PARTICLES IN THE BIOTECH

PRODUCT LIFE CYCLE: ANALYSIS,

IDENTIFICATION AND CONTROL

Dr Tara Sanderson, Formulation Services Manager, SGS M-Scan

2

KEY MESSAGES

Why is it important to characterise and control particles in

the product?

What different types of particles are often seen in the

product?

Summary of mechanisms of proteinaceous particle

generation

Overview of instrumentation useful for particle analysis

Higher risk areas of particle generation in a drug

development program and routes of control

Case study: Reformulation of a mAb showing significant

aggregation following shipment and temperature

excursions – useful HTS techniques to incorporate

3

WHY DO WE NEED TO CONTROL PARTICLE

LEVELS?

Potential to cause immunogenic responses

Regulators require demonstrable limitation, control and

identification of product-related impurities

Can impact product stability and shelf life

4

WHAT IS THE IMPACT IF PARTICLE

GENERATION IS NOT CONTROLLED?

Decreased shelf life and / or alternative storage has an

overall impact on cost and profitability of the drug product

Regulators will require further characterisation and

evidence of clearance

If aggregation is significant, process changes or

reformulation may be required - Time and cost

implications

Following reformulation, comparability studies are required to

determine impact on continued use of reference standard and

suitability of method validations

Significant time and cost impacts if method validations need

repeating or new Ref Std required

Additional batches / new stability studies required

5

TYPES OF PARTICLES

There are various types of particles that may be present in

biotech products

Non-Proteinaceous:

Fibres: e.g. container closure shards, shedding from filters

Particulates that shed from packaging: glass / plastics

Delamination: Plastic: Rubber:

Silicone oil from syringes:

6

TYPES OF PARTICLES

Proteinacious aggregates: visible and subvisible

Particle Size Particle Nature

~>100µm Visible particles

~1-100µm Sub-visible particles

>10nm – 1µm Oligomers

7

COMPARISON OF DP PROTEINACEOUS VS

NON-PROTEINACEOUS PARTICLES

Silicone oil particles from a syringe Protein aggregation in DP vial

8

Fragments Monomer Oligomers Subvisible Particles Visible Particles

SEC / SEC/MALS

SV-AUC

LO / MFI

NATIVE-PAGE

DLS / Nanoparticle Tracking

Analysis (Nanosight)

AF4

Visual Appearance

Resonant Mass Measurement

ANALYSIS OF PARTICLES

1mn 10mn 100nm 1µm 10µm 50µm >100µm

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Protein / protein interactions: electrostatic interactions /

hydrophobic interactions / covalent bonding from free thiols

or exposed internal thiols

Air / liquid interface / container interactions: partial

unfolding of the molecule

Protein / contaminant interactions: critical nucleus –

catalyst for aggregation formation

IN MOST CASES AGGREGATION EVENTS OCCUR AS A RESULT OF

PARTIAL CONFORMATIONAL CHANGES

MECHANISMS BEHIND PARTICLE FORMATION?

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• Control through Sequence design: Technologies available for evaluation of aggregation propensity

• Free thiols

Sequence • Low pH hold

• Filtration / column selection

• Include in-process aggregate analysis

Expression and

Purification

• Inadequate formulation design: Ensure aggregation assessed upon agitation and F/T

Formulation

• Include continued sub-visible particle testing as part of characterisation & comparability studies

• Reformulate

Characterisation

• Agitation of liquids

• Ensure shipment studies and excursions studies completed: alternative condition

• Reformulate

Shipments

• Thawing may

show particles – ensure before and after tests performed

• Filter before fill

• Reformulate

Drug Product Fill

• Route of administration: Assess with in-use studies

• Reformulate

Release

• Measure particle trends

• Characterise any particles generated

• Reformulate

Stability Studies

POTENTIAL ROUTES FOR AGGREGATION &

CONTROL

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CONTROL THROUGH FORMULATION –

CASE STUDY

Case Study: IgG1, pI 9.6, ~150 kDa, formulated in 20mM PO4, 125mM NaCl, pH.7

IgG1 candidate was found to have higher than specification aggregation upon shipment and F/T

Challenges: Time and material constraints

Aim: To reformulate to control aggregation during shipment and potential temperature excursions

Formulation Design Strategy:

Employ preformulation characterisation on control and agitated material to determine degradation pathway and choose required methods for screening approach

Employ pH screen / followed by excipient screen using agitation and F/T degradation to define the optimum formulation

Sample Treatment

To mimic problem: Samples were degraded using conditions equivalent to the worst case shipment and temperature excursions that could be observed for the product-specific shipment route:

24h agitation at ambient / 3 x cycles in thermal cycling unit from -20°C to 40°C.

Degraded protein compared to control protein

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PREFORMULATION CHARACTERISATION

Analysis Control Degraded

Primary

structure:

NR Peptide

mapping-MS

for SS-

bridges

No scrambling

observed,

expected IgG1 SS-

bridge pattern

SS-bridge scrambling

observed

Charge

profile: icIEF

pI 9.3-9.6, 6

isoforms

pI 9.3-9.6, 6 isoforms

Equivalent profile to native

Secondary

structure:

FTIR

α-helix: 0%

β-sheet: 42%

α-helix: 0%

β-sheet: 43%

Equivalent profile

to control

Overall

tertiary

structure:

Near-UV CD

Equivalent profile

to degraded

Equivalent profile to

control, but some

differences observed

~ 280nm

13

PREFORMULATION CHARACTERISATION

Conformational stability: Intrinsic Fluorescence: 9-50µl, 96 well plate format

35

30

25

20

15

10

5

0

Inte

nsity /

10

3 c

oun

ts

500450400350300250

Wavelength / nm

Optim 2

-5000

0

5000

10000

15000

20000

25000

30000

35000

40000

320 370 420

Flu

ore

scen

ce in

ten

sit

y (

au

)

Wavelength (nm)

untreated

treated

Clariostar BCM, Barycentric Mean

14

Oligomers

Analysis Control Degraded Material

consumption

Visual

appearance

Clear, colourless Opalescent,

colourless

0.5mL

SE-UPLC Monomer: 98.7%

Aggregate: 1.2%

Fragment: 0.2%

Monomer: 95.1%

Aggregate: 1.5%

Fragment: 3.4%

5 µg

96 well plate format

SV-AUC Monomer: 82.2%

Dimer: 6.5%

Trimer: 5.8%

Pentamer: 2.1%

Hexamer: 3.4%

Monomer: 80.1%

Dimer: 7.6%

Trimer: 5.2%

Pentamer: 3.3%

Hexamer: 3.8%

40 µL

1mg/mL at 400µL

PREFORMULATION CHARACTERISATION:

AGGREGATION

AU

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

0.011

0.012

Minutes

4.20 4.40 4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20 6.40 6.60 6.80 7.00 7.20 7.40 7.60 7.80 8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60 9.80 10.00 10.20 10.40 10.60

Aggregates

Fragments

280 nm Monomer

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Analysis Control Degraded Material

consumption

DLS

Peak 1

Peak 2

Mean Radius (nm): 5.5

Mean MW: 182kDa

% Intensity: 100%

ND

Mean Radius (nm): 2.7

Mean MW: 34kDa

% Intensity: 34.7%

Mean Radius (nm): 34.4

Mean MW: 13,248kDa

% Intensity: 65.3%

20µl

384 well plate format

PREFORMULATION CHARACTERISATION

Control Aggregated Control Aggregated

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PREFORMULATION CHARACTERISATION

≥2µm ≥5µm ≥10µm ≥25µm ≥50µm ≥100µm

LO Untreated 1730 515 110 15 0 0

MFI Untreated 12378 1486 187 31 0 0

LO Treated 27525 25080 19565 5340 635 10

MFI Treated 61224 61661 30396 3042 363 25

0

10000

20000

30000

40000

50000

60000

70000 N

um

ber

of

Part

icle

s p

er

mL

Particle Size (µm)

LO Untreated

MFI Untreated

LO Treated

MFI Treated

6000 / container 600 / container USP<788>

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CONCLUSIONS FROM THE PREFORMULATION

CHARACTERISATION

Conclusions from the preformulation characterisation:

Aggregation – irreversible SS-bridge scrambling occuring

but no apparent charge based changes (deamidation /

oxidation)

No significant changes to 2°, minimal 3° structure or

conformational structure changes detected

Significant changes in particle numbers, with the majority

observed higher than 2µm

Screening Tools:

SE-UPLC & DLS

In addition, for lead candidates: Particle counts, DSC,

Intrinsic fluorescence

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PH SCREEN

Buffers salts and excipients selected based on the the et the route of administration and degradation profile

pH screen: from pH 3.5 to 7.5

Buffer ions containing 125mM NaCl: citrate, acetate, glutamate, succinate, histidine (25mM)

Samples agitated and treated to 3 x F/T cycles to choose optimum pH and buffer salt.

SE-UPLC & DLS utilised: total material consumed: 160ul (1.6mg) / total preparation & screen time: 48h

Optimal pH and buffer ion: 25mM succinate, pH 6.5 containing 125mM NaCl

Excipient screen: excipients selected for conformational stability & surfactants to reduce surface charge interaction

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EXCIPIENT SCREEN

From pH Screen: 25mM succinate, 125mM NaCl, pH6.5

Design Factors for DOE:

2 % Trehalose

His, Pro, Glu, Arg, Gly: 0 – 67 mM

Tween 20, Poloxamer 188: 0.01% to 0.1%

44 combinations

Screened using SE-UPLC: 5µg / degraded sample,15h

analysis time

DLS with heat ramp from 20-60°C: 20 µl / undegraded

sample, 3h analysis time

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RESULTS FROM EXCIPIENT SCREEN

SEC

DLS (60°C)

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LEAD CANDIDATE SELECTION AND ANALYSIS

DOE Lead candidates selection:

R25: 67mM Gly, 67mM Arg, 0.01% Tween 20, 0.05% Poloxamer 188

R25b: 67mM Gly, 67mM Arg, 0.06% Tween 20

R30: 67mM Pro, 22mM Gln, 0.1% Poloxamer 188

R38: 67mM Pro, 22mM Gln, 67mM Gly, 0.1% Tween 20, 0.1% Pol188

Predictive analysis using undegraded material by intrinsic fluorescence and DSC for thermal and conformational stability

Particle counts for >2 µm particles

Temperature Ramps: 20°C – 100°C, domain Tm’s and Tm onset and Tagg compared

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Tm2 Fab

Tm1 Fc, CH2 Tm3 Fc, CH3

DSC Thermograms

LEAD CANDIDATE SELECTION

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LEAD CANDIDATE SELECTION

(TM ONSET DATA )

Optimal candidates from DSC

Ranking:

1: R25

2: R25b

3: R38

4: R30

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LEAD CANDIDATE SELECTION: INTRINSIC FLUORESCENCE & SLS: OPTIM 2 WITH HEAT RAMP

FROM 20-95°C

Tm1

Tm2

Tagg 266nm

Tagg 473nm

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LEAD CANDIDATE SELECTION:

PARTICLE COUNT RESULTS

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Ranking from Conformational Analyses:

1 R25: 67mM Gly, 67mM Arg, 0.01% Tween 20, 0.05% Poloxamer 188

2 R25b: 67mM Gly, 67mM Arg, 0.06% Tween 20

3 R38: 67mM Pro, 22mM Gln, 67mM Gly, 0.1% Tween 20, 0.1% Poloxamer 188

4 R30: 67mM Pro, 22mM Gln, 0.1% Poloxamer 188

Ranking from Particle Counts:

1 R25: 67mM Gly, 67mM Arg, 0.01% Tween 20, 0.05% Poloxamer 188

2 R25b: 67mM Gly, 67mM Arg, 0.06% Tween 20

3 R38: 67mM Pro, 22mM Gln, 67mM Gly, 0.1% Tween 20, 0.1% Poloxamer 188

4 R30: 67mM Pro, 22mM Gln, 0.1% Poloxamer 18

Final Selection: 25mM Succinate, 125mM NaCl, 67mM Gly, 67mM Arg, 0.01% Tween 20, 0.05% Poloxamer 188, pH 6.5

FINAL SELECTION

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SUMMARY

Traditional screening tools, such as SEC & DSC are useful

methods to employ in a formulation screen, but it is also

critical to ensure larger aggregates are also investigated in

combination with these.

It is also critical to use methods that allow analysis of the

full range of aggregates and subvisible particles otherwise

a significant degradation pathway may not be properly

evaluated.

Ever increasing constraints on material availability and

shorter time to decision point means that more sensitive /

high throughput instrumentation is required for effective

early screening.

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ACKNOWLEDGEMENTS

SGS M-Scan Formulation and Biophysical team:

Aoife Bolger

Marisa Barnard

Inigo Rodriguez-Mendieta

Zeb Younes

David Miles

Stella Chotou

Jon Phillips

Fabio Rossi

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Life Science Services Dr. Tara Sanderson

Formulation Services Manager

SGS M-Scan Ldt Phone: +44 (0)118 989 6940

Berlin & Taunusstein

E-mail : tara.sanderson.@sgs.com

Web : www.sgs.com/lifescience

THANK YOU FOR YOUR ATTENTION