High Throughput Screening of a Monoclonal Antibody: Practical Considerations. DDF Summit, August 2017

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1 High Throughput Screening of a Monoclonal Antibody: Practical Considerations Summit, August 2017

2 Agenda 1. Background 2. Tecan liquid handler, description 3. Components (materials of construction) considerations 4. Excipient grades (compatibilities & impurities) 5. mab concentrations and solution handling 6. Lyophilized formulation capabilities 7. mab formulation screen Case study 8. Summary 9. Questions 2

3 Background What is/are the objective(s) of a high throughput screening approach to formulation development? 1) Generate a large data set 2) Derive formulation information based on the outputs from screening a relatively large number of variables 3) Make data-based decisions about formulations that will optimally stabilize a protein product. The generation of reliable data that is suitable for making a formulation decision is key. 3

4 Tecan Liquid Handler Waste/ wash 4

5 Liquids excipients, materials of construction 1. Plates, dispensing needle coatings, excipients Sugar Buffer 5

Plastics Component compatibilities Polystyrene Negatively charged Non-specifically binds protein Concentration dependence Polypropylene Hydrophobic Degradation, adherence Oxidation of Met, Trp groups

6 Plastics Component compatibilities Polystyrene Negatively charged Non-specifically binds protein Concentration dependence Polypropylene Hydrophobic Degradation, adherence Oxidation of Met, Trp groups Proteins Hydrophobic groups can adhere to plastics Problematic at low concentrations Plate Color/ Clear plastic Visual examination of contents vs light protection Plate coatings For reduction in non-specific binding Flexibility, temperature resistance, bottom geometry Polycarbonate Typically low binding 6

7 Impurity Detection Methods (in Excipients) 1. If testing is not performed for a specific heavy metal, the most common source of evidence nowadays come from a limit test being carried out. After treatment, the heavy metal is complexed with thioacetamide or precipitated as a sulphide. Then, one compares the resulting colouring of the sample solution against that from a reference lead solution. 2. These limit tests still form the majority of testing for heavy metals in the current national and international Pharmacopoeias (e.g. Ph. Eur or USP <231>). Thereby, it is possible however to make only a semiquantitative statement about the total contents of heavy metals in the sample, and in addition - and additionally, only for those heavy metals that actually form dark coloured complexes or sulphides. 3. Spectroscopic tests are to be found only in individual monographs and methods (e.g. nickel in polyols and oils, lead in sugar) to date. 4. There is no sign of any changes in the near future with regard to the methods in the Ph. Eur. and JP. This is in contrast to the US Pharmacopoeia

Excipients Grades and Potential issues Product Specifications TEST Meets ACS Reagent Chemical Requirements Sodium Phosphate Monobasic Monohydrate (Avantor) SPECIFICATION Trace quantities of Copper,

8 Excipients Grades and Potential issues Product Specifications TEST Meets ACS Reagent Chemical Requirements Sodium Phosphate Monobasic Monohydrate (Avantor) SPECIFICATION Trace quantities of Copper, Zinc, Nickel and Iron can cause IgG1 mab fragmentation. mabs, 2011 May-Jun; 3(3): For Laboratory,Research,or Manufacturing Use Assay (NaH₂PO₄ H₂O) ph of 5% Solution at 25 C Insoluble Matter Chloride (Cl) ACS - Sulfate (SO₄) Calcium (Ca) % <= 0.01 % <= 5 ppm <= % <= % Potassium (K) Heavy Metals (as Pb) <= 0.01 % <= % Limit Test Trace Impurities - Iron (Fe) <= % Meets Reagent Specifications for testing USP/NF monographs 8

9 Comparison of traced metal- induced fragmentation of an IgG molecule Size exclusion chromatography analysis of metal spiked samples. Specific hinge cleavage by CuII is significantly higher compared to other metals such as MgII, MnII, ZnII, FeIII and NiII under the same solvent conditions (phosphate-buffered saline at 37 C). 9

10 Other excipients Polysorbates (20 and 80) Contain peroxides and can oxidize AA side chains during heat/ light exposure. (Met, Trp Oxidation potential) Can adsorb to and react with active substance (some mabs, albumin conjugates, proteins with relatively high hydrophobicity indices). Sucrose Degradation to monomers fructose and glucose can result in glycation of proteins through the Maillard reaction. Reduction to monomer species usually occurs at acidic ph (~ph 4-5). 10

cryo/lyo protectants Evaluate Cake characteristics Fill

11 Tecan Lyophilization Block Uses of Lyo Block Screen protein (minimize quantity) Simultaneous screening of multiple cryo/lyo protectants Evaluate Cake characteristics Fill volumes Solids concentrations Stability Excipient: protein ratios 11

12 mab concentrations and solution handling Concentrations (10-100mg/mL) Lower concentration limit avoids surface adsorption, allows for efficient screening. Solution buffer exchanges by centrifugation methods. Higher concentration (depending on material availability) allows for evaluation of high concentration effects on stability. Solution buffer exchanges by dialysis. Tecan Filling needles Teflon coated maximize protein/ excipient transfer Ease of cleaning needle surfaces between and after dispensing Eliminates well-to-well carry over of solutions. 12

13 HTF ph and Buffer screen of a Monoclonal Antibody A case study 13

14 Study Objective Identify a target ph and buffer species that would best stabilize an IgG1 molecule against thermal degradation. Analytical tests used for evaluation Absorbance (360nm) evaluation of light scattering due to particle formation SE HPLC Purity assay - monitor monomer, aggregate and fragment cief Purity assay evaluate changes in the charge variant profile SPR Potency assay ligand binding Tests were chosen based on availability of Plate-based analytical methods 14

15 Study Design HTF screen - ph and buffer species Test article mab BDS at 100mg/mL, ph Buffer (50mM) ph test range of (0.5pH unit increments) Buffer species: Acetate, Citrate, Succinate, Histidine, Phosphate Statistical Designs (e.g. DoE) Not used (i.e. All combinations were tested.) Design expert Plate Randomization Data Analysis. 1, ) Acetate, 2) Citrate 3, 4, ) Acetate, 4) Citrate, 5)Succinate 6, 7, 8, ) Acetate, 7) Citrate, 8) Succinate, 9) Histidine 10, 11, 12, ) Citrate, 11) Succinate, 12) Histidine, 13) Phosphate 14, ) Histidine, 15) Phosphate ) Phosphate 15

fouling at high concentrations (100mg/mL) poor buffer exchange Dialysis Pros and Cons Similarities with buffer exchange

16 Buffer and Sample preparation Tecan options Centrifugation (Pros and Cons) Similar to centriprep buffer exchanges but in 96-well plate format Relatively quick Buffer exchanges complete for low concentrations of proteins (~50mg/mL) Membrane fouling at high concentrations (100mg/mL) poor buffer exchange Dialysis Pros and Cons Similarities with buffer exchange processes relying on equilibration of charge species e.g TFUF Can be used for high concentration proteins (e.g. 100mg/mL) Duration (Days) 16

17 Plate design Order of addition programmed into the robotic liquid handler ph -> Std Order Random Buffer -> Acetate Citrate Acetate Citrate Succinate Acetate Citrate Succinate Histidine Citrate Succinate Histidine Phosphate Histidine Phosphate Phosphate Order Plate 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM 50mM

18 Plate prep Samples added to 96 wells (16 different buffer/ph combinations with 6 replicates) Plate sealers prevent evaporation Samples stored at 40 C for 2 weeks prior to analysis 18

19 Analytical method considerations Analytical testing was done at refrigerated conditions where possible Replicates were randomized across the plate to reduce analytical variability Samples were diluted in formulation buffer to minimize degradation during analysis Analytical methods: A320, SE-HPLC, cief, SPR 19

20 Star Plots Used for analysis of results Feature of Design Expert Star plots How to use Identify analytical methods to use If a degradant is measured the inverse of the result is used If a main peak is measured the result is used Individual results and inverse results are normalized to initial The area represented by the results are plotted in a star-design Looking for maximum area to indicate protein stability over time Advantages Allows rapid evaluation of multiple results Visual comparisons Facilitates decision-making Responses evaluated Inverse A320 SE-HPLC %monomer Inverse SE-HPLC %aggregate Inverse SE-HPLC %fragment cief %main SPR Maximum area in the star plot represented best condition for thermal stability 20

21 Star Plot results Feature of Design Expert Responses evaluated Inverse A360 SE-HPLC %monomer Inverse SE-HPLC %aggregate Inverse SE-HPLC %fragment cief %main SPR Buffer ph Combinations - Week 2 Responses AcepH5 AcepH5_5 AcepH6 CitpH5 CitpH5_5 CitpH6 CitpH6_5 HistpH6 Maximum area in the star plot represented best condition for thermal stability HistpH6_5 HistpH7 PhospH6_5 PhospH7 PhospH7_5 SucpH5_5 SucpH6 SucpH6_5 Legend A320 Biacore SEC Agg SEC Frag SEC Main cief Main 21

Gibbs Donnan effects At high concentrations, chemical potential and Gibbs- Donnan effect play a role in sample preparation. Acetate ph 5.0 was closer to ph 5.5 when measured Acetate ph 5.

22 Gibbs Donnan effects At high concentrations, chemical potential and Gibbs- Donnan effect play a role in sample preparation. Acetate ph 5.0 was closer to ph 5.5 when measured Acetate ph 5.5 was closer to ph 6.0 when measured Additional studies were performed with direct buffer exchanges to confirm acetate results. Results confirmed. Message: At high protein concentrations, and depending on target ph, protein self-buffering effects modify the efficiency of buffer exchanges. Must be taken into account sample prep. 22

23 Summary 1. Tecan liquid handler, - described the layout, liquid handling and plate characteristics 2. Components (materials of construction) considerations adherence to plastics, compatibility 3. Excipient grades (compatibilities & impurities) Potential metal impurities and excipient degradation issues 4. mab concentrations and solution handling working concentration range of tecan 5. Lyophilized formulation capabilities Lyo block characteristics 6. mab formulation screen Case study 23

24 Acknowledgements Biopharmaceutical Product Sciences and BioAnalytical Sciences, Statistical Sciences, R&D GSK Melissa Shuman Jim Ludlow Nicole Lundberg Andrea Frangiosa Laurie Pompeo Jennifer Woods Harnath Doddapenani Becky Wood Jennifer Dally Tom Wrzosek Conference organizers Thank you for your attention 24

25 Questions

Highly automated protein formulation development: a case study

Application Note Highly automated protein formulation development: a case study Introduction and background Therapeutic proteins can be inherently prone to degradation and instability, and they often pose