Formulation Aspects in Biosimilar Development. Kedar S. Gokhale, PhD. Lupin Ltd. (Biotech)

Transcription

1 Formulation Aspects in Biosimilar Development Kedar S. Gokhale, PhD. Lupin Ltd. (Biotech)

2 Typical Protein Formulation Components of a protein formulation Active Ingredient Buffer Tonicity modifier Stabilizer Anti-Aggregating Agent Bulking Agent/Lyo Protectant Active Ingredient: Concentrations ranging from ~ mg/ml Primary Container Closures: Vials, PFS, Cartridges, Dual Chamber Syringes Secondary Container: Device, Auto injectors

3 Buffering Ranges Buffer Phosphate Histidine Citrate Succinate Buffering Range Acetate 3.6 to 5.6 Typically isoelectric point for monoclonal antibodies is around 8-9. Most formulations are around ph 5-6 Approximately Two units away from the pi

4 Other Excipients Component Tonicity Modifier Stabilizer Anti Aggregating Agents Examples Sodium Chloride, Dextrose Sugars, Polyols, Amino Acids Surfactants, Amino Acids Bulking Agents/Lyo Protectants Sugars, Polyols The formulation should be isotonic (290 mosmols) Sugars/Polyols are usually added at around 5-10 % Anti-aggregating agents around 5-10 mm Surfactants around % Viscosity modifiers might be needed for high concentration formulations

5 Pre-Formulation Typical Time frame ~ 3 months High throughput screening, Use of DOE IPMG input: FTO analysis Steps for Pre-formulation Selection of optimum ph/buffer, e.g. range 3-9 Selection of stabilizer/polyol Selection of surfactant

6 Stress Temperature: Accelerated Conditions Stress conditions Freeze Thaw at various freezing and thawing temperatures Agitation Shaking on an orbital shaker. Temperature variations End of stability ph Buffer capacity Oxidation: Peroxide, Heat Deamidation: High and low ph LMW impurities (Clips): Heat Aggregation: Agitation, Heat

7 High Throughput Analytics DSC: Thermal/conformational stability DLS: Aggregation and poly-dispersity Structural Changes FTIR Circular Dichroism Fluorescence UV

8 Analytical Tools Attribute Appearance Color Concentration Analytical Methods Visual Observation Visual Observation (USP/EP standards) UV, Elisa Surface Charge/Charge Variants IEF, IEX HPLC, HI HPLC, RP HPLC, Size Variants Activity CE-SDS, SDS PAGE, SEC, DLS, AUC, visible and sub visible particles, Turbidity Bioassay (In-vivo, In Vitro)

9 Impact of Formulation on Syringeability Rheological characterization and injection forces of concentrated protein formulations: An alternative predictive model for non-newtonian solutions, Allmendinger A. et al, European Journal of Pharmaceutics and Biopharmaceutics, Volume 87, Issue 2, July 2014, Pages Rheological and syringeability properties of highly concentrated human polyclonal immunoglobulin solutions, Burckbuchler V. et al, Eur J Pharm Biopharm Nov;76(3):351-6

10 Conc. Vs. Viscosity

11 Force Required for 30 G needle

12 Needle Gauge comparison

13 Long Term Studies Based on pre-formulation studies finalize 3 leading candidates Place on long term stability 6-9 months Real Time, Stress and Accelerated Testing to be done to monitor all CQAs Final Formulation is now decided! What next?

14 Formulation: Excipient Concentration Cell Culture Expansion Clarification Chrom. Conc. and TFF Chrom. Conc. and TFF DS Actual formulation occurs after the at the UF/DF stage. After the UF/DF ((Ultrafiltration - Diafiltration) process at times a decrease or increase in the concentration of excipients is observed Charged Interaction Between protein and charged excipients Causes uneven distribution of charged excipient molecules Donnan effect is characterized by reduced excipient levels in DS (Drug Substance) after UF/DF process

15 Case Study Miao, F. et al, Theoretical Analysis of Excipient Concentrations During the Final Ultrafiltration/Diafiltration Step of Therapeutic Antibody, Biotechnol. Prog., 2009, Vol. 25, No. 4 Four therapeutic monoclonal antibodies produced (products A, B, C, D) were used in these diafiltration studies Impact of ph, molarity on histidine content was measured

16 Effect of ph on Histidine Concentration Experiment conducted at ph 5.3, 65 mg/ml of protein concentration, 10 mm histidine Concentration of histidine in permeate and retentate starts to diverge after 2 dia volumes ph remains stable after 3 dia volumes

17 Effect of ph on Histidine Concentration Experiment repeated at ph 7.6 which is close to the pi value of histidine No charge is present on histidine Concentration of histidine in permeate and retentate remains the same

18 Effect of Protein Concentration (Product A)

19 Effect of Protein Concentration (Product B)

20 Effect of Protein Concentration (Product C)

21 Effect of Protein Concentration (Product D)

22 Points to consider for Excipient Concentration The decrease in excipient concentration of charged species is reported in histidine, chloride and acetate buffer The molarity of the buffer and concentration of the protein play an important role Increase in molarity reduces the impact of Donnan effect Increase in concentration of protein increases the impact of Donnan effect. The TFF buffer excipient concentration should be adjusted based on the predicted loss and finalized based on actual data.

23 Summary IPMG input to determine FTO Pre-formulation Studies 3 Months Selection of excipients High Throughput Screening DOE Stress Studies Long Term Stability 6-9 months CQA testing Excipient concentration

24 Acknowledgements Dr. Dhananjay Patankar, Syngene International Dr. Rustom Mody, Lupin Biotech.

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