Hudson Valley LyoMAC Webinar April 18, Elizabeth M. Topp, Ph.D. Professor, Dept. of Industrial and Physical Pharmacy Purdue University

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1 Hudson Valley LyoMA Webinar April 18, 2018 Elizabeth M. Topp, Ph.D. Professor, Dept. of Industrial and Physical Pharmacy Purdue University 1

Recombinant protein drugs are often stored as amorphous powders. Bulk API storage Final product Of 107 protein drug products approved by US FDA 2006-2016, 46 (43%) are solids.

2 Recombinant protein drugs are often stored as amorphous powders. Bulk API storage Final product Of 107 protein drug products approved by US FDA , 46 (43%) are solids. a Produced by lyophilization or spray drying. Degradation can occur during lyophilization and storage. Few analytical methods for protein conformation in solids, low resolution, semi-quantitative. a enterwatch: FDA-approved drugs by year. 2

3 In solution MR (2-D) ircular dichroism (D) Infrared (FTIR) IR Raman Fluorescence U.V. absorption H/D exchange X-ray crystallography In solids FTIR IR Raman Fluorescence ssmr H/D exchange Low resolution Semi-quantitative 3

4 Basic principles of HDX MS analysis H/D Exchange Quench and Digest Quenching locks in deuterium and unfolds the protein Digestion localizes the information 4

5 Intensity Solid-state HDX (sshdx-ms) Reconstitute in quench buffer (5 o, ph 2.5) ESI-MS Protein structure in amorphous solids? m/z Pepsin digestion 5

6 Myoglobin (holo ) 6

7 et Deuterium Uptake Effect of RH on deuterium uptake Intact protein: Myoglobin / mannitol 1:1, 5 o Time (hr) 7

8 et Deuterium Uptake Effect of RH on deuterium uptake Intact protein: Myoglobin / sucrose 1:1, 5 o Time (hr) HDX in sucrose is less than mannitol despite similar or greater water content protein structure/dynamics or spatial heterogeneity in water content 8

9 Myoglobin peptide map Pepsin digestion A B G L S D G EWQQ V L VWGKV E A D I AGHGQE V L I R L F T GH P E T L E K F DK F KH L K D E F T E A EMK A S E D L K K H G T VV L T A L GG I L K K K G H H E A E L K P L A Q S HA T KHK I P G I K Y L E F I S D A I I H V L H S KH P GD F GADAQG A MT K A L E L F R D I AA K Y K E L G F QG H 9

Myoglobin in D 2 O solution 5 o Deuterium uptake 91-100%

10 Myoglobin in D 2 O solution 5 o Deuterium uptake % 81-90% 71-80% 61-70% 51-60% 41-60% 31-40% 21-30% 11-20% 0-10% 10

11 Lyophilized Myglobin: Mannitol (1:1 w/w), 43% RH, 5 o Deuterium uptake % 81-90% 71-80% 61-70% 51-60% 41-60% 31-40% 21-30% 11-20% 0-10% 11

12 Lyophilized Myglobin: Sucrose (1:1 w/w), 43% RH, 5 o Deuterium uptake % 81-90% 71-80% 61-70% 51-60% 41-60% 31-40% 21-30% 11-20% 0-10% 12

13 Ingredients Myoglobin (Mb) Sucrose Formulations MbA MbB Mb MbD MbE 1.7 mg/ml, 45% w/w 1.7 mg/ml, 45% w/w Mannitol mg/ml, 45% w/w 1.7 mg/ml, 45% w/w 3.4 mg/ml, 90% w/w mg/ml, 45% w/w al - - Potassium Phosphate, (ph 7.0) 0.4 mg/ml, 10% w/w 0.4 mg/ml, 10% w/w 0.4 mg/ml, 10% w/w 3.0 mg/ml, 80% w/w mg/ml, 45% w/w 0.4 mg/ml, 10% w/w mg/ml, 10% w/w 0.4 mg/ml, 10% w/w B.S. Moorthy et al., Mol. Pharm., 11/6: ,

14 MbA MbB 24h Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 14

15 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 15

16 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 16

17 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 17

18 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 18

19 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 19

20 % Loss of Monomer % Loss of Monomer % Loss of Monomer % Loss of Monomer A A % Deuterium Uptake % Deuterium Uptake A fast /( fast + slow ) A fast /( fast + slow ) 20

21 % Loss of Monomer % Loss of Monomer % Loss of Monomer % Loss of Monomer Band Intensity for α-helix B1 B Band Position for α-helix (Wavenumber cm -1 ) Band Intensity for α-helix B2 B Band Position for α-helix (Wavenumber cm -1 ) 21

22 Monoclonal Antibody (mab) an sshdx be applied to mabs? Fastest growing class of protein drugs Includes antibody fragments and drug conjugates (ADs) High molecular weight (to 150,000 Da or more) Lyophilized for bulk drug storage or final product 22

23 mab Formulations an sshdx be applied to mabs? omposition Formulation Basic composition Mannitol (mg/ml) Sucrose (mg/ml) F mg/ml mab1 in buffer at ph 6.0 F2-80 F F Knowing only this, rank order the storage stability of the four formulations using sshdx-ms. B.S. Moorthy et al, Mol. Pharmaceutics, 2018, 15 (1), pp

24 mab Stability and Solid Properties FTIR band intensity Sucrose content Moisture content Glass transition, Tg 24

25 mab Stability and sshdx 5 o, 960 days 25 o, 960 days 40 o, 960 days 50 o, 180 days 25

26 Process effects E.M. Moussa et al., Pharm. Res., January

27 Use sshdx-ms: To screen solid formulations of protein drugs and select the most stable To characterize process changes and confirm comparability To evaluate novel drying methods Instead of storage stability studies in process scale-up and tech transfer (in progress) 27

sshdx can be performed in powders using D 2 O(g) as a deuterium source. Rate and extent of sshdx are affected by RH, excipient type and amount, temperature.

28 sshdx can be performed in powders using D 2 O(g) as a deuterium source. Rate and extent of sshdx are affected by RH, excipient type and amount, temperature. For Mb and a mab in lyo powders, sshdx parameters are highly correlated with the extent of aggregation on storage. sshdx may interrogate the H-bond network in the amorphous solid state. 28

29 Purdue University (current) Dr. Karthik handrababu Dr. Mohamed AbouGhaly Yuan hen Iris ho athan Wilson Harshil Renawala Raj Kammari Lia Bersin Rishabh Tukra Ethan hau Roche / Genentech Dr. Andrea Allmendinger Dr. Dan Zarraga Dr. Ben Walters Dr. Lokesh Kumar Dr. Kathleen Abadie AbbVie Dr. Steve Schultz Ms. Sherry Kim Purdue University (past) Dr. Andreas Sophocleous Dr. Balakrishnan S. Moorthy Financial support: IH RO1 R01GM085293, PhRMA Foundation Fellowship (AS), Purdue University, Roche/Genentech, Inc., IST AMTech 29

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33 D 2 O 33

Biotherapeutics. Biopharmaceutical Sciences Group Waters Corporation Waters Corporation 1

Biotherapeutics. Biopharmaceutical Sciences Group Waters Corporation Waters Corporation 1 Characterization of the Higher-Order Structure of Biotherapeutics Biopharmaceutical Sciences Group Waters Corporation 2011 Waters Corporation 1 FDA desire for specific innovations in biotherapeutic analysis