Current Regulatory and Scientific Perspectives on non-viable Particles in Biotech Products Anthony Mire-Sluis Vice President, Corporate, Product and Device Quality
The Formation and Nature of Particles
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An overview of visible particles in protein products • Most, if not all, protein products contain some degree of visible particles • These visible particles can be identified as follows: – Foreign particles associated with the manufacturing process – ‘expected’ (e.g. cellulose, polyester) – Foreign particles not associated with the manufacturing process – ‘unexpected’ (e.g. hair, paint fleck etc.) – Protein particles characteristic of the product (e.g. pure protein, protein/silicon etc.) • Vials/syringes are subjected to manual or automated inspection in order to reduce the number of vials/syringes containing visible particles in released lots 3
Not all Aggregates or Particles are the Same….
P G
P G
P G
P G
P G
P G
P G
Non Dissociable Dimer
Dissociable Dimer
P P G G
P P G G
P P G G P P G G P P G G
P P G G P P G G P P G G
P P G G
P P G G
P P G G
P P G G
P P G G
P P G G
Non Dissociable Aggregate
P P G G
P P G G
P P G G
P P G G
P P G G
P P G G P P G G
P P G G
P P G G
P P G G
P P G G P P G G P P G G
P P G G
P P G G P P G G P P G G
P P G G
P G
P P G G P P P P G G G G P P G G
P P G G P P G G P P G G
P P G G
P P G G P P P P G G G G P P G G
P P G G P P G G
P P G G P P P P G G G G P P G G
P P G G
P P G G P P P P G G G G
P P G G
Subvisible Particles
P P G G
P P P P P P P P P P G GP P P G G P PP P G GP P G GP P G GP P P P P P G G G G P PG G PG PG G PG GPG G P GP G P GP G G G P P P GP G P P P GP G P P P PG G P PG G G GG G G G G G PG PG G G P P G G P P P P P P G GP P P G G P PP P G GP P G GP P G GP P P P P P G G G G G G G G GG G G G G G G G P P P P P P P P P P P P P P P P P P P P G G G G G G G G G G G G G G G G G G G G P P P P P P P PP P P P P P P P P P G G G G G G P PG G G G P PG G P PG G P PG G P PG G GP GP P P G G G G P P P P P P P P P P PG PG P P P GP G G G G G G G G G G G G P G G P G P GP GP G PG P G PG P P P P P P P P P P P P GP GP PG PG G P G P GP G PG PG G G G G G G G G G G P P P P P P P P P P P G G G G G PG P G G G PG P G G G GP P G G G GP P G G P P G G G G P P G PG P G G G GP P P P P P P P P P P P P P G G G G G G G G G G G G G G G G G G P P P P P PP P P PP P P P PPPP P P P P P P P P P P G G G G G GG G G GG G G G G GG G G G G G G G G G G G P P P P PP PP P P PP PP P P PP PP P P PP PP P P P P G G G G GG GG G G GG GG G G G G G G G G G G G G G G G G P P P P P P P P P P G G G G G G G G G G
P P G G P PP P G GG G P P G G P P G G
P P G G
P P G G
Visible Extraneous Particles
Visible Protein Particles 4
Multiple Pathways Can Lead to Protein Aggregation IgG Protein Aggregates by FPIA Imaging
NI1I2Uaggregate(A1) A2 A3 N=Native state, I=intermediate state, U=Unfolded state and A=aggregate state
Reversible and irreversible reactions can occur Solvent conditions, temperature, protein concentration, etc. Determinine which state the majority of the protein will exist Roberts et al., Biotechnol Bioeng, 98 (2007) 927-38
Manufacturing Process Steps and Product Fermentation Purification Formulation Storage Shipping Administration
Stress Conditions Heat Freeze-thaw Cross-linking Protein concentration Formulation change – pH, salt Chemical modification Mechanical Stress Surface effects Nano-particles
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Protein Particle Images Showing Distinct Morphological Features Depending on how they were Created untreated 2 – 10 µm
agitate-so+ 2 – 5 µm
pH 11
metal
2 – 20 µm
2 – 40 µm
H2O2 2 – 5 µm
stir-20h 2 – 20 µm
syringe-so-
syringe-so+
2 – 40 µm
2 – 50 µm
stir-3d
65C/pH 8.5
2 – 60 µm
2 – 130 µm
90C 2 – 220 µm
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Protein aggregates range in size (1 million fold) Definitions by size: • Aggregates (Oligomers):10 nm – 0.1 µm • Particles: - Submicron: 0.1 to 1 µm - Subvisible: 1 to 125 µm - Visible: ≥125 µm (Amgen) 10 to 150 µm - capillary clogging concern for IV dosage if >10 um
0.001µm
0.01µm
0.1 µm
1 µm
10µm
100µm
1000 µm
10,000 µm
Currently there are no generally accepted definitions for particulates; these can be defined based on either size or mechanism of formation 7
Regulatory Expectations For Visible Particles Are Changing • Pharmacopeia requirements for visible particles remain contradictory – EP Monograph on MAb - “…without visible particles unless otherwise justified” – EP Monograph on Parenteral Preparations - “…and practically free from particles” – USP-Foreign and Particulate Matter - “…is essentially free from visible particulates” •
Particulate matter is defined as ‘mobile randomly sourced, extraneous substances, other than gas bubbles, that cannot be quantitated by chemical analysis….’
– JP Foreign Insoluble Matter Test for Injections - “…and free from readily detectable foreign insoluble matters”
• The USP is currently updating Chapter ‘Injections’ and two new Chapters and ‘Visible Particulate in Injections’ – will define what ‘essentially free’ means – will be an informational chapter regarding sources of particles and detection methods 8
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Subvisible Particles were previously only addressed due to USP/EP Chapters on Particulate Matter • EP chapter on ‘Particulate Contamination: Sub-visible Particles and USP Particulate Matter in injections contained guidance on acceptability of >10 and >25 micron particles (6000 and 600 per container) • Method used routinely is light obscuration using a HIAC (High Accuracy Counter) machine
• Essentially created to control levels of extrinsic particles • Did not cover sub visible protein particles in biotechnology products • No other regulatory guidance existed for subvisible particles apart from the pharmacopoeias 9
Light Obscuration Principle
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Principle: Light Obscuration
Applicable size range: 1 – 102 m
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Regulatory Expectations For Subvisible Particles Changed and Continue to do so • In 2009, the FDA published their expectations about smaller subvisible protein particles between 1-10 microns • FDA required reporting down to 2 microns – requiring modification of current light obscuration HIAC technology, especially for comparability studies • Following a CaSSS/FDA meeting in January 2010, the agency requires both HIAC and an orthogonal method (e.g. MFI that visualizes particles) to characterize subvisible particles, unless a correlation between the two can be established • Most recently, during discussions for biosimilars, the agency is requesting analysis down to 0.1 microns using NanoSighttype technology 12
Amgen Developed A Small Volume Light Obscuration (SVLO) Method to Measure Sub Visible particles Protein Solutions • Benefits: – Test individual units (as much as possible) – Reduced sample volume 5mL •
Release and stability testing: () 5 mL/test –
•
Characterization & investigation testing: 5 mL/test
Qualification and validation: < 0.5 L
– Extend to 7 size channels: ≥ 2, 5, 10, 15, 20, 25, 50 m – Modify & improve sample handling procedure to reduce bubbles – Improve performance compared to the compendial method
• Intended use for drug products: – Release and stability testing – Process characterization – Investigations
We expect the USP SV-HIAC chapter to be compendial in late 2012 and will extend to EP and JP as part of compendial harmonization A new informational chapter on subvisible particle testing is being written 13
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Concerns Regarding Particles in Biotechnology Products
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The Potential for Extrinsic Particles to Cause Harm has not been Clearly Demonstrated at Present • There are no systematic clinical evaluations of the effects of particulates in pharmaceutical/biological products. • The route of administration contributes to the nature of potential hazards. Distribution of particles into the circulation with direct intravenous administration could potentially be associated with a greater theoretical risk of serious adverse events such as thrombosis. – It is physically possible that particles could occlude a pulmonary artery when administered intravenously.
– Particles may stimulate activation of pro-coagulant mechanisms such as platelet aggregation and clotting factors. • In contrast, subcutaneous administration would engender a greater potential concern about injection site reactions and immunogenicity. – Particles may be intrinsically irritating, resulting in a local inflammatory reaction at the site of a subcutaneous injection. – Cutaneous granulomas may be formed following subcutaneous administration of poorly soluble, persistent and undegradable particles. 15
Proposed Mechanisms for Protein Aggregate Induction of Immunogenicity Naïve T cell
Activated T cell
Helper T cell
TCR Cytokines
MHC
Resting B cell
APC
+
Activated B cell Aggregated Protein 16
Do we believe the subvisible particles of therapeutic proteins are immunogenic? One can not ignore the work carried out with vaccines, where aggregation clearly increases immune responses in humans Also there have been multiple cases where the reduction of aggregation (e.g. HGH, interferon, IVIg, HSA, Factor VIII) caused a reduction of the immunogenicity of the product in humans In addition, the analysis of the antibodies produced in response to certain products have been shown to bind to the aggregated form and not the monomeric form
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Protein Particles More Immunogenic than Soluble Proteins for T Cell Responses by ~1000X (Rock KL et al 1993) 250 Soluble OVA
CPM x 10–3
200
OVA-Fe beads
150 100 50 0 10–2
10–1
100
101
Antigen (g/ml) 18
102
103
Do we believe the subvisible particles of therapeutic proteins are immunogenic? If so is this a generic property, or specific to the protein being administered?
Non human studies have shown that aggregated material can induce immune responses, but can be confounded by the way that the aggregates are formed, resulting in subtly different types of aggregates The nature of the product and the patient population can definitely influence its propensity to induce an immune response: – – – – –
Endogenous counterpart or not How close to ‘natural’ sequence Product impurities (e.g. host cell proteins) Dosing regimen (single use or chronic, SQ or IV etc.) Patient population (immunosuppressed, immune dysfunction)
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Summary • Expectations and scientific data are continuously evolving in regards to subvisible and visible particles • One needs to differentiate between intrinsic protein particles and extrinsic foreign particles • Measurement and Characterization of particles during product development should occur to help understand their nature and any potential impact
• Strategies should then be developed that define acceptable product quality, protect patients and meet business needs
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Acknowledgements • • • • • •
Linda Narhi Raquel Dompenciel Erwin Freund Monica Pallitto Deborah Shnek Shawn Cao
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