Aggregation Analysis of

Innovator and Biosimilar

Therapeutic Proteins using FTIR

Presenter’s Name: 18 pt
Presenter’s Title: 16 pt
4/8/23

DE60292147
 FTIR spectroscopy
Basics
The electromagnetic spectrum just beyond the visible portion of light

IR light causes molecular vibrations

– Each chemical bond naturally vibrates at a specific characteristic frequency
– IR light is absorbed when it matches the vibrational frequency of a chemical bond

2 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
FTIR spectroscopy
Benefits

Sampling
– Small amount of sample required
– No sample preparation (in most cases) and no consumables
– Non-destructive
– Versatile: Can be applied to solids, liquids and gases
Usability
– Fast and easy – Results in seconds
– Simple to operate
– Economical
– Flexible: Can be used for qualitative (Identification) and
quantitative analysis (Quantification)

3 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
The Cary 630 FTIR
Rapidly delivers qualitative and quantitative information
– Material identification
– Quantification
– and many more applications ….

Cary 630 FTIR and MicroLab Software

– Intuitive and easy-to-use
– Simple picture guidance through the entire analytical
workflow
– Instantly receive color-coded, actionable results
– Meets the performance criteria of global
pharmacopeia
– Supports US FDA 21 CFR Part 11, EU Annex 11, etc.
compliance

4 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Compact, intuitive, reliable
There is no FTIR spectrometer like the
Cary 630 FTIR

DE60292147
 Cary 630 FTIR is used in pharma labs
A compact yet powerful tool
Versatile
– Modular design to be prepared for a wide range of
samples and applications
– Interchangeable sampling accessories
– No alignment required

Cary 630 engine Reliable

DialPath TumblIR – Field-proven, rugged components, non-humidity
sensitive ZnS optics
– Warranty 10-10-5

Pharmacopeia ready
Diffuse Reflectance Transmission ATRs – Meet global regulatory requirements
– Optional 21 CFR Part 11 data integrity software
… and many more
package available

6 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Agilent’s Cary 630 FTIR
The modular design

Click it together
– Sampling modules simply clicked to the front
– No need to power down to switch between modules
– Permanently aligned optics
– Zero-user alignment
– Software automatically recognizes the attached
module and sets the correct parameters
– Specifically engineered for optimal performance and
intuitive handling

7 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Cary 630 FTIR and MicroLab Software
The full solution- Designed for non-spectroscopists

The intuitive and easy-to-use design of the Cary 630 FTIR is matched by the Agilent MicroLab software

Learn the software in minutes

Step-by-step guidance using instructive
pictures and an intuitive software design
allow easy navigation through the entire
workflow, for less training and fewer user
mistakes.

Instantly receive color-coded, actionable results

8 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Applications
(Bio)Pharma

Applications
– (Raw) material identification (excipients, APIs, preparations, packaging)
– QA/QC of finished goods (isomer assays, polymorphism, batch-to-batch
comparison, supplier assessment, intermediates)
– Quantification of API in formulations (assays following monographs)
– Identifying counterfeits
– Pharmaceutical Packaging Material QC (USP 661.1)
– Quantification of cannabinoids

9 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Be confident
The Cary 630 and MicroLab Pharma

Instrument Performance
The Cary 630 meets the performance requirements of
global pharmacopoeia such as the European, US,
Indian, and Japanese Pharmacopoeia.
White Paper: 5994-2339EN

Data Integrity
The MicroLab Pharma software features built-in
technical controls that ensure the security of your
data, control access, and facilitate compliance as
defined by US FDA 21 CFR Part 11, EU Annex 11
and similar national electronic record regulations.
White Paper: 5991-6024EN

10 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Application in (Bio)Pharma
Measurement of rituximab aggregation in concentrated samples using the Agilent Cary 630 FTIR spectrometer

11 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Aggregation of Monoclonal Antibodies (mAbs) at High Concentrations
Protein aggregation
– Occurs during manufacturing or storage
– When exposed to stressful conditions such as low
pH, high temperature, high concentration
– Impacts their efficacy, potency and safety

Monitor protein aggregation

– Size Exclusion Chromatograph (SEC) is gold
standard – but samples need diluting before
analysis
– FTIR is suitable for aggregation studies in high
concertation samples
– App note demonstrates the use of the Cary 630 FTIR
to analyze aggregation of Innovator and Biosimilar
mAbs at 50 mg/mL – quickly and easily without
dilution

12 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Experimental
1) Materials & sample preparation

Innovator and Biosimilar rituximab purchased locally

mAb samples were concentrated using Vivaspin 500 spin columns

(10kDa MWCO; Sartorius)

Concentrations of mAbs measured Steps:

Extinction coefficient of 1.7 mL.mg-1.cm-1 used to mAbs concentrations
at 280 nm using Cary 60 UV-Vis Estimated concentration of Innovator and Biosimilar rituximab is 50mg/mL

Formation buffer:
5.35 mg/mL sodium citrate monobasic pH 6.5 (CAS 18996-35-5)
Formulation buffer used for kinetic studies 9 mg/mL NaCl
0.7 mg/mL polysorbate 80 (CAS 9005-65-6, Sigma-Aldrich)

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Experimental
2) Workflow for the analysis of innovator and biosimilar rituximab

Thermal stress Sample analysis Data analysis

Parameters Value
Spectral Range (cm-1) 4000 to 650
Background Scans 140
Sample Scans 140
Resolution (cm-1) 4
Zero Fill Factor None
Apodization Triangular
Phase Correct Mertz
Aggregation Pipette 10 µL of the thermally Sampling Technology ATR
Incubate each sample for 15 min stressed mAb onto the Cary 630
at temperatures ranging from 20 ATR sample interface
Use MicroLab Expert software
to 90oC
(version 1.1. 0.1) for data
Use the MicroLab software for
analysis
Aggregation kinetics measurements
Incubate at 60oC temperature and
take measurements after 0.5, 1,
2, 3, and 4 h

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Results
Temperature induced aggregation
– Temperature-dependent changes to the secondary structure of the innovator and biosimilar were compared
by FTIR at 25°C & 75°C
– Amide I band coming from intramolecular b-sheets secondary structure of innovator mAb shifted from
1,638 cm-1 (25°C) to 1,616 cm-1 (75°C) indicating aggregation (Figure A)

Second derivative of IR spectra show significant change in Amide I band due to aggregation (Figure B)

15 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Results
Melting temperature (Tm) of mAbs
– Ratio of absorbance at 1,616 cm-1 and 1,638 cm-1 for innovated and biosimilar were compared as
temperature increased
– Tm is defined as crossover point at which absorbance at 1,616 cm-1 becomes greater than 1,638 cm-1

Abs (1616 cm-1)/Abs (1638 cm-1)

1.4
1.3 Innovator Biosimilar

1.2
1.1
Ratio

1
0.9
Tm Innovator – 70.2oC
0.8

0.7
Tm Biosimilar – 71.8oC

0.6

0.5
20 30 40 50 60 70 80 90
Temperature (°C)

Tm of the innovator and biosimilar estimated as 70.2°C and 71.8°C

Agreed well with reported Tm of rituximab of 71.6 °C, measured by Differential Scanning Calorimetry

16 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Results
Effect of formulation buffer on aggregation kinetics
– Incubated mAbs in formulation buffer and in water
– Incubated each at 60°C (lower than Tm) and take measurements at 0.5,1, 2, 3 & 4 h
– First-order kinetics for aggregation assumed
– Slope of the line used to determine k1 and t1/2

-6
Without formulation buffer With formulation buffer
-6.2
k1 (h-1) t1/2 (h) k1 (h-1) t1/2 (h)
-6.4
Innovator 0.0611 11.34 0.0487 14.34
-6.6
f(x) = − 0.0486975129937764 x − 6.62171526970271 Biosimilar 0.0681 10.17 0.0322 21.52
-6.8
LN Absorbance

-7
Innovator without formulation
-7.2 buffer
Formulation buffer protects against
-7.4
degradation (lower k1 and higher t1/2)
-7.6
f(x) = − 0.0610504829108975 x − 7.60962976595885
-7.8

-8
0 1 2 3 4 5
Timepoint (h)

17 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147
Summary
Study protein aggregation using the Cary 630 FTIR with ATR module
– A simple and easy-to-use platform
– Aggregation in highly concentrated samples can be quickly performed without extensive sample preparation
– Observed an excellent agreement between measured and reported thermal parameters for mAbs
– The Cary 630 can be used to provide orthogonal information on protein stability and folding, useful for formation
development biopharma laboratories

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Thank you!

19 Aggregation Analysis of Innovator and Biosimilar Therapeutic Proteins using FTIR DE60292147