bioworld taiwan danny chou feb292016

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….

Dissociable Dimer

Non Dissociable Dimer

Non Dissociable Aggregate

Subvisible Particles

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

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

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)

0 200 400 600

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

• 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)*

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

7169 4512 26719122

7213 2776 1823 946 3582 372

-3.E+04

2.E+04

7.E+04

1.E+05

2.E+05

1-2um 2-4um 4-6um 6-8um 8-10um > 10um > 25um

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

0 100 200 300 400 500 600

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

Control Shaken

Glass A Glass CGlass B MySafill

Shaking Experiment - Flow Microscopy

-10000

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)

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)

8-10um

10-25um

greater than 25um

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: pharmd98@gmail.com

Phone (USA): 303-483-3690

Compassionbiosolution.com

bioworld taiwan danny chou feb292016

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