Drug Substance Process Challenges- A Vaccine Perspective. May 17, 2016 Justin Moran

Transcription

1 Drug Substance Process Challenges- A Vaccine Perspective May 17, 2016 Justin Moran

2 Outline Background Vaccines versus monoclonal antibodies Case study: Development of a protein antigen 2

3 A Legacy of Achievement in Preventing Disease Wyeth Lederle Praxis Biologics Pfizer Pfizer Smallpox vaccine developed at company s Lancaster County vaccine farm A combined vaccine for diphtheria, tetanus and pertussis in young children Lederle First to license a conjugate-based vaccine for H. influenzae type b Lederle Wyeth Lederle First 13-valent pneumococcal conjugate vaccine for infants and young children First vaccine approved by FDA to prevent serogroup B Meningococcal disease Pfizer Diphtheria antitoxin is first FDA-licensed product manufactured at company s Pearl River site First to license oral form of live trivalent poliovirus vaccine in the United States First to license a meningococcal serogroup C conjugate vaccine Prevnar 13 approved for adults 50 years and older 3

4 What Makes a Good Vaccine Vaccines are composed of single or multiple antigens Want to attack different cycles of infection or different strains May be either proteins, viruses, plasmids, polysaccharides/conjugates or a combination Can be manufactured with consistent physical and chemical properties Induces high avidity functional antibodies (bactericidal or opsonic) Is clinically safe with no inherent biological properties or toxicity 4

5 Important Factors in Vaccine Process Development For protein antigens, DS development is similar to other recombinant protein development Vaccine antigens do not have biological activity Process must be locked when material is made for toxicology studies Significant early investment Very short timelines from antigen selection to process lock Process must not only produce high purity antigens but also be robust/scalable 5

6 Differences in Process Development Between Vaccines and mabs 6

7 Difference between Vaccines and mabs Expression System Vaccines: Large variety (E. coli, native organism, mammalian, yeast, etc) mabs: CHO 7

8 Difference between Vaccines and mabs Expression System Vaccines: Large variety (E. coli, native organism, mammalian, yeast, etc) mabs: CHO, NSO Level of Expression Vaccines: Variable 3mg/L-3g/L, typically <1g/L mabs: 3-5g/L 8

9 Difference between Vaccines and mabs Expression System Vaccines: Large variety (E. coli, native organism, mammalian, yeast, etc) mabs: CHO, NSO Level of Expression Vaccines: Variable 10mg/L-3g/L, typically <1g/L mabs: 3-5g/L Fermentation Time Vaccines: Microbial systems 1-2 days mabs: days 9

10 Difference between Vaccines and mabs Expression System Vaccines: Large variety (E. coli, native organism, mammalian, yeast, etc) mabs: CHO, NSO Level of Expression Vaccines: Variable 10mg/L-3g/L, typically <1g/L mabs: 3-5g/L Fermentation Time Vaccines: Microbial systems <24hr mabs: days Release Vaccines: Most are expressed intracellular, need to lyse cells mabs: Secreted 10

11 Difference between Vaccines and mabs Capture Step Vaccines: Depends on protein mabs: Protein A 11

12 Difference between Vaccines and mabs Capture Step Vaccines: Depends on Protein mabs: Protein A Platform Process Vaccines: Difficult due to differences in protein attributes mabs: Typically 2-3 column process 12

13 Difference between Vaccines and mabs Capture Step Vaccines: Depends on Protein mabs: Protein A Platform Process Vaccines: Difficult due to differences in protein attributes mabs: Typically 2-3 column process Viral Clearance Vaccines: Not an issue for microbial systems mabs: Required, must demonstrate LRV 13

14 Difference between Vaccines and mabs Capture Step Vaccines: Depends on Protein mabs: Protein A Platform Process Vaccines: Difficult due to differences in protein attributes mabs: Typically 2-3 column process Viral Clearance Vaccines: Not an issue for microbial systems mabs: Required, must demonstrate LRV Material Requirements (typical doses) Vaccines: 2-100ug mabs: mg 14

15 Process Development for a Protein Antigen 15

16 What is C. difficile? C. difficile is a Gram-positive, spore-forming, anaerobic pathogen 1,2 Source: Image courtesy of CDC PHIL, image ID 9999, Lois S. Wiggs. C. difficile spores are highly resistant to heat and commonly used hospital cleaning agents, such as general-purpose detergents and ethanol-based hand sanitizers, and can persist on hard surfaces for months 3-6 C. difficile can produce toxins that may cause disease 1 1. Centers for Disease Control and Prevention. Frequently asked questions about Clostridium difficile for healthcare providers [updated 6 March 2012]. 2. Centers for Disease Control and Prevention. Emerging infections program [updated August 2015]. 3. Fawley WN et al. Infect Control Hosp Epidemiol. 2007;28: McFarland LV et al. N Engl J Med. 1989;320: Fekety R et al. Am J Med. 1981;70: Rodriguez-Palacios A et al. Anaerobe. 2010;16:

17 CDI Background C. difficile Infection (CDI) Primary cause of antibiotic related infectious diarrhea in elderly Increase in incidence, severity and recurrence in recent years Caused by exotoxins A (308kDa) and B (270kDa) Glucosyltransferase domain (GT) Autocatalytic processing domain (CP) Translocation domain Cell binding domain Glycosylation of Rho family GTPases by cytotoxic glucosyltransferase (GT) results in apoptosis and cell death Develop a prophylactic vaccine to prevent CDI 17

18 Significant Challenges to Manufacturing Toxins Significant challenges to large-scale manufacturing of toxins Exposure to toxins Decontamination of facilities of heat-resistant spores Low levels of expression Separation of toxin A from toxin B Take advantage of molecular biology Construct strains unable to form spores Site directed mutation to neutralize previously defined cytotoxicity determinants GT activity Autoproteolytic release Recognition of Rho GTPase substrates Donald et al Microbiology (2013) 18

19 Challenges to Upstream Process Development Selection of production host E. coli Lots of experience with other proteins Issues with expression of large proteins C. diff low levels of expression not suitable for commercial process HCPs not as big of an issue Overcoming low levels of expression (10mg/L) in native host 10-fold increase in cell density Glucose supplementation and ph control Use of fdx promoter Lack of PaLoc locus allowed for production of toxoids separately 19

20 Comparable growth and titers achieved for both TxdA and TxdB Growth (OD600) Toxoid production (mg/l) Growth (OD600) Toxoid production (mg/l) Toxoid A Toxoid B Fermentation time (hours) Fermentation time (hours) 20

21 Challenges to Downstream Process Development Physical properties of the proteins Toxoid A MW: 307kDa 6 cysteine residues (no disulfides) pi 5.6 Toxoid B MW: 270kDa 8 cysteine residues (no disulfides) pi 4.3 Limit exposure to high shear to prevent aggregation Limit ph of buffers to <7.5 to minimize disulfide formation 21

22 Purification Process for Toxoid A Toxoid A Recovery-Flocculation followed by centrifugation/depth filtration Removes HCP and nucleic acid impurities Improves clarity of column load Capture-HIC Eliminates need for buffer exchange after flocculation and before polishing step Polishing-CEX Low ph minimizes disulfide formation UFDF Exchange into final buffer Yield ~60% 22

23 SDS-PAGE of Toxoid A in-process samples Lane 1. Lysate 2. Floc centrate 3. HIC load 4. HIC pool 5. CEX pool 6. Final 7. MW markers 23

24 Purification Process for Toxoid B Toxoid B Recovery-Centrifugation Flocculation and depth filtration not possible due to low pi Capture-AEX Low pi allows AEX to be performed at neutral ph Take advantage large beads to allow insoluble impurities to flow through Polishing-HIC Eliminates the need for buffer exchange after AEX column UFDF Exchange into final buffer Yield ~60% 24

25 SDS-PAGE of Toxoid B in-process samples Lane 1. Lysate 2. AEX load 3. AEX pool 4. HIC pool 5. Final 6. MW markers 25

26 Residual cytotoxic activity still observed Genetic mutations reduced cytotoxic activity by ~4log Toxin A (0.9 ng/ml), Toxoid A (9 ug/ml) Toxin B (0.009 ng/ml), Toxoid B (30 ng/ml) Would like to reduce cytotoxicity to <1000 ug/ml Approaches to reduce cytotoxicity Genetic modification Difficult since source of residual cytotoxic activity is not know Chemical modification Formalin inactivation Used historically to inactivate toxins Modification unstable which results in reversion Other chemical methods 26

27 Use of EDC and NHS to inactivate toxins Advantages Used commercially to conjugate a variety of molecules to proteins Small molecules that can be easily removed Short reaction times Reactions can be performed at room temperature Forms a stable amide bond Forms cross links and other modification Disadvantages Has never been used to inactivate toxins Unknown impact on key epitopes Any regulatory concerns 27

28 Inactivation with EDC/NHS resulted in desired reduction in cytotoxic activity without reversion EC50 (ug/ml) Time (weeks) TxdA-EDC TxdA-FI 0 >1000 > > > > > EC50 (ug/ml) Time (weeks) TxdB-EDC TxdB-FI 0 >1000 > > > > >

29 Retention of key epitopes after inactivation Relative Percent Binding measured using 10 anti-txda mabs CDIFA mabs TxdA-EDC 91±1 92±2 97±2 97±2 96±2 93±2 95±2 97±2 92±2 92±1 Relative Percent Binding measured using 8 anti-txdb mabs CDIFB mabs TxdB-EDC 87±1 84±3 81±1 83±4 76±3 101±4 41±4 83±3 All antibodies used in this study were shown to be neutralizing and targeted various domains Percent binding is relative to genetically modified toxoids Pfizer Confidential 29

30 Serum of hamsters immunized with inactivated toxoids neutralize wild type C. difficile toxins The 50% neutralization titers for both A and B were >10 4 (defined as the reciprocal of serum dilutions that neutralize 50% of the toxins). Sera was collected two weeks post dose 3 30

31 Summary Due to process lock at reg tox, significant early investment in process development is needed Process used to make material for toxicology studies must not only yield high purity but also be robust/scalable Due to differences in physical properties, platform purification processes not typically applicable Since vaccines contain multiple antigens, must be able to switch between proteins, polysaccharides, etc Vaccine to treat CDI Developed fermentation/purification processes that yield high purity antigens Used innovative method to reduce residual cytotoxic activity Demonstrated ability to induce neutralizing antibodies 31

32 Acknowledgements Fermentation Jason Lotvin Sujata Patel-Brown Purification Eugene Vidunas Antony Mathews Michele Weaver Ping Cai Eun Hee Koh Hailey Yuan Zi-rong Zheng Analytical Karen Xu Marjolaine Carriere Bio-assay J. Erik Johnson Management Mark Ruppen Annaliesa Anderson Kathrin Jansen Michael Pride 32