ORTHOGONAL PARTICLE CHARACTERIZATION TECHNIQUES …...Archimedes –High Resolution SVP...

© Malvern Panalytical 2017

ORTHOGONAL PARTICLE CHARACTERIZATION TECHNIQUES FOR BIO-APPLICATIONS:

AN INTRODUCTION TO DLS (DYNAMIC LIGHT SCATTERING), NTA (NANOPARTICLE

TRACKING ANALYSIS) AND RMM (RESONANT MASS MEASUREMENT) -

Dr. Markus Epe

Field Application Scientist, Europe

[email protected]

+49 171 330 6151

mailto:[email protected]

© Malvern Panalytical 2017 15 May 20182 Title of the presentation

AGENDA

Application Examples

Technology Overview

What is Resonant Mass

Measurement (RMM) and where

can I use it?

Orthogonal technologies

Protein Aggregation Pathways

and Subvisible Particles


PROTEIN

AGGREGATION

PATHWAYS

AND

SUBVISIBLE

PARTICLES

15 May 20183Title of the presentation


Protein Aggregation

SubVis / Vis

Particles

Particle CountsProtein Structure

Hydrodynamic Size

Adapted from: Chi et al., 2003. Pharm. Res. 20:1325


PROTEIN AGGREGATES – WHY SHOULD ANYONE CARE?

• Immunogenicity• Aggregates may result in an unwanted immune response in the patient’s body

• Loss of efficacy

• Other adverse effects

• Loss of efficacy• Unfolded proteins do not have the same therapeutic effect

• Enumeration of particulates is REQUIRED by the US FDA and other regulatory

agencies• USP<787>, USP<788>, USP<790>


SUBVISIBLE PARTICLES

• Subvisible particle counts provide the most sensitive detection of protein

aggregation.

• Formation of particles (nano & micro) is an early step on aggregation

pathway.

• Even trace levels of particles can impact subsequent stability of protein

solutions; Particles beget particles.


THE PARTICLE PROFILE

• Extrinsic Particle: not part of the formulation, packaging or

assembly process. True contamination unexpected in drug

product (hair, clothing fragments, insects, etc.)

• Intrinsic Particle: non protein particle that is associated with

the container/closure (including delivery systems such as

silicone oil), formulation components, manufacturing or

packaging process.

• Inherent Particle: particles that originate from the drug

product (protein therapeutic itself or formulation components).

Narhi et al. 2015 JPS. 1899-1908


MANY FACTORS IMPACT PARTICLE PROFILES

• Manufacturing process• Pumps

• Tubing

• Disposable technology

• Configuration• Concentration

• Fill Volumes

• Container/closure/devices

• Formulation/excipients

• Shipping and handling

• Particle detection methods

• .....

“The presence of visible particulate matter is one of the top 10 reasons for the recall of parenteral products”Analysis of Aggregates and Particles in Protein

Pharmaceuticals. Jiskoot and Mahler, 2012

So far in 2016, FDA has recalled 9 products for particular content


Particle Characterization Activities

Nanoparticles (1 nm – 1 µm)• SEC-MALS

• Taylor-Dispersion Analysis (Viscosizer TD)

• Nanosight (NTA)

• RMM (Archimedes)

• Validation of product specific methods

• Product/Process Characterization

• Biosimilarity/Comparability

• Stability testing

• Forced degradation testing

• Formulation testing

• Batch release testing

• Orthogonal USP <788>, USP <787> testing

• Raw Material ID

• Customized Image analysis

• Classification of particles (silicone oil droplets

vs. protein, etc.)

• Raman chemical ID of particles (subvisible and

visible)

Subvisible Particles (1 µm – 100 µm)• Archimedes (≤ 6 µm)

• Light Obscuration (HIAC)

• Micro-Flow Imaging (MFI 5200)

• Raman Microscopy (Morphologi G4-ID)

Visible Particles (≥ 100 µm)• Visual Inspection

• Raman Microscopy

Orthogonal Technologies


RESONANT

MASS

MEASUREMENT

(ARCHIMEDES)


• „Weights“ particles in a fluid and differenciates the „heavier“ from the „lighter“ ones e.g. Protein,

Nanomaterials from Oil or lipids

• Gives you size, weight and number of particles in solution

RMM – why using this…..

› DLS can give you size range but only qualitative information

› RMM offers Quantitative information about stability of proteins which is

appreciated by authorities

› RMM helps to characterize proteins and define stability parametres

› To have a look into coating procedures of Nanomaterials

› Look into ultrafine bubbles (<1µm): recently published as having

influence on stability for proteins in solution

RMM – what it does..


OVERVIEW SIZE RANGES FOR ANALYTICS


Archimedes – High Resolution SVP Characterisation

› Resonant Mass Measurement

› High resolution particle counts /

concentration

› Protein aggregates: 200 nm – 5 µm


Archimedes : Distinguishing Protein and Silicone

›Syringes and other containers

commonly introduce oil droplets into

therapeutic protein formulations

›Presence of oil droplets can

compromise measurements of the

quantity of protein aggregates

By measuring buoyant mass, Archimedes can distinguishbetween particles are negatively buoyant, and those that arepositively buoyant.


APPLICATIONEXAMPLES


Archimedes: Characterising Protein Aggregates

›Assess impact of stress of

SubVis particles

› Impact of shear stress on

protein aggregates

›Obtain particle size

distribution profile

›Compare different stress

conditions – Force Deg.

Control

Shear-stress


1994: Betaseron, IFN-β 1b (Bayer HealthCare)

1996: Lyophilized Avonex, IFN-β 1a (Biogen Idec)

2002: Rebif, IFN-β 1a (Merck Serono) - Liquid version of Avonex

2009: Extavia (Novartis), IFN-β 1b

Case Study – Comparison of IFN-β Products for Multiple Sclerosis

From: Barnard et al, J. Pharm. Sci. (2013): Vol.102, 915-928



Total Particles

ARCHIMEDES : DISTINGUISHING PROTEIN AND SILICONE


Archimedes : Distinguishing Protein and Silicone


Total Particles Protein

Oil


ARCHIMEDES APPLICATIONS: COATING

M. R. Nejadnik, W. Jiskoot. 2014. “Measurement of the Average Mass of Proteins Adsorbed to Nanoparticle by

Using a Suspended Microchannel Resonator” Pharmaceutics, Drug Delivery and Pharmaceutical Technology


RESONANT MASS MEASUREMENT: APPLICATION SUMMARY

Determining total

counts of protein-

aggregates / particles

and in a specific size

range (200 nm-5µm)

Determining surface

coating on particles by

“weighting” them: weight

difference is due to

coating applied

Determining count

depending on positive

and negative buoyoncy:

look into stabilization of

solutions

Counting particles– analyzing mixtures of aggregates

(50 nm – 5 µm range)

Looking into Nanobubble/ ultra

fine bubble-Applications:

determine bubbles from other

Materials in solution


SUMMARY ARCHIMEDES

• Gives quantitative data about protein aggregation in subvisible range (200 nm-5 µm for

Proteins) and provides ortholog information to other technologies like Morphological Imaging

and ID, MicroFlow Imaging, Light Obscuration, NTA in addition to qualitative information from

DLS

• Small sample consumption ~100 µl

• Various applications: protein-aggregation, nanomaterials, coatings, milk skimming,

stabilisation of emulsions / nanoparticle formulations etc.

Quantifying protein aggregates and silicon oil by physical properties

ORTHOGONAL PARTICLE CHARACTERIZATION TECHNIQUES …...Archimedes –High Resolution SVP...

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