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 customercontact@freeslate.com

© 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