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Analytical Analysis and Sample Prep At each time point the vials are removed from the incubator. The system first performs visual inspection, pH, and viscosity analysis and then prepares daughter plates for other analytics. The data is collected into LEA and mapped back to the original formulation.
THE CM3 BIOLOGICS FORMULATION SYSTEM Freeslate’s CM3 for Biologics Formulation System (BFS) shown below automates formulation development from start to finish and can be configured with a variety of elements for a variety of workflows.
Automated Protein Formulation: From Formulation to Buffer Exchange, Stress Testing and Analysis Jos de Keijzer, Julien Merillon
Freeslate, Inc., Sunnyvale, California
Freeslate, Inc. 415 Oakmead Parkway Sunnyvale, CA 94085 United States
+1 408.773.4000 www.freeslate.com [email protected]
© 2015 Freeslate, Inc. All rights reserved. Freeslate is a registered trademark. All other brands or product names mentioned are trademarks owned by their respective organizations.
ABSTRACT Buffer and excipient screening studies are important to formulation development of biopharmaceuticals. Preparation of protein drug substances into suitable liquid formulations is often accomplished by buffer exchange using a variety of methods that are both time consuming and labor intensive. In this poster, we describe a novel and automated high throughput system based on UF/DF technology for buffer exchange of liquid protein formulations. In addition to the buffer exchange module, the different modules for formulations and the analytical integrated instruments will be discussed. Example of automated workflows and case studies are presented to demonstrate the utility of the new product.
Analytical Side
Automated Viscosity PDT injecting sample into the HTV Measurement Module
Analytical Side 1 ● Wyatt DynaPro PlateReader II DLS
● Ixion Centrifuge
● Molecular Devices SpectraMax UV/Vis
● Thermo Scientific Microplate sealer
● Inheco Incubator Shaker
Analytical Side 2 ● Wyatt DynaPro DLS
● Molecular Devices SpectraMax UV/Vis
● Thermo Scientific Microplate sealer
● Inheco Incubator Shaker for accelerated stress at 37 ˚C
● Liconic Incubators for plate storage at 5 ˚C, 25 ˚C and 37 ˚C
Formulation Robot Deck ● Visual Inspection Station
● Viscosity Station
● Plate rack (2x)
● Vortexing Station
● Cooled Storage Bay
● Pipette Tip Storage (2x)
● Wash Station
Formulation Robot Arm ● Multi Channel Liquid Dispense
● Positive Displacement Pipette Arm
● Multi Channel pH Probe
● Vial and Plate Gripper
Automated Visual Inspection Particle Size 80 µm and up Color assessment Turbidity measurement
Automated pH 4 solid state ISFET pH probes
NEW OPTION: Buffer Exchange Addition of a buffer exchange unit for automated buffer exchange
The BFS consists of two robotic systems connected by a carousel. The robot on the right side ,or Formulation side, of the system has multiple arms and tools that perform liquid handling and vial and plate transfers. It also has various elements such as heating, cooling, and mixing stations along with analytical tools including visual inspection, pH, and viscosity. The robot on the left side, or Analytical side, of the system can be physically integrated with a variety of instruments for incubation and analytics.
Formulation Side
Stress conditions are captured and linked to
formulation composition and downstream analytical data
NEW AUTOMATED BUFFER EXCHANGE The BFS now offers an automated buffer exchange module. This innovative and proprietary micro UF/DF system uses pressure based filtration and allows for automated buffer exchange and concentration.
50
60
70
80
90
100
0 2 4 6 8 10
%M
ain
peak
- SE
C
Time (weeks)
9 week stability of mAb 1 after buffer exchange
Freeslate
Stock
Amicon
RESULTS AND CONCLUSIONS With the BFS system a formulation team can complete a 24-vial 3-month DOE stability study with less hands on time. This complete study, fully inclusive of analytical setup, automation execution, data analysis, and reporting, required only 0.25-0.5 FTE ( Full Time Employee) over the three months.
DLS Results ● Cumulant radius increases dramatically in 40 °C stressed samples ● Increase in radius is only observed in formulations stressed at 40 °C
How it Works
AUTOMATED STABILITY CASE STUDY A formulation study was designed to evaluate the stability of a mAb in 12 different formulations at two different protein concentrations. The study design was created in LEA (Lab Execution and Analysis Software) and then executed on the BFS.
4x6 vial rack array
Protein @ 210 mg/ml
Protein @ 50 mg/ml
Saline 25 °C • 45 days • 90 days
4 °C • 45 day • 90 day
40 °C • 45 days • 90 days
T0
pH
0
1
2
3
F13
F15
F17
F19
F21
F23
T0
T25C,45d
T25,90d
Viscosity
Dynamic Light Scattering
Color Matching
01020
F1 F5 F9 F13
F17
F21
Particle Count 0
200
400
F1 F4 F7 F10
F13
F16
F19
F22
Turbidity
HPLC and CE
Sample Prep
Vial IDs pH Visible Particle Color Turbidity DLS HPLC
VIS, pH, and Viscosity
After 9 weeks post-processing, the SEC data indicate the Freeslate results are comparable to
Amicon and the stock protein solution.
DLS 50 mg/ml
Viscosity 50 mg/ml
210 mg/ml
● Viscosity changes at 50 mg/ml give no indication of the significant viscosity changes that occur at 210 mg/ml
● Viscosity differences due to formulation composition, mAb concentration, and absence or presence of excipients are easily evaluated.
DLS 210 mg/ml
Color Analysis at T0
Color Analysis at 40°C, 90 days
Color Analysis
● F01 –F12 - 210 mg/ml, F13-F22, 24 - 50 mg/ml, F23 – 10 mg/ml ● Significant differences in color due to concentrations are observed.
Add ~450 µL DS or DP to each well using liquid handler
Formulation A
Formulation B Formulation C
Formulation D
Formulation D
Formulation E
Formulation F
Formulation G
Repeat until 99% Exchanged 2–10 hours
Add back removed volume to each well
Measure volume
Measure volume, system calculates volume removed
Pressure
Filtrate to waste
A280 DLS
A280
SEC
iCIEF
CE-SDS-MW