 4
WHAT IS THE IMPACT IF PARTICLE
GENERATION IS NOT CONTROLLED?
Decreased shelf life and / or alternative storage has an
overall impact on cost and profitability of the drug product
Regulators will require further characterisation and
evidence of clearance
If aggregation is significant, process changes or
reformulation may be required - Time and cost
implications
Following reformulation, comparability studies are required to
determine impact on continued use of reference standard and
suitability of method validations
Significant time and cost impacts if method validations need
repeating or new Ref Std required
Additional batches / new stability studies required
5. 5
TYPES OF PARTICLES
There are various types of particles that may be present in
biotech products
Non-Proteinaceous:
Fibres: e.g. container closure shards, shedding from filters
Particulates that shed from packaging: glass / plastics
Delamination: Plastic: Rubber:
Silicone oil from syringes:
9. 9
Protein / protein interactions: electrostatic interactions /
hydrophobic interactions / covalent bonding from free thiols
or exposed internal thiols
Air / liquid interface / container interactions: partial
unfolding of the molecule
Protein / contaminant interactions: critical nucleus –
catalyst for aggregation formation
IN MOST CASES AGGREGATION EVENTS OCCUR AS A RESULT OF
PARTIAL CONFORMATIONAL CHANGES
MECHANISMS BEHIND PARTICLE FORMATION?
10. 10
• Control through
Sequence design:
Technologies
available for
evaluation of
aggregation
propensity
• Free thiols
Sequence • Low pH hold
• Filtration /
column selection
• Include in-
process
aggregate
analysis
Expression
and
Purification
• Inadequate
formulation design:
Ensure
aggregation
assessed upon
agitation and F/T
Formulation
• Include continued
sub-visible particle
testing as part of
characterisation &
comparability studies
• Reformulate
Characterisation
• Agitation of
liquids
• Ensure shipment
studies and
excursions
studies
completed:
alternative
condition
• Reformulate
Shipments
• Thawing may
show particles –
ensure before
and after tests
performed
• Filter before fill
• Reformulate
Drug Product Fill
• Route of
administration:
Assess with
in-use studies
• Reformulate
Release
• Measure particle
trends
• Characterise any
particles generated
• Reformulate
Stability Studies
POTENTIAL ROUTES FOR AGGREGATION &
CONTROL
11. 11
CONTROL THROUGH FORMULATION –
CASE STUDY
Case Study: IgG1, pI 9.6, ~150 kDa, formulated in 20mM PO4, 125mM NaCl, pH.7
IgG1 candidate was found to have higher than specification aggregation upon
shipment and F/T
Challenges: Time and material constraints
Aim: To reformulate to control aggregation during shipment and potential
temperature excursions
Formulation Design Strategy:
Employ preformulation characterisation on control and agitated material to
determine degradation pathway and choose required methods for screening
approach
Employ pH screen / followed by excipient screen using agitation and F/T
degradation to define the optimum formulation
Sample Treatment
To mimic problem: Samples were degraded using conditions equivalent to the worst
case shipment and temperature excursions that could be observed for the product-
specific shipment route:
24h agitation at ambient / 3 x cycles in thermal cycling unit from -20°C to 40°C.
Degraded protein compared to control protein
12. 12
PREFORMULATION CHARACTERISATION
Analysis Control Degraded
Primary
structure:
NR Peptide
mapping-MS
for SS-
bridges
No scrambling
observed,
expected IgG1 SS-
bridge pattern
SS-bridge scrambling
observed
Charge
profile: icIEF
pI 9.3-9.6, 6
isoforms
pI 9.3-9.6, 6 isoforms
Equivalent profile to native
Secondary
structure:
FTIR
α-helix: 0%
β-sheet: 42%
α-helix: 0%
β-sheet: 43%
Equivalent profile
to control
Overall
tertiary
structure:
Near-UV CD
Equivalent profile
to degraded
Equivalent profile to
control, but some
differences observed
~ 280nm
15. 15
Analysis Control Degraded Material
consumption
DLS
Peak 1
Peak 2
Mean Radius (nm): 5.5
Mean MW: 182kDa
% Intensity: 100%
ND
Mean Radius (nm): 2.7
Mean MW: 34kDa
% Intensity: 34.7%
Mean Radius (nm): 34.4
Mean MW: 13,248kDa
% Intensity: 65.3%
20µl
384 well plate format
PREFORMULATION CHARACTERISATION
Control Aggregated
Control Aggregated
17. 17
CONCLUSIONS FROM THE PREFORMULATION
CHARACTERISATION
Conclusions from the preformulation characterisation:
Aggregation – irreversible SS-bridge scrambling occuring
but no apparent charge based changes (deamidation /
oxidation)
No significant changes to 2°, minimal 3° structure or
conformational structure changes detected
Significant changes in particle numbers, with the majority
observed higher than 2µm
Screening Tools:
SE-UPLC & DLS
In addition, for lead candidates: Particle counts, DSC,
Intrinsic fluorescence
18. 18
PH SCREEN
Buffers salts and excipients selected based on the
the et the route of administration and degradation profile
pH screen: from pH 3.5 to 7.5
Buffer ions containing 125mM NaCl: citrate, acetate,
glutamate, succinate, histidine (25mM)
Samples agitated and treated to 3 x F/T cycles to choose
optimum pH and buffer salt.
SE-UPLC & DLS utilised: total material consumed: 160ul
(1.6mg) / total preparation & screen time: 48h
Optimal pH and buffer ion: 25mM succinate, pH 6.5
containing 125mM NaCl
Excipient screen: excipients selected for conformational
stability & surfactants to reduce surface charge interaction
19. 19
EXCIPIENT SCREEN
From pH Screen: 25mM succinate, 125mM NaCl, pH6.5
Design Factors for DOE:
2 % Trehalose
His, Pro, Glu, Arg, Gly: 0 – 67 mM
Tween 20, Poloxamer 188: 0.01% to 0.1%
44 combinations
Screened using SE-UPLC: 5µg / degraded sample,15h
analysis time
DLS with heat ramp from 20-60°C: 20 µl / undegraded
sample, 3h analysis time
27. 27
SUMMARY
Traditional screening tools, such as SEC & DSC are useful
methods to employ in a formulation screen, but it is also
critical to ensure larger aggregates are also investigated in
combination with these.
It is also critical to use methods that allow analysis of the
full range of aggregates and subvisible particles otherwise
a significant degradation pathway may not be properly
evaluated.
Ever increasing constraints on material availability and
shorter time to decision point means that more sensitive /
high throughput instrumentation is required for effective
early screening.
28. 28
ACKNOWLEDGEMENTS
SGS M-Scan Formulation and Biophysical team:
Aoife Bolger
Marisa Barnard
Inigo Rodriguez-Mendieta
Zeb Younes
David Miles
Stella Chotou
Jon Phillips
Fabio Rossi
29. 29
Life Science Services Dr. Tara Sanderson
Formulation Services Manager
SGS M-Scan Ldt Phone: +44 (0)118 989 6940
Berlin & Taunusstein
E-mail : tara.sanderson.@sgs.com
Web : www.sgs.com/lifescience
THANK YOU FOR YOUR ATTENTION