CMC strategy forum Japan

Evaluation of protein aggregates/subvisible particles

in therapeutic protein injections

National Institute of Health SciencesDivision of Biological Chemistry and Biologicals

Hiroko Shibata

Risk source Event Consequence

Protein aggregates/subvisible particles

Production of anti-drug antibodies

Influence on PKInfluence on PD Type I AllergyType III Allergy

Protein aggregates/subvisible particles ⇒one of the factors related to immunogenicity⇒mostly identified as a CQA for therapeutic protein

Analytical procedures for protein aggregates/subvisible particlesRelevance of protein aggregates/subvisible particles to immunogenicity

Issues

2

Immunogenicity and protein aggregates/subvisible particles

Current situation for the evaluation of protein aggregates/subvisible particles

100µm10µm1µm100nm10nm1nm

JP Insoluble particulate matter testfor DP, ≥ 10µm, ≥25µmLight obscuration, Microscopy

JP Foreign particulate matter testfor DP, ≥ 100µm???Visual inspection

Size exclusion chromatographyImpurity test for DS, SP

protein aggregates/subvisible particles

Standardization of evaluation methods for protein aggregates/subvisibleparticles that are not determined by the current specification test

Analytical methods; Flow ImagingResonant Mass MeasurementNanoparticle Tracking AnalysisLaser DiffractionCoulter counter, Flow Cytometry

Flow Imaging (FI)Light Obscuration (LO)

Size and Number of particles are determined by value and number of pulse signal

Protein aggregates are transparent and have low refractive index compared to polystyrene standard particles that used for the calibration of LO, thus LO does not allow accurate quantification of protein aggregates.

Size, Number and Shape of particles are determined by capturing an each particle in a flowing sample

Particles are defined based on contrast between particles and dispersion liquid, thus FI has high sensitivity to detect nearly transparent particles.

Issues for the determination of particulate matter in therapeutic protein injections

USP Stimuli Article 36(3) A Light-obscuration Method Specific for Quantifying Subvisible Particles in Protein TherapeuticsUSP Stimuli Article 36(1) Flow Microscopy : Dynamic Image Analysis for Particle Counting

Flow imaging (FI) method has emerged as a powerful strategyfor analysis of subvisible particles.

・FI method has not yet been standardized.・FI method has not yet been listed in any compendium.

However…

We conducted collaborative study in attempt to assessthe applicability of standardization of FI method.

・The detectability for highly transparent particles.

・The difference of analytical performance between

Inter-

Intra-

Method (LO, FI)

Laboratory

Instrument (FlowCam, MFI)

To elucidate…

Objective of collaborative study

Shared samples : three subvisible particle preparationsNPAS 90; Heat-induced IVIG (Sanglopor) at 90 ºCNPAS 70; Heat-induced IVIG (Sanglopor) at 70 ºC*These samples were suspended in 0.5% HPMC solution.ETFE; Ethylene-tetrafluoroethylene particles. Sample was kindly provided by Dr. Dean C. Ripple

LaboratoriesLO; 12 laboratories, FI; 10 laboratories

Replication4 times × 3 days / laboratory

Study Design

Cooperative determination of particle sizes and counts in shared samples using FI and LO

Representative digital images of particles IVIG heated at 90 ºC IVIG heated at 70 ºC Not proteinaceous

Dark particles Transparent particles Dark and transparent particles

Transparency：NPAS 70 > ETFE > NPAS90Aspect ratio：NPAS 70 > ETFE > NPAS90

There were some dataset with high variance.

Overall, most of the medians of particle count in each laboratory were within 80-120% of the Lab A-F or Lab G-J.

✓

✓

✓

✓

✓

✓

✓Consistent

MFI：6 Labs（A－F）FlowCam：4 Labs（G－J）

Inter-laboratory comparison of particle counts : Flow imaging

Inter-laboratory comparison of particle counts: Light obscuration

KL-04：6 Labs (A,C,F,I,J,K)HIAC： 3 Labs (B,E,L)Others：3 Labs

Overall, there were no remarkable differences in particle counts among instruments and laboratories.

There were some dataset with high variance.

* *

*

*

*

*

*

*

* Significant difference

Inter-instrument comparison of particle counts: FlowCam vs MFI

Difference in particle counts Between FlowCam and MFINPAS 70 > ETFE > NPAS90

Transparency or Aspect ratioNPAS 70 > ETFE > NPAS90

Comparison among methods (FI vs LO)

Difference in particle counts Between FI and LONPAS 70 > ETFE > NPAS90

Transparency or Aspect ratioNPAS 70 > ETFE > NPAS90

Distribution of subvisible particles analyzed by MFI, FlowCam, and LO.

↓

↓

↓

Conclusion Comparison among laboratories

Our collaborative measurements using FI show that consistent results were obtained by the instrument from the same manufacture in all samples, and there were no apparent differences among laboratories except for several results with high variability or particle count.

Comparison between manufactures (MFI vs FlowCam)FlowCam exhibited the results similar to those of MFI, although FlowCam measured a relatively higher number of particles compared to MFI.

Comparison between methods (LO vs FI)LO underestimated the counts and size, especially for highly transparent and irregularly shaped particles.

Considering our data, setting FI as a release test would be feasible to precisely analyze the sizes and counts of particles in therapeutic protein injections. While further discussion is necessary, it is conceivable that FI can be listed in a pharmacopoeia as a test method.

Masato Kiyoshi, Hiroko Shibata*, Akira Harazono, Tetsuo Torisu, Takahiro Maruno,Michiko Akimaru, Yuuka Asano, Mai Hirokawa, Keisuke Ikemoto, Yukari Itakura,

Takafumi Iwura, Aya Kikitsu, Takashi Kumagai, Naoki Mori, Hiroaki Murase, HirotakaNishimura, Atsushi Oda, Taiichiro Ogawa, Takuma Ojima, Shinji Okabe, ShuntaroSaito, Satoshi Saitoh, Hiroyuki Suetomo, Kazuhiro Takegami, Momoko Takeuchi,

Hidehito Yasukawa, Susumu Uchiyama, Akiko Ishii-Watabe

Collaborative Study For Analysis Of Subvisible Particles Using Flow Imaging andLight Obscuration: Experiences In Japanese Biopharmaceutical Consortium

Journal of Pharmaceutical Sciences

Accepted Date: 7 August 2018

AMED-HS: Challenges for the future

Toward standardization of flow Imaging

Ability of flow imaging method to distinguish

protein aggregates from silicone oil particles

System suitability

Validation of analytical procedures

Acceptance criteria

Other methods…

Current situation: FDA Guideline 2014Immunogenicity Assessment for

Therapeutic Protein ProductsB. Product-Specific Factors That Affect Immunogenicity

Product Aggregates and Measurement of AggregatesRecommendation

Strategy to minimize protein aggregate formation

Assessment of the range and levels of

subvisible particles (2 – 10µm) initially and during shelf life

Risk assessment of the impact of subvisible particles

on the clinical performance

Characterization of particles in smaller size ranges (0.1 – 2 µm)

Analytical methods; orthogonal techniques, quantitative method

However…. FDA does not recommend any specific techniques/methods in this guideline

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100µm10µm1µm100nm10nm1nm

Size exclusion chromatography (SEC)Protein aggregates/subvisible particles

Field Flow Fractionation（FFF）

Analytical Ultracentrifugation（AUC）

Dynamic Light Scattering（DLS）

Flow Imaging（FI）

Laser Diffraction（LD）

Nanoparticle Tracking Analysis（NTA）

Resonant Mass Measurement（RMM)

No single method can cover full range of particulate matter size⇒ Need standardized evaluation method

with combination of some of these techniques

JP Insoluble Particulate Matter testLight Obscuration （LO）

Electrical Sensing Zone （Coulter principle）

Flow Cytometry

Issue for evaluation of protein aggregates/subvisible particles

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Acknowledgement

NIHSDr. Akiko IshiiDr. Akira Harazono Dr. Masato Kiyoshi

Osaka UniversityDr. Susumu UchiyamaDr. Tetsuo Torisu

AMED-HS members (2017)AstellasChugaiDaiichi SankyoJCRKyowa Hakko KirinMitsubishi TanabeMochidaNippon KayakuOnoTakedaToray Research CenterU-Medico

Thank you for your attention