NIST/University of Maryland Biomanufacturing Summit Rockville, MD, June 25, 2015

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1 IST/University of Maryland Biomanufacturing Summit Rockville, MD, June 25, 2015 Elizabeth M. Topp, Ph.D. Dane O. Kildsig hair and Department Head Dept. of Industrial and Physical Pharmacy, Purdue University 1

2 Recombinant protein drugs are often stored as amorphous powders. Bulk API storage Final product Of nine protein drug products approved by US FDA in 2011, five are solids. 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. 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 hemical definition of amorphous solids is possible and can be used to rationally design solid formulations of protein drugs. urrent paradigm: chemical environment in amorphous solids is unknowable, protein conformation in solids can only be measured with low resolution, trial-and-error informed by limited physical characterization is the only possible approach to formulation development. 4

5 onformation and excipient interactions with peptide-level resolution Y. Li et al., Biotech. Bioeng., 97/6: , 2007 Y. Li et al., Anal. Biochem., 366:18-28, 2007 Y. Li et al. Pharm. Res., 25/2: , 2008 S. Sinha et al., Biophys. J., 95/12: , 2008 A. Sophocleous et al., Mol. Pharm., 9/4: , 2012 A. Sophocleous et al., Mol. Pharm., 9/4: , 2012 B.S. Moorthy et al., Mol. Pharm., 11/6: , 2014 B.S. Moorthy et al., JoVE, (98), e52503,

6 A chemical reaction, in which labile hydrogen atoms exchange with deuterium. 2R-H + D 2 O 2R-D + H 2 O Reaction is quenched at low ph and low temperature. The increase in mass (+1 amu) can be detected using mass spectrometry (L-MS, L-MS/MS). The rate and extent of HDX provides information on protein structure and dynamics in aqueous solution. Due to back exchange, only deuteration of backbone amide groups is detected. 6

7 Basic principles of HDX MS analysis H/D Exchange Quench and Digest Quenching locks in deuterium and unfolds the protein Digestion localizes the information 7

8 HDX for lyophilized solids (sshdx) Expose powder to D 2 O vapor Store at controlled T, RH * Protein solution Lyophilize or spray dry * * Solid powder Excipient solution + solvent + solvent Analyze HSQ-MR ESI-MS FTIR 15 Intensity Intensity 1 H m/z cm -1 8

9 POTETIAL BEEFITS Information on protein structure, with peptide-level resolution. Screen formulations and/or processes for effects on protein structure. Obtain fundamental information on protein structure and dynamics in amorphous solids. UKOWS Will it work? Will D 2 O(g) sorption kinetics affect the results? Are HDX results predictive of storage stability? How should the results be interpreted? Obtain fundamental information on amorphous solids (spatial, dynamic heterogeneity) with protein as a reporter. 9

10 Myoglobin (holo ) 10

11 o detectable secondary structural differences observed post-lyophilization by FTIR Signal Intensity native-like α-helix peak at 1655 cm Wavenumber (cm -1 ) Second-derivative FTIR spectra of Mb in lyophilized solids containing mannitol (solid line) or sucrose (dashed line). 11

12 Water sorption and water content Myoglobin / mannitol or sucrose 1:1, 5 o Water ontent (g water/g dry solid) sucrose mannitol Time (hr) Water ontent (g H 2 O/g dry solid) Mannitol Sucrose Relative Humidity (%) Water vapor sorption is essentially complete in ~ 1 h. At low RH, water content of mannitol and sucrose formulations are equal. At high RH, water content in sucrose formulation is greater than in mannitol. 12

13 Effect of RH on deuterium uptake Intact protein: Myoglobin / mannitol 1:1, 5 o et Deuterium Uptake Time (hr) 13

14 Effect of RH on deuterium uptake Intact protein: Myoglobin / sucrose 1:1, 5 o et Deuterium Uptake Time (hr) HDX in sucrose is less than mannitol despite similar or greater water content protein structure/dynamics or spatial heterogeneity in water content 14

15 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 15

16 Peptic fragments Myoglobin / mannitol 1:1, 5 o Deuterium Uptake (%) Increasing RH D 2 O Solution A

17 Peptic fragments Myoglobin / sucrose 1:1, 5 o Increasing RH D 2 O Solution 17

18 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% 18

19 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% 19

20 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% 20

21 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: ,

22 MbA MbB 24h Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 22

23 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 23

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

25 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 25

26 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 26

27 MbA MbB Mb MbD MbE Deuterium Level < 10% < 20% < 30% < 40% < 50% < 60% < 70% < 80% < 90% > 90% 27

28 % Loss of Monomer A1 25 % Loss of Monomer A % Deuterium Uptake % Deuterium Uptake % Loss of Monomer A fast /( fast + slow ) % Loss of Monomer A fast /( fast + slow ) 28

29 % Loss of Monomer % Loss of Monomer Band Intensity for α-helix B1 B Band Position for α-helix (Wavenumber cm -1 ) % Loss of Monomer % Loss of Monomer Band Intensity for α-helix B2 B Band Position for α-helix (Wavenumber cm -1 ) 29

30 sshdx can be performed in powders using D 2 O(g) as a deuterium source. sshdx rates are much slower than vapor sorption. Rate and extent of sshdx are affected by RH, excipient type and amount, temperature. For Mb in lyo powders, sshdx parameters are highly correlated with extent of aggregation on 1-yr storage. 30

31 Relative contributions of protein conformation, protein dynamics and excipient interactions to sshdx results. Solution HDX Solvent exposure onformation Dynamics Solid-state HDX D 2 O exposure onformation Dynamics; local? Matrix / excipient interactions? 31

32 Mapping protein-protein interactions and the sidechain environment of proteins in amorphous solids L. Iyer et al., Mol. Pharm., 10: , 2013 B.S. Moorthy et al., JoVE, in press L. Iyer et al., Mol. Pharm., in preparation 32

33 Developed as an alternative and complement to HDX. Eliminates back exchange. Used to study protein-protein interactions (PPI) in cells. A variety of chemical crosslinking strategies, with triggers initiating reaction. Here, we evaluate photo-activatable crosslinkers in amorphous lyophilized solids. 33

34 Photolytic labeling Excipient hν Excipient Amino acid hν Amino acid Side chain + HS Side chain hν Side chain E 34

35 Photoamino acids Photoleucine (pleu) OOH H 2 pleu UV 2 OOH H 2 carbene p-benzoyl phenylalanine (pbpa) OOH H 2 pbpa H UV OOH H 2 OH ketyl radical 35

36 Diazirine reacts in solids Mb + pleu +sucrose (1:2 w/w), 365 nm U 1L 2L 3L 4L 5L 6L ormalized intensity 1:100 1:20 1:50 Formulation Deconvoluted mass (Da) L. Iyer et al., Mol. Pharm., 10: ,

37 Workflow MS analysis for adducts formed + Mb MS analysis for sites of labeling + excipients, lyophilize 37

38 Types of adducts Protein-protein adducts Protein-water adducts Protein-excipient adducts ESI L-MS and MS/MS, trypsin digestion Detection (mass list) Up to four missed cleavages (trypsin) Dead-end modifications ( 2 loss without adduct) Multiple SDA (0-4 labels per peptide) Adducts of two peptides 38

39 Tryptic Peptides (52-57) (60-78) (83-96) ( ) ( ) A B D E F G H /64-78 * 63-78/64-79 * Protein-protein interactions Lyo Mb, no excipient ( ) ( ) (83-96) (60-78) (52-57) A B D E F G H /64-78 * 63-78/64-79 * Map shows peptidepeptide adducts detected in 1, 2 or 3 injections Symmetric about the diagonal Many interactions involving the E and F helices and - terminus detected Detected in 1/3 injections Detected in 2/3 injections Detected in 3/3 injections 39

40 Tryptic Peptides (52-57) (60-78) (83-96) ( ) ( ) A B D E F G H /64-78 * 63-78/64-79 * Protein-protein interactions Lyo Mb, + raffinose ( ) ( ) (83-96) (60-78) (52-57) A B D E F G H /64-78 * 63-78/64-79 * Interactions somewhat more distributed than in excipient-free formulation. Detected in 1/3 injections Detected in 2/3 injections Detected in 3/3 injections 40

41 ( ) ( ) (83-96) (60-78) (52-57) A B D E F G H Tryptic Peptides /64-78 * 63-78/64-79 * (52-57) (60-78) (83-96) ( ) ( ) A B D E F G H /64-78 * 63-78/64-79 * Protein-protein interactions Lyo Mb, + Gdn Hl Many more interactions detected. Detected in 1/3 injections Detected in 2/3 injections Detected in 3/3 injections 41

42 ( ) (83-96) (60-78) (52-57) A B D E F G H Tryptic Peptides /64-78 * 63-78/64-79 * A B D E F G H Water and excipient interactions KEY Peptide/water adducts A Mb alone, lyo B Mb alone, soln Mb + raffinose, lyo D Mb + raffinose, soln E Mb + Gdn Hl, lyo F Mb + Gdn Hl, soln Peptide/raffinose adducts G Mb + raffinose, lyo H Mb + raffinose, soln Detected in 1/3 injections Detected in 2/3 injections Detected in 3/3 injections 42

43 Photolytic labeling can be performed in solid powders using diazirine chemistry. SDA-labeled Mb forms adducts with protein, water and excipient (raffinose) in lyo solids. Formulation affects the adducts formed. Method allows water replacement hypothesis to be tested. 43

44 For discussion Protein aggregates Amorphous forms of small molecule drugs Excipients HDX PLL Protein conformation Matrix interactions Formulation effects, screening Process effects, screening Properties of amorphous solids DESIG Lyophilized powders DESIG an we have all the answers yesterday? Hong-Ren Wang, Vertex 44

45 Purdue University (current) Dr. Balakrishnan S. Moorthy Lavanya Iyer Saradha handrasekhar Jainik Panchal Hamed Ghomi (with M. Lill) Ehab Moussa Anshul Mishra Yuan hen Iris ho Reham our Geoffrey Federspiel Dr. Fred Regnier Dr. Markus Lill Dr. hiwook Park Purdue University (past) Dr. Andreas Sophocleous Dr. Jun Zhang Dr. Bo Xie Esben Bertelsen Jun Xu Daniel Epling Serene Macaraig Jon Oh University of Kansas Dr. Yunsong (Frank) Li Dr. Sandipan Sinha Dr. Lei Zhang Mette Thing Steele Reynolds Brock Roughton Dr. Kyle amarda Dr. Jennifer Laurence Dr. David Weis Dr. Todd Williams, Director, KU-MSL KU Mass Spectrometry Service Laboratory Other Dr. Patrick onnelly, Vertex Pharmaceuticals Financial support: IH RO1 R01GM085293, PhRMA Foundation Fellowship (AS), FDA HHSF , AbbVie Inc., IPTE ritical Path Manufacturing Sector Initiative U01FD004275, Purdue University, Baxter, Inc., Pfizer, Inc., MedImmune, Inc., Roche/Genentech, Inc., IST AMTech, enter for Pharmaceutical Processing Research (PPR) 45

46 Topp Lab, May 23,

47 47

48 Argireline acetate HDX can occur at any labile hydrogen. Back exchange is rapid for side chains; only HDX of peptide bonds is detected. In larger proteins, HDX occurs slowly for H- atoms involved in secondary structure or buried in the core. HDX reports the exposure of the backbone to exchange. 48

49 Folded protein Unfolded protein Unfolded, deuterated protein Folded, deuterated protein D 2 O Two kinetic processes: (i) protein unfolding and refolding, (ii) deuterium exchange. Two kinetic extremes: Exchange much faster than refolding (EX1: k 2 >> k -1 ) Refolding much faster than exchange (EX2: k -1 >> k 2 ) 49

50 Myoglobin HDX kinetics 50

51 HDX theory EX2 and EX1 limits Broadening of peak width may be observed instead if populations not well resolved. most situations less common k obs = k 1 *k 2 /k -1 = K op *k 2 k obs = k 1 51

52 EX2/EX1-like peak width broadening Example: Phe138-Gly153 in Mb 9 Phe138-Gly153 Peak Width (Da) Time (hr) Mannitol Solution control Sucrose Greater peak widths in mannitol formulations suggest mixed EX1/EX2 behavior, structural perturbation. onsistent with greater deuterium uptake overall. 52

53 Data analysis - sspll A B Bar graph of (A) peptide-peptide adducts, (B) peptide-water adducts and () peptideraffinose adducts detected by L-MS. White bars represent Mb-SDA lyophilized in the absence of excipients (blank), grey bars represent Mb-SDA lyophilized with raffinose and black bars represent Mb-SDA lyophilized with Gdn Hl. X 1n values were counted for peptides assigned to 8 groups. Bars represent mean normalized X 1n values (X 1n* ) ± SD (n=3). ote that in the abscissa for panel (), Group (6) spanning residues Lys 78 -Lys 98 was expanded to Lys 78 -Lys 102 to accommodate peptide Lys 79 -Lys 102 that was found to form raffinose adducts. 53