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Aggregation Properties of Therapeutic Proteins Revealed by
New Analytical Methods
Danny K. Chou, PharmD, PhD
President, Compassion BioSolution, LLC
Biologics world Taiwan 2016
25th, February 2016
Presentation Outline
• How protein aggregation occurs and the dominant forces that control the formation of aggregates and particulates.
• How does protein aggregation affect development and commercial viability of biopharmaceuticals
• How do you monitor and control formation of aggregates and particles? What is the state-of the-art analytical approach?
• Why proper integration of formulation, container-closure, and analytical technology is essential to the success of a biologics development program.
Success Drivers in Biologic Drug Development
• Thanks to the favorable clinical profile of biologics
and increasing market demand the growth in
development of biopharmaceuticals is already
surpassing that of conventional drugs.
• Along with this trend the challenges of
biopharmaceutical development has become a
significant barrier to entry and sustainable
commercial success
• Commercial success requires both innovative
technical development and management of unique
challenges associated with the nature of biologics
• One of the these key technical challenges is protein
aggregation
Why Are Proteins so Difficult to Develop?
Putting Things Into Perspective With Respect
to Size of Biologic Molecules
*NEJM 2011
What is Protein Aggregation and Why is
it Important?
• Protein aggregates: “High molecular weight
proteins composed of multimers of natively
conformed or denatured monomers”
(Rosenberg, 2006)
• Aggregates can reduce biological activity, or
worse, induce immune response, but the
mechanism is still not very well understood
• Immunogenicity is a major product SAFETY
concern
Protein Aggregation – Mechanisms
• Protein therapeutics are highly complex in terms of size, structure
and function.
• Structural flexibility presents a higher risk for physical instability as
well as a major regulatory concern on product quality and safety.
Krishnamurthy et al. BioProcess International, 2008
Phenomenon of Protein Aggregation –
What Do We Know at the Present?
• Both conformational and colloidal stability play a role
Chi et al., Pharm. Res. 20:1325, 2003
Contributing Factors to Formation of
Soluble Protein Aggregates and Particles
Bioprocessing from start to end
Physical/chemical Stresses: pH, ionic strength, temperature, chemical modification, light, agitation, mechanical shock, freeze-thaw, etc.
Air/Solid-Liquid Interfaces: Protein contact with tubings, pumps, pipes, vessels, filters, columns, etc.
Foreign Particles: Stainless steel, glass, plastic, rubber, tungsten, silicone oil, etc.
Fermentation/cell culture
PurificationFilling
Packaging Shipping Storage
Administration
Not all Aggregates or Particles are the same….
PG
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Dissociable Dimer
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Non Dissociable Dimer
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Non Dissociable Aggregate
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Subvisible Particles
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Visible Protein ParticlesVisible Extraneous Particles
Why is protein aggregation relevant to
biopharmaceutical development and
manufacturing?
• Protein aggregation has long been suspected as a having a role in safety and efficacy of biologics
How subvisible particles became a key focus for
regulators throughout the globe
Subvisible Particles- a highly visible topic
Orthogonal Techniques that Cover Various Particle Size Ranges
0.001 um 0.01 um 0.1 um 1 um 10 um 100 um 600 um
SEC, AUC
DLS Flow microscopy
HIAC / Light Obscuration
Visual
1 nm 10 nm 100 nm 1000 nm
Subvisible aggregates
Silicone droplets
Nano-emulsions &
suspensionsVisible aggregates
Emulsions & suspensions
Glass, rubber, plastic, etc.,
particles
RMM
Nanosight
Extended characterization per USP<1787>
Technique Size Range
Light obscuration 2-300 um
Electrical senzing zone (Coulter) 0.4-1600 um
Laser diffraction 0.1-3500 mm
Light microscopy 0.3 um to 1 um
Flow imaging analysis 1 um-100 um for
size distribution;
5-100 um for
morphology
Electron microscopy (EM): Scanning EM,
scanning transmission EM, and transmission
EM
A to mm
Fourier Transform Infrared (FTIR)
microspectroscopy
10 um to 1 mm
Dispersive-Raman microspectroscopy 0.5 um to 1 mm
Electron microscopy (EM) with energy-
dispersive X-ray spectrometry (EDS)
A to mm for
imaging
Size and distribution
Size and morphology
Characterization
Criteria for Ideal Methodology
• Detects SVPs ranging from 0.1 – 100 µmSize Range
• Ideal if it allows for validation and setting acceptable limits.
Particle Count
• Protein Aggregate vs Silicone Oil Droplet vs External Inclusions (Metal, rubber etc.)
Particle Type
• Recording of particle image; Provision for visual identification and analysis.Image of Particle
• Stable Aggregates vs Dilution-dependent Transient aggregates.
No prior sample manipulation
While This Field Continues to Evolve
There is an Opportunity in Front of Us
• SVP testing can be applied during process development to optimize processing conditions and reduce impurities
• Proper integration of orthogonal technique is a powerful way to improve formulation robustness and assess drug product- delivery device compatibility
Case 1: Use of Subvisible Particulate (SVP)
Analysis During Process Development
Background:
• IgG monoclonal antibody (mAb A)
• Manufactured at 2k L scale for Phase I and Phase II
clinical testing
• 3 Column Purification Platform
• Affinity, Cation, Anion
• Virus filtration in final position prior to UF/DF
Validation Study of VF Performance (mAb A)
0
100
200
300
400
500
600
0 200 400 600
VP
ro F
lux
(LM
H)
Volumetric Throughput (L/m2)
1% XMuLV Run 11% XMuLV Run 2Decoupled No Virus1% MVM Run 11% MVM Run 2Coupled No Virus
• Achieved only 53% of target throughput
• Only non-spiked coupled train (with pre-filter) exceeded target
• Decoupled trains displayed both cake formation and pore plugging type fouling
• Visible particle formation post transfer of feed into reservoirs
• Conclusion: FAIL. Requires revalidation
Concentration mg/mL 7.9
*Target Throughput L/m2
(g/m2) > 318 (2510)
**Achieved Throughput L/m2
( g/m2)168
(1330)
* Target based on production scale** Achieved based on worst VF performance
Key Goals for Viral Filtration Validation Study of
mAb A
• Overcoming filter fouling
– Minimize factors linked to particulate formation
• Optimize strategies for handling VF load when conducting VF validation
• Measuring sub visible particles (SVP)
– Establish SVP analytical techniques and apply to VF loads
– Correlating SVP formation and distribution as function of VF handling practices
Detection Method: Flow Microscopy
• Utilizes optical system similar to microscope
• Detects particle sizes of 1 μm to >100 mm diameter
• Captures real time images of particles in fluid as it passes through a flow cell
• Distribute sizes based on equivalent spherical diameter (ESD) or area based diameter (ABD)*
*Fluid Imaging Technologies (© 2010). Imaging Particle Analysis – Technology. Retrieved September 4, 2013, from
http://www.fluidimaging.com/
EXPERIMENT “Gently” handled VF Load was tested directly on FlowCAM “Non Gentle” handled VF load experienced turbulence (3x pour and swirl) to mimic validation handling practices, then tested on FlowCAM
FlowCAM Data Acquisition : How is it Done?
Quantitative Particle Detection of mAb A
During Viral Filtration
• Particle concentration increased as a result of turbulent, “non gentle” conditions• Handling conditions were linked to membrane fouling; therefore new methods
need to be developed to minimize particle formation in an effort to increase process efficiency while reducing cost
222761
61594
7169 4512 26719122
1005
69267
7213 2776 1823 946 3582 372
-3.E+04
2.E+04
7.E+04
1.E+05
2.E+05
2.E+05
1-2um 2-4um 4-6um 6-8um 8-10um > 10um > 25um
Part
icle
s p
er
mL (
P/M
L)
Particle Size ( μm)
Non Gentle
Gentle Handling
1-2 μm 2-4 μm 4-6 μm 5-8 μm 8-10 μm >10 μm >25 μm
Orthogonal Particle Detection Method: DLS
Dynamic Light Scattering
• DLS (dynamic light scattering, also known
as quasi-elastic light scattering), uses light
scattering of a laser beam and very fast
decay measurement to determine the
hydrodynamic radius and polydispersity of
the species present.
• It can measure radii down to 0.5 nm and
determine radii for two populations with
radii at least 5-fold different, with best
results if 10-fold different
Kuebler S. “Characterizing stable protein formulations.” Genetic Engineering & Biotechnology News 27.20 (2007). Accessed September 4,2013 from http://genengnews.com.
EXPERIMENT “Gently” handled VF feed was loaded directly onto microplate for DLS testing “Non Gentle” handled VF load experienced turbulence (3x pour and swirl) to mimic validation handling practices prior to testing Over the duration of one hour, DLS data was collected
“Gently” handled samples
5-6 nm
“Non-Gently” handled samples
5-6 nm
Aggregates of mAb
• DLS can be used as an orthogonal approach for SVP detection
• Detected differences in handling conditions (5-6 nm mAbs vs. large particles)
• Non-monomer particles were detected as a result of “non gentle” conditions
• 20nm VPro pore size, may be increasingly susceptible to membrane fouling
due to handling conditions
Qualitative Particle Detection of VF Load
Impact of Process Change on Process
Efficiency and Cost for mAb A
• By modifying formulation of in-process material and handling techniques, particle formation was mitigated, enabling flux to maintain > 100 LMH and target throughputs were exceeded • With 1% spikes of both feeds, was able to show adequate log clearance
0
50
100
150
200
250
300
350
400
450
0 100 200 300 400 500 600
VP
ro F
lux
(LM
H)
Volumetric Throughput (L/m2)
1% MVM N=1
1% MVM N=2
1% XMuLV N=1
1% XMuLV N=2
Concentration mg/mL 7.0
*Target Throughput L/m2
(g/m2)> 318 (2510)
**Achieved Throughput L/m2
( g/m2)517
(3620)
* Target based on production scale** Achieved based on worst VF performance
Case 2: Use of SVP Analysis During
Combination Product Development
• Drug product-container compatibility is a critical
factor in the successful development of biologic
combination products
• SVP testing was conducted to evaluate stability of
a high concentration mAb formulation (mAb B), in
different brands/types of pre-filled syringes (PFS)
Glass PFS Plastic PFS
Brand Brand X MySafill®
PackagingMaterial
Glass(borosilicate)
Plastic(Cyclo Olefin Polymer )
Pros Scratch resistant, Transparent
Low protein adsorption*,Tungsten not required,
Retractable needle,
Cons Breakage, Tungsten,
Alkali oxide, Negatively charged Surface
Higher leachable profile,More easily scratched
*In selected cases
Glass PFS and MySafill® (a new polymer PFS with
integrated safety feature) were directly compared with respect to their impact on stability of mAb B
What is Different about MySafill®?
Same injection technique as the conventional pre-filled syringe; retraction of needle is activated by pressing the plunger rod after completion of injection
Courtesy of Medical Chain International
Shaking Experiment – Visual Observation
Active
Control
Glass PFS A
Glass PFS B
Glass PFS C MySafill
Active
Shaken
Glass PFS A
Glass PFS B
Glass PFS C
MySafill
Shaking Experiment – SEC-HPLC
96.0
96.4
96.8
97.2
97.6
98.0
98.4
98.8
% M
AIN
PEA
K
Control Shaken
Glass A Glass CGlass B MySafill
Shaking Experiment - Flow Microscopy
-10000
0
10000
20000
30000
40000
50000
60000
70000
PA
RTI
CLE
CO
NC
. (P
AR
TIC
LES/
ML)
Flow Microscopy (Average of 2 Consecutive Runs)
Control Shaken
Glass PFS A Glass PFS B Glass PFS C MySafill PFS Placebo
Morphology of Particles is Important
for IdentificationGlass PFS Agitated in bad formulation Glass PFS Agitated in good formulation
MySafill Agitated in bad formulation MySafill Agitated in good formulation
Effect of Stress Method on Aggregate
MorphologyImages of mechanical stress-induced
particles in IgG solution
Images of thermal stress-induced
particles in IgG solution
Container Material & Formulation Impact on
Subvisible Particle Formation upon Agitation
(Flow Microscopy)
0
2000
4000
6000
8000
10000
12000
14000
16000
BD Glass Syringe (noagitation)
BD Glass Syringeagitated (No PS 20)
Glass Syringe agitated(with PS 20)
MySafill (no agitation) MySafill agitated (noPS 20)
MySfaill agitated (withPS 20)
2-4um
4-6um
6-8um
8-10um
10-25um
greater than 25um
Part
icle
Co
nce
ntr
atio
n
(par
ticl
es/m
L)
Glass, No Agitation Glass-PS, Agitation Glass+PS, Agitation Plastic, No Agitation Plastic-PS, Agitation Plastic+PS, Agitation
Proper integration of formulation, delivery device, and analytical technology
is essential
The ‘Triad’ of Biologics Drug
Product Development
Formulation
To AchieveStability and
Process
Efficiency
New Analytics
Enable
True Product
Characterization
Innovative
Delivery
Technology
IV to SC
Improved patient care and chance of commercial success
Current Situation
The Future of Biologics Marketplace The ‘pinnacle’ is not as hard to reachas it may seem
The Best Selling Brand of a Biologic in AsiaLack of proper integration in biologic drug product development will manifests itself
Conclusions
• New technologies are enabling better understanding of protein aggregation pathway as well as early detection, which is the first step towards effective control of this key quality attribute
• Protein aggregate/subvisible particle analysis should begin ‘upstream’ of drug product development (stable drug product begins with stable drug substance)
• One can speed up drug product development and ensure long term sustainability by optimizing biologics formulation and integrating it with delivery device and analytical technology.
• The increased regulatory and market expectation for high quality biopharmaceuticals creates opportunities for those who fully embrace drug product formulation, analytical, and drug delivery expertise. These are the backbone of every successful commercial product!
Thank you!
Compassion BioSolution, LLCDanny K. Chou, PharmD, PhD
E-mail: [email protected]
Phone (USA): 303-483-3690
Compassionbiosolution.com