BTEC: Analytical Services and Capabilities Nathaniel Hentz, - PowerPoint PPT Presentation

BTEC: Analytical Services and Capabilities Nathaniel Hentz, Assistant Director Analytical

What is BTEC? • Simulated cGMP facility • Education and training • Process and analytical services • First and largest in the world

Advisory board

Unique educational programs Minimize training Multi-disciplinary done by industry • Undergraduate, graduate, industry • Hands-on experiences • Upstream, downstream, • Simulated cGMP analytical • Regulatory compliance • Small, intermediate, • Industry focused large scales curriculum • Industry experienced instructors

BTEC strategic plan: Economic development • University courses, programs • Industry courses & events • State-of-art facility • Collaborate with industry • National, international leadership in biomanufacturing • Outreach to K-12 • Support community colleges

Facility features • 63,000 gsf labs • 9,000 gsf classrooms • $39 MM infrastructure • $15 MM equipment • $6 MM/y operating

EDUCATION AND TRAINING

Academic programs Chemical & Food, Bioprocessing & Biomolecular Engr. Nutrition Sciences Biomanufacturing Bioprocessing Degree Concentration (BBS) BTEC Biomanufacturing Minors (Undergrad, Grad) Undergraduate Certificate Post-Graduate Certificate Graduate BIOM Microbiology Biotechnology (BIT) Professional Master in Microbial Biotechnology (MMB)

Professional development courses Tracks Biomanufacturing Bioprocess Development Bioprocess Engineering Analytical Technologies Customized courses also available

FDA training Contract ($455,000 over 5 years) • – 4 hybrid online/hands-on courses • Upstream bioprocessing • Downstream bioprocessing • QC/Analytical • Aseptic processing – Cohorts of 16 inspectors trained annually – FDA approved execution of Option Year #4 (2011-2012)

BARDA (DHHS) training Contract ($860,000 Year 1, renewable • over 5 years) – cGMP Influenza Vaccine Manufacturing • Regulatory/quality systems • Facilities and utilities • Upstream bioprocessing • Downstream bioprocessing • QC/Analytical • Aseptic processes – Three cohorts of 12 students – 11 countries; 3-week sessions

BIOPROCESS AND ANALYTICAL SERVICES

Bioprocess and analytical services capabilities BTEC offers: Faculty with expertise in biomanufacturing topics • Staff with hands-on biomanufacturing experience • Facilities and equipment ideal for this type of work • Students eager to learn • Flexibility in the types of projects • – “Risk - free environment” – Smaller projects that contract organizations not likely to take on

Analytical Services • Analysis and Testing – Amino acid analysis on spent media (UPLC) – Small molecule concentration in chicken plasma (HPLC) – Isoflavone concentration in soybean extract (HPLC) – Antiviral activity (qPCR) – Environmental monitoring (bioburden, coliform, LAL) • Assay Development – Protein expression (Western Blot) • Process Development Support – SDS-PAGE – Bioburden – Endotoxin (gel-clot) – Microbial ID

BTEC Analytical Capabilities Quantitation Purity • HPLC • Electrophoresis – Reverse phase – Microchip – SEC – SDS-PAGE – Cation exchange – Capillary gel electrophoresis • ELISA • HPLC • Capillary electrophoresis Microbial • Environmental monitoring Characterization • MALDI-TOF • Bioburden • LC-MS • Endotoxin • MALLS • Identification • Circular dichroism Optimization and Validation • Amino acid analysis (UPLC) • Total organic carbon (TOC)

BTEC Analytical Case Studies Quantity, purity, degradation, Endotoxin modification, GFP & stability, identity Bioburden Quantity, purity, mAb aggregation, charge heterogeneity Bacitracin Quantity, variant distribution, potency Influenza Vaccine Quantity, purity, potency EM and Clean Contamination Enumeration, ID

Method Development: Bacitracin mAU 254nm,4nm (1.00) Column: Phenomenex Kinetix C18, 2.6 µm, 22.5 150 x 4.6 mm Bacitracin A 20.0 Mobile Phase: 20 mM phosphate, pH 6.0 in 17.5 MeOH (53%) and MeCN (6%) 15.0 Isocratic: 0.6mL/min 12.5 Sample Temp: 4  C 10.0 Bacitracin B Column Temp: 40  C 7.5 5.0 Bacitracin F 2.5 0.0 10.0 15.0 20.0 25.0 min HPLC method fully validated per ICH Q2(R1)

Characterization: Charge Heterogeneity 30mV Detector A Ch1:280nm Parent Protein G affinity Column: Dionex MAbPac SCX-10, 250 x 4.6 25 purified mAb Mobile Phase A: 20 mM MES, 60 mM NaCl, 20 pH 5.6 15 Mobile Phase B: 20 mM MES, 300 mM NaCl, Acidic Basic pH 5.6 10 Gradient:10% B (0-2 min), 10-55 %B (2-32 5 min), 55% B (32-37 min), 55-100 %B (37-42 min), 100% B (42-47 min), 100-10% B (47-48 0 10.0 15.0 20.0 25.0 30.0 35.0 min min), 10%B (48-63 min) 30mV Detector A Ch1:280nm Purified mAb + Column Temp: 30  C 25 carboxypeptidase B 20 15 10 Carboxypeptidase B cleaves 5 C-terminal Lys and Arg 0 10.0 15.0 20.0 25.0 30.0 35.0 min

Characterization: Charge Heterogeneity mV Detector A Ch1:280nm Crude mAb 10.0 Column: Dionex MAbPac SCX-10, 250 x 4.6 7.5 Mobile Phase A: 20 mM MES, 60 mM NaCl, pH 5.6 5.0 Mobile Phase B: 20 mM MES, 300 mM NaCl, 2.5 pH 5.6 Gradient: 10% B (0-2 min), 10-55 %B (2-32 0.0 10.0 15.0 20.0 25.0 30.0 35.0 min min), 55% B (32-37 min), 55-100 %B (37-42 mV min), 100% B (42-47 min), 100-10% B (47-48 Detector A Ch1:280nm Crude mAb + min), 10%B (48-63 min) 10.0 carboxypeptidase B Column Temp: 30  C 7.5 5.0 2.5 0.0 10.0 15.0 20.0 25.0 30.0 35.0 min

Characterization: Charge Heterogeneity mV Detector A Ch1:280nm 25.0 Protein G affinity 22.5 purified mAb 20.0 Column: Dionex MAbPac SCX-10, 250 x 4.6 17.5 15.0 Mobile Phase A: 20 mM MES, 60 mM NaCl, pH 5.6 12.5 10.0 Mobile Phase B: 20 mM MES, 300 mM NaCl, pH 5.6 7.5 5.0 Gradient: 10% B (0-2 min), 10-55 %B (2-32 min), 55% 2.5 B (32-37 min), 55-100 %B (37-42 min), 100% B (42-47 0.0 min), 100-10% B (47-48 min), 10%B (48-63 min) 10.0 15.0 20.0 25.0 30.0 35.0 min mV Detector A Ch1:280nm Column Temp: 30  C 25.0 Purified mAb 22.5 + PNGase 20.0 17.5 15.0 12.5 PNGase (Peptide N-Glycosidase F) cleaves 10.0 between the innermost GlcNAc and Asn 7.5 residues of N-linked glycoproteins. 5.0 2.5 0.0 10.0 15.0 20.0 25.0 30.0 35.0 min

Characterization: Aggregation uV 4500 Parent mAb monomer 4000 3500 3000 High MW: mAb 2500 aggregates 2000 1500 1000 Lower MW: Heavy chain 500 0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

Protein Stability mAU mAU 280nm,4nm (1.00) 20.0 280nm,4nm (1.00) Forced Degradation: 30 Purified green fluorescent 17.5 GFP + H 2 O 2 at 4 hr 25 protein (GFP) 15.0 20 12.5 10.0 Oxidized 15 7.5 10 Parent 5.0 5 2.5 0 0.0 -2.5 -5 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 min 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 min mAU mAU 280nm,4nm (1.00) Forced Degradation: 280nm,4nm (1.00) 17.5 7 Forced Degradation: GFP + H 2 O 2 at 24 hr 15.0 6 GFP + UV 254nm 12.5 5 Oxidized 10.0 4 3 7.5 Parent 2 5.0 1 2.5 0 0.0 20.0 22.5 25.0 27.5 min 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 min

Protein Stability Forced Degradation Overlay uV Column: Kinetex C18, 150 x 4.6, 2.6- 30000 Parent  m, 100Å 25000 Mobile Phase A: 0.5% TFA in H 2 O Mobile Phase B: 0.5%TFA in MeCN 20000 Oxidized Gradient:5% B (0-0.2 min), 5-50% B 15000 (0.5-29min), 50-80% B (29-31 min), 80% B (31-33 min), 80-5% B (33-34.5 10000 min), 5% B (34.5-39 min) UV 5000 Column Temp: 40  C 0 22.5 25.0 27.5 min

Protein Identification: Peptide Mass Fingerprinting Cleave protein with trypsin for >4hr at 37  C 1. Data: Tryp4h_GFP_05Aug2011_0001.G16[c] 5 Aug 2011 15:56 Cal: CAL_peptide-070810 8 Jul 2010 14:59 Shimadzu Biotech Axima Assurance 2.8.1.20080410: Mode Linear, Power: 85, Blanked, P.Ext. @ 6000 (bin 98) %Int. 205 mV[sum= 27859 mV] Profiles 1-136 Smooth Av 5 141-156 216-238 2592 100 42-85 (MSO) 86-96 1946 5220 1266 80 141-162 1944 110-122 1268 2615 60 74-107 (MSO) 102-131 1968 1479 2608 1289 169-209 1970 141-158 1-52 (MSO) 97-109 1480 127-128 5242 3472 40 46-85 5490 2202 2619 4479 5236 1287 1544 1077 3729 4749 4143 2621 5254 3116 3495 4500 20 1311 5512 3927 3751 4771 4166 2945 3130 3516 0 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 m/z Sequence Coverage: 93.2%

Characterization: Protein biotinylation Biotinylate GFP by coupling to 1  amines; 1. 2. Cleave with trypsin Data: Tryp4h_GFPb_05Aug2011_0001.G15[c] 5 Aug 2011 15:54 Cal: CAL_peptide-070810 8 Jul 2010 14:59 Shimadzu Biotech Axima Assurance 2.8.1.20080410: Mode Linear, Power: 85, Blanked, P.Ext. @ 6000 (bin 98) %Int. 148 mV[sum= 17121 mV] Profiles 1-116 Smooth Av 5 1943 100 1945 80 2429 1613 5220 4478 1266 1947 1772 60 1268 2433 1616 1969 1774 5446 2437 1269 1477 1950 40 973 4483 1770 3069 2446 1265 5450 2911 5242 3118 3324 5469 3956 5233 4494 20 3472 3715 4031 4254 0 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 m/z Biotinylation Sites: Lys 41, 101, 107, 113, 156, 158

Microbial ID Decision Tree Obvious Contamination? Yes No Bioreactor Centrifuge Plate (pellet) Yes No ID? Incubate Microscope Plate ID? ID? Environment No Yes Yes No Incubate Gram Stain MALDI Database? ID Yes No MicroSeq ID