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 done by industry

• Hands-on experiences
• Simulated cGMP
• Regulatory compliance
• Industry focused

curriculum

• Industry experienced

instructors

Multi-disciplinary

• Undergraduate,

graduate, industry

• Upstream, downstream,

analytical

• Small, intermediate,

large scales

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 BTEC

Biomanufacturing Minors (Undergrad, Grad) Undergraduate Certificate Post-Graduate Certificate Graduate BIOM

Chemical & Biomolecular Engr.

Biomanufacturing Concentration

Food, Bioprocessing & Nutrition Sciences

Bioprocessing Degree (BBS)

Microbiology

Professional Master in Microbial Biotechnology (MMB)

Biotechnology (BIT)

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

• f Option Year #4 (2011-2012)

BARDA (DHHS) training

• Contract ($860,000 Year 1, renewable
• ver 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

• rganizations 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

• HPLC

– Reverse phase – SEC – Cation exchange

• ELISA
• Capillary electrophoresis

Characterization

• MALDI-TOF
• LC-MS
• MALLS
• Circular dichroism

Purity

• Electrophoresis

– Microchip – SDS-PAGE – Capillary gel electrophoresis

• HPLC

Microbial

• Environmental monitoring
• Bioburden
• Endotoxin
• Identification

Optimization and Validation

• Amino acid analysis (UPLC)
• Total organic carbon (TOC)

BTEC Analytical Case Studies

Quantity, purity, degradation, modification, stability, identity Quantity, purity, aggregation, charge heterogeneity Quantity, variant distribution, potency Quantity, purity, potency

Endotoxin & Bioburden

GFP mAb Bacitracin Influenza Vaccine EM and Contamination

Enumeration, ID Clean

Method Development: Bacitracin

Column: Phenomenex Kinetix C18, 2.6 µm, 150 x 4.6 mm Mobile Phase: 20 mM phosphate, pH 6.0 in MeOH (53%) and MeCN (6%) Isocratic: 0.6mL/min Sample Temp: 4C Column Temp: 40C

10.0 15.0 20.0 25.0 min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 mAU 254nm,4nm (1.00)

Bacitracin A Bacitracin F Bacitracin B

HPLC method fully validated per ICH Q2(R1)

10.0 15.0 20.0 25.0 30.0 35.0 min 5 10 15 20 25 30mV

Detector A Ch1:280nm

10.0 15.0 20.0 25.0 30.0 35.0 min 5 10 15 20 25 30mV

Detector A Ch1:280nm

Protein G affinity purified mAb Purified mAb + carboxypeptidase B

Characterization: Charge Heterogeneity

Parent Acidic Basic

Column: Dionex MAbPac SCX-10, 250 x 4.6 Mobile Phase A: 20 mM MES, 60 mM NaCl, pH 5.6 Mobile Phase B: 20 mM MES, 300 mM NaCl, pH 5.6 Gradient:10% B (0-2 min), 10-55 %B (2-32 min), 55% B (32-37 min), 55-100 %B (37-42 min), 100% B (42-47 min), 100-10% B (47-48 min), 10%B (48-63 min) Column Temp: 30C

Carboxypeptidase B cleaves C-terminal Lys and Arg

10.0 15.0 20.0 25.0 30.0 35.0 min 0.0 2.5 5.0 7.5 10.0 mV

Detector A Ch1:280nm

10.0 15.0 20.0 25.0 30.0 35.0 min 0.0 2.5 5.0 7.5 10.0 mV

Detector A Ch1:280nm

Crude mAb Crude mAb + carboxypeptidase B

Characterization: Charge Heterogeneity

Column: Dionex MAbPac SCX-10, 250 x 4.6 Mobile Phase A: 20 mM MES, 60 mM NaCl, pH 5.6 Mobile Phase B: 20 mM MES, 300 mM NaCl, pH 5.6 Gradient:10% B (0-2 min), 10-55 %B (2-32 min), 55% B (32-37 min), 55-100 %B (37-42 min), 100% B (42-47 min), 100-10% B (47-48 min), 10%B (48-63 min) Column Temp: 30C

10.0 15.0 20.0 25.0 30.0 35.0 min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 mV

Detector A Ch1:280nm

10.0 15.0 20.0 25.0 30.0 35.0 min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 mV

Detector A Ch1:280nm

Protein G affinity purified mAb Purified mAb + PNGase

Characterization: Charge Heterogeneity

Column: Dionex MAbPac SCX-10, 250 x 4.6 Mobile Phase A: 20 mM MES, 60 mM NaCl, pH 5.6 Mobile Phase B: 20 mM MES, 300 mM NaCl, pH 5.6 Gradient:10% B (0-2 min), 10-55 %B (2-32 min), 55% B (32-37 min), 55-100 %B (37-42 min), 100% B (42-47 min), 100-10% B (47-48 min), 10%B (48-63 min) Column Temp: 30C

PNGase (Peptide N-Glycosidase F) cleaves between the innermost GlcNAc and Asn residues of N-linked glycoproteins.

Characterization: Aggregation

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min 500 1000 1500 2000 2500 3000 3500 4000 4500 uV

Parent mAb monomer High MW: mAb aggregates Lower MW: Heavy chain

20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 min

• 5

5 10 15 20 25 30 mAU 280nm,4nm (1.00) 20.0 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 min

• 2.5

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 mAU 280nm,4nm (1.00)

Forced Degradation: GFP + H2O2 at 4 hr

Protein Stability

Parent Oxidized

Purified green fluorescent protein (GFP)

20.0 22.5 25.0 27.5 min 1 2 3 4 5 6 7 mAU 280nm,4nm (1.00) 21.0 22.0 23.0 24.0 25.0 26.0 27.0 28.0 29.0 min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 mAU 280nm,4nm (1.00)

Forced Degradation: GFP + H2O2 at 24 hr

Parent Oxidized

Forced Degradation: GFP + UV254nm

22.5 25.0 27.5 min 5000 10000 15000 20000 25000 30000 uV

Column: Kinetex C18, 150 x 4.6, 2.6- m, 100Å Mobile Phase A: 0.5% TFA in H2O Mobile Phase B: 0.5%TFA in MeCN Gradient:5% B (0-0.2 min), 5-50% B (0.5-29min), 50-80% B (29-31 min), 80% B (31-33 min), 80-5% B (33-34.5 min), 5% B (34.5-39 min) Column Temp: 40C

Protein Stability

Parent Oxidized UV

Forced Degradation Overlay

Protein Identification: Peptide Mass Fingerprinting

Sequence Coverage: 93.2%

20 40 60 80 100 %Int. 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500

m/z

205 mV[sum= 27859 mV] Profiles 1-136 Smooth Av 5 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)

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86-96 110-122 97-109 141-156 141-158 216-238 102-131 46-85 42-85 (MSO) 1-52 (MSO) 127-128 74-107 (MSO) 169-209 141-162

1. Cleave protein with trypsin for >4hr at 37 C

Characterization: Protein biotinylation

Biotinylation Sites: Lys 41, 101, 107, 113, 156, 158

1. Biotinylate GFP by coupling to 1 amines; 2. Cleave with trypsin

20 40 60 80 100 %Int. 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500

m/z

148 mV[sum= 17121 mV] Profiles 1-116 Smooth Av 5 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)

1943 1945 2429 1613 5220 4478 1266 1947 1772 1268 2433 1616 1969 1774 5446 2437 1269 1950 1477 4483 973 1770 3069 2446 1265 5450 5242 2911 3118 3324 5469 5233 3956 4494 3472 3715 4031 4254

Microbial ID Decision Tree

Obvious Contamination?

Yes No

Centrifuge (pellet)

Yes No

Plate Incubate Microscope Plate Incubate Gram Stain

Yes No

MALDI Database? ID?

No Yes

ID MicroSeq ID ID?

No Yes

ID?

Bioreactor Environment

Microbial Identification by MALDI-TOF

Bacillus thuringiensis Unknownus bacterius Status: isolating microbial flora, identifying, banking and building MALDI database