Protein Formulation Workflow by pengxiuhui

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									Protein Formulation
          Workflow
 In-house Development Capability
Rational for Micro-scale, Parallel
Protein Formulation Workflow

       “There is the need for more complex and orthogonal analytical
     technologies to detect all of the chemical and physical instabilities that
        may result for large molecules during formulation development”




 > Parallel formulation development to increase experimental breadth
 > Micro-scale systems to reduced drug substance requirements
 > Automation to reduce human error and increase throughput
 > Electronic data management to improve communication and analysis
 > Reduce the barrier to screening to move formulation development earlier
   into the product development process
    Protein Formulation Workflow

                                   Sample Conditioning Chambers
UV-Vis / Florescence Reader
& LabChip GEL SDS
                                             Chromatography
                                             Analytical Systems
Plate Stackers and
Label Applicators




                                        Dynamic Light
 Robotic Sample                         Scattering Station
 Preparation and Analytical
 Work-up stations
High Throughput
Protein Formulation Process
                                                 Automated Formulation System
      Protein Solution                                 (Sample volumes as
                                                         low as 125 µL)           Agitation Testing
         Preparation
                                   For                                          PhotoStability Studies
                                   surfactant
                                                                                Freeze Thaw Cycling
                                   screening
                                                    Formulation/                        DSC
                                                  Sample Preparation
                                                       Robot
                                                  Details: Final Formulation        Environmental
Formulation Prep / Dialysis
                                                   Adjustment, Plate/Vial             Chambers
         Station
                                                  handle, Image Samples,               Details: 4
Details: Prepare buffer exchange                   Analytical Sample Prep           Temperatures, 4
 solutions and perform dialysis                       with weights, pH                time points


                                                                                      Analytical
  Sterile Filtration / Filling /                                                       Stations
       Capping Station                                                             UV-Vis, FI / SEC /
                                                                                     RPLC / IEC
                                                Lyophilization                     GelElectrophoresis
                                                   Station                             Osmometer
High Throughput
Protein Formulation Process

         Design Library



            Prepare
          Formulations



         Stress Testing


          Analytical
       Sample Preparation


        Sample Analysis




       Data Processing,
         Visualization
       Report Generation
                  Protein Formulation Solubility Results
                  High-Throughput vs Traditional Manual-based Measurements

                                                                       No Surfactant                                                                                                                                                                                                0.05wt% P20

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              Protein Solubility versus pH with 150mM NaCl as Tonicity
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       Modifier

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         25

                                                                                                                                                                                                                                                                                                                                                                                                                                                             No Tonicity Modifier
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         20
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 •Manual Study
                                                                                                                                                                                                                                                                                                                                                                                                                                                             50mM NaCl                                                                           (Multi-week)




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Solubility (mg/mL)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         15
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 •HT Approach Study
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Manual Multi-Week
                                                                                                                                                                                                                                                                                                                                                                                                                                                             150mM NaCl                                                                                  High-Throughput Study
Recombinant Protein Concentration                                                                                                                                                                                                                                                                                                                                                                                                                                                                        10                                      (2-3 days)
       17.8 .. 20.1                                                                                                                                                                                                                                                                                                                                                                                                                                          250mM NaCl
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          5
       15.6 .. 17.8
       13.4 .. 15.6
       11.3 .. 13.4                                                                                                                                                                                                                                                                                                                                                                                                                                          2.5wt% Sorbitol
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          0
        9.1 .. 11.3                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           pH 4     pH 5     pH 6     pH 7      pH 8
        6.9 .. 9.1
        4.7 .. 6.9                                                                                                                                                                                                                                                                                                                                                                                                                                           5.0wt% Sorbitol
        2.5 .. 4.7
        0.3 .. 2.5
                                                                                                                                                                                                                                                                                                                                                                                                                                                             1.5wt% Glycine                                   Protein Solubility versus pH with 5% Sorbitol as Tonicity
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      Modifier
Units: mg/mL
                                                                                                                                                                                                                                                                                                                                                                                                                                                             3.0wt% Glycine                              25



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         20                                       •Manual Study
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  (Multi-week
                                                                                                                                                                       10mM Sodium Phosphate Buffer, pH 7
                                                                                                                                                                                                            10mM Sodium Phosphate Buffer, pH 8




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    Solubility (mg/mL)
                                    10mM Sodium Acetate Buffer, pH 4

                                                                        10mM Sodium Acetate Buffer, pH 5
                                                                                                           10mM Histidine Buffer, pH 6




                                                                                                                                                                                                                                                                                                                                                                                   10mM Sodium Phosphate Buffer, pH 7
                                                                                                                                                                                                                                                                                                                                                                                                                        10mM Sodium Phosphate Buffer, pH 8
                                                                                                                                         10mM Histidine Buffer, pH 7




                                                                                                                                                                                                                                                 10mM Sodium Acetate Buffer, pH 4

                                                                                                                                                                                                                                                                                    10mM Sodium Acetate Buffer, pH 5
                                                                                                                                                                                                                                                                                                                       10mM Histidine Buffer, pH 6

                                                                                                                                                                                                                                                                                                                                                     10mM Histidine Buffer, pH 7




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         15
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  •HT Approach Study
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         Manual Multi-Week


                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         10
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  (2-3 days)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         High-Throughput Study




                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          5



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              pH 4     pH 5     pH 6      pH 7      pH 8
    Protein Formulation Surfactant Screen
    DLS vs SEC-HPLC Results

Average Radius from Dynamic Light Scattering         Size Exclusion Chromatography: Percent Area of Aggregates Peak




                                               Protein A
                                               P20
                                               P80
                                               F68
    Summary

Workflow Functionality
•  4x more formulations with same amount of protein
          – Formulation volumes as low as 125 µl
•  Fully automated Analytical systems and Sample preparation
          – HPLC ( Size Exclusion, Ion Exchange, Reversed Phase, HIC)
          – UV-vis and Fluorescence Spectrometry.
          – Dynamic Light Scattering
          – LabChip Gel Electrophoresis
          – Osmometer
•  Fully Automated Sample/Equipment Tracking and Record Keeping
          – Bar codes to track Vial racks
          – Future: RFID tags/label to track mobile phases, columns, analytical equipment.
          – Future: Ultrasound and Optical sensors for rack/vial/cap detection and error handling
Applications
•    Solubility screens mapping short-term thermodynamic solubility for high concentration formulations
•    Automated sample preparation and analysis for HPLC, DLS, Chip Electrophoresis, pH, osmometry on
     microscale samples
•    Agitation and freeze-thaw short-term tests (non aseptic) for stability assessment at the microscale
•    Preformulation studies in final container-closure systems in an aseptic environment
      • 12 to 48 different formulations (250mg to 1g of protein for the entire study)
      • Stresses: temperature, photolytic, oxidative, agitation, freeze-thaw
      • At least 4 time points for each sample at each stress condition

								
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