BIOSIMILAR PROFILING SUMMER 2018 ANDREA DETLEFSEN

Transcription

1 BIOSIMILAR PROFILING SUMMER 2018 ANDREA DETLEFSEN

2 PROTEIN DRUGS Therapeutic proteins are one of the fastest growing categories of pharmaceuticals Ex: mabs, recombinant proteins As patents expire, biosimilars will enter the market Biosimilars are difficult to make and get approval for due to complicated synthesis processes and strict FDA regulations Data must demonstrate that the new molecule is similar to, or interchangeable with, a biological reference product already approved by FDA Protein biosimilars: Opportunities and challenges. (n.d.). Retrieved from

3 HANDFUL OF PROTEIN DRUGS & BIOSIMILARS Protein drugs and their approved biosimilars: Eprex EpoetinAbseamed, Retacrit, Binocrit, Epoetin alfa Hexal, Salipo Treats anemia Neupogen G-CSF, FilgrastinHexal, biograstim, Nivestim, Zarzio, Ratiograstim & Filgrastim Ratiopharm, Tevagrastim Bone marrow stimulant Genotropin hgh Stimulates cell growth/regeneration Humatrope Omnitrope, Vltropin Stimulates cell growth/regeneration Kirchoff, C. F., Wang, X., Conlon, H., Anderson, S., & Ryan, A. M. (2017). Biosimilars: Key regulatory considerations and similarity assessment tools. Wiley Biotechnology and Bioengineering, 12(114https:// Retrieved from

4 BIOLOGICS, BIOSIMILARS, AND BIOEQUIVALENTS Biologic a biological product produced by or containing parts of a living organism Vaccines, blood, allergens, recombinant proteins Biosimilar a product similar to an existing biologic with no difference in clinical safety Different from generics, refers to small molecules only Bioequivalent pharmaceutical equivalent of another drug Same active ingredient, dose, route of administration, strength/concentration, comparable bioavailability Aschenbrenner, D. S. (2014). What's in a name? Biologic, biosimilar, bioequivalent, bioavailable. Drug Watch, 114(8),

5 BIOSIMILARS Undergo analytical, animal, and clinical studies Analytical: similarity to reference biologic Animal: assess toxicity Clinical: immunogenicity, pharmacokinetics, pharmacodynamics Unacceptable differences: safety, purity, potency Acceptable differences: shape, color, flavor, preservative, mechanism of action, expiration time, stabilizer/buffer Any differences will be highly evaluated Center for Drug Evaluation and Research. (n.d.). Biosimilars - Biosimilar Development, Review, and Approval. Retrieved from

6 PROJECT GOAL & TECHNIQUES Investigate a new biosimilar profiling service for Novatia using well established, existing techniques Use several model systems relevant to the field Mass Spec disulfide mapping NMR protein folding evaluation

7 MODEL SYSTEMS: RIBONUCLEASE A Small: 13.7 kda 8 Cys & 4 disulfides Function: chop RNA into bits Chosen because: both native and scrambled samples can be purchased, small, stable, many disulfides

8 MODEL SYSTEMS: SOMATOTROPIN (HGH) Size: 22.1 kda 4 Cys, 2 disulfides Function: stimulates growth and cell reproduction/regeneration Chosen because: used as medication for growth deficiencies & other disorders, used in a study by Thermo Scientific demonstrating Pepfinder disulfide mapping capabilities, small, fewer disulfide bonds

9 MODEL SYSTEMS: MSQC4 Size: 150 kda 34 Cys, 16 disulfides Function: n/a Chosen because: complex, has many disulfides, standard used in Novatia s lab, mabs are common type of protein drug

10 MS METHODS Disulfide mapping

11 METHODS Digests: Proteinase K, Trypsin Soluble SMART digest Proteinase K digest unsuccessful, fragments were too small Other preparation: Reduction, alkylation, ribonuclease scrambling Instrument methods/solvents LTQ: initial screening Velos: high resolution analysis Standard approach: data dependent high resolution scan followed by MS/MS on top 5 Pepfinder processing

12 PEPFINDER PROCESSING WORKFLOW Process non-reduced data with pepfinder Reprocess & include reduced data as a reference Generate a theoretical digest & calculate expected masses of disulfide linked peptides Make a combined list of ion lists from both processing runs in Excel Remove all entries without identification or confidence levels Remove duplicates in monoisotopic mass category Filter list to obtain only entries that include SS bonds Compare resulting list with expected disulfide peptides

13 RIBONUCLEASE A (NATIVE & SCRAMBLED) METHODS Soluble SMART digest protocol using 50ug of RNAse Dissolve in 200uL SMART digest buffer, add 5 ul SMART digest trypsin Incubate 70C with 1400rpm rotation for 15min Split samples into two aliquots Add 0.5uL TCEP to one of each sample Let sit at RT for 30min Add 10uL 5% TFA Dilute to 5uM

14 RIBONUCLEASE A RAW DATA Native Ribonuclease A Native Ribonuclease A reduced Scrambled Ribonuclease A Scrambled Ribonuclease A reduced

15 Theoretical Digest (KR-) K1-K E2-K F8-R Q11-K S32-R N34-K D38-R C40-K N62-K N67-R E86-K Y92-K T99-K H105-V Expected SS Bonds Expected Peptide Mass 26:84 (Q11-K31:N67-R85) :95 (C40-K61:Y92-K98) :110 (C40-K61:H105-V124) :72 (N62-K66:N67-R85) :84, 65:72 (Q11-K31:N62-K66:N67-R85) :95, 58:110 C40-K61:Y92-K98:H105-V124) RIBONUCLEASE RESULTS Native RNAse Total of 67 peptides with disulfide bonds identified All expected peptides were represented Of identified peptides with disulfide links, MS area of unexpected peptides was 3.67% Scrambled RNAse Total of 154 peptides with disulfide bonds identified More identified due to variation in scrambling Of identified peptides with disulfide links, MS area of unexpected peptides was 100%

16 SOMATOTROPIN METHODS Soluble SMART digest protocol using 50ug of somatotropin Dissolve in 200uL SMART digest buffer, add 5 ul SMART digest trypsin Incubate 70C with 1400rpm rotation for 15min Split sample into two aliquots Add 0.5uL TCEP to one aliquot Let sit at RT for 30min Add 10uL 5% TFA Dilute to 5uM

17 SOMATOTROPIN RAW DATA Somatotropin Somatotropin reduced

18 SOMATOTROPIN RESULTS Theoretical Digest (KR-) F1-R L9-R A17-R L20-K E39-K Y42-R E65-K S71-R I78-R S95-K D116-R L128-R T135-K Q141-K F146-K N159-R K168-K D169-K V173-R I179-R S184-F Expected SS Bonds Expected Peptide Mass 53:165 Y42-R64 N159-R :189 (I179-R183:S184-F Native somatotropin Total of 11 peptides with disulfide bonds identified All expected peptides were represented All identified peptides were expected with the exception of some with missed cleavages

19 MSQC4 METHODS Soluble SMART digest protocol using 50ug of MSQC4 Dissolve in 200uL SMART digest buffer, add 5 ul SMART digest trypsin Incubate 70C with 1400rpm rotation for 15min Split samples into two aliquots Add 0.5uL TCEP to one of each sample Let sit at RT for 30min Add 10uL 5% TFA Dilute to 5uM Additional methods included alkylation with IAA before digestion Was unsuccessful

20 MSQC4 RAW DATA MSQC4 MSQC4 reduced

21 MSQC4 RESULTS Light chain 22: : Heavy chain 22: : : : :230 & 233: Native MSQC4 Total of 51 peptides with disulfide bonds identified All expected peptides were represented Of identified peptides with disulfide bonds, MS area of unexpected peptides was 3.09% Both inter and intra-chain scrambling observed Light chain C22 participated in 77% of identified scrambled peptides Light to Heavy light 217:heavy

22 MSQC4 RESULTS & ALKYLATION ATTEMPT Alkylation of MSQC4 before digestion and was attempted, however alkylated species were not present in the ion list Looking for +57 species, especially of peptide contacting C22 Additionally, C22 in the light chain was still the biggest participant in scrambling

23 DISULFIDE MAPPING SUMMARY Overall successful: Unexpected scrambling in native conformation found at 4% or less of total MS area Able to differentiate between scrambled and native ribonuclease Similar results to Thermo Scientific analysis of somatotropin Able to detect characteristic scrambling of MSQC4 in the midst of expected peptides Caveats: Difficult to differentiate between % naturally occurring scrambling, scrambling during sample processing, and false results in Pepfinder processing Requires reduced and nonreduced runs Requires expected disulfides to be known, or a sample of the reference biologic comparison If not given, only a rough estimation of scrambling may be provided

24 NMR METHODS Folding structure analysis

25 METHODS Experiment: 2D-HMQC Scrambling Ribonuclease Reduce and denature RNAse Incubate at RT in open container to allow for cysteines to re-oxidize Isolate and lyophilize scrambled protein

26 What it s called: 2D HMQC 1 bond N-H correlation experiment ABOUT THE EXPERIMENT Why it was chosen: well established, commonly used in structure determination of RNAse and other proteins Sensitive to changes in the chemical environment in the vicinity of N-H bonds How long it takes: 5 hrs Using SOFAST HMQC and natural isotopic abundance of 15N Buffer used: 50mM sodium acetate, ph 4.55 Parella, T. (n.d.). 2D HSQC Experiment. Retrieved from Schmeider, P. (n.d.). The SOFAST-HMQC - theorie and application.

27 PROTON SPECTRUM: NATIVE RIBONUCLEASE A Aromatic C-H, Amide, Side chain N-H Alpha C-H Side chain C-H

28 PROTON SPECTRUM: SCRAMBLED RIBONUCLEASE A Aromatic C-H, Amide, Side chain N-H Alpha C-H Side chain C-H

29 NATIVE & SCRAMBLED RIBONUCLEASE A Native Aromatic C-H, Amide, Side chain N-H Native Scrambled Scrambled Aromatic C-H, Amide, Side chain N-H

30 2D NATIVE RIBONUCLEASE A SPECTRUM Proton Native Ribonuclease A Nitrogen

31 2D SCRAMBLED RIBONUCLEASE A SPECTRUM Proton Scrambled Ribonuclease A Nitrogen

32 2D NATIVE & SCRAMBLED RIBONUCLEASE A SPECTRUM Proton Native & scrambled Ribonuclease A overlay Nitrogen

33 NMR CONCLUSIONS Significant difference between scrambled and native confirmation RNAse Shown in both Proton and 2D N-H spectra Most evident in the amide/aromatic C-H/side chain N-H region Also evident in alpha C-H and C-H side chain regions Secondary structure (a helices, b sheets) vs unfolded, stretched out Confirmed the use of this well established technique Homemade scrambled RNAse is possible A vigorous exercise in amicon filtration & dissolving urea

34 OVERALL CONCLUSIONS Successful? Could this work as a new service? What would be required from customers? How would we market it?/what exactly is the service offering? Improvements SOFAST HMQC C-H correlation Quantitative comparison of 2D N-H spectra

35 THANK YOU! Thanks to Mark and Dave for their expertise in MS and NMR, respectively, and everyone for a great summer! QUESTIONS?