Download Kempbio PRISM Talk 12-9

How waves of innovation in biotechnology shaped a small
business venture.
Chris Kemp
Kempbio, Inc. 5119 Pegasus Ct. Suite P. Frederick, MD 21704
PrincetonInternational SchoolofMathematics andScience
December 9,2016
A Riddle for You
When do multiplication and division
mean the same thing?
When You are Studying Biology!
POP QUIZ !
POP Quiz !
• Name Three Profoundly Destabilizing Scientific Ideas
from the 20th Century
– Transformed culture, language, politics and society
• Hint #1
– One was from chemistry/physics
– One dealt with information
– One was from Biology
POP Quiz !
• Name Three Profoundly Destabilizing Scientific Ideas
from the 20th Century
– Transformed culture, language, politics and society
• Hint #1
– One was from chemistry/physics
– One dealt with information
– One was from Biology
• Hint #2
– All are basic “building blocks”
– All are the least divisible unit of a larger form
POP Quiz: Answer
• The Atom (matter)
• The Byte (digitized information)
• The Gene (biological information)
Taken from: The Gene An Intimate History by Siddhartha Mukherjee
How waves of innovation in biotechnology shaped a small
business venture.
Chris Kemp
Kempbio, Inc. 5119 Pegasus Ct. Suite P. Frederick, MD 21704
PrincetonInternational SchoolofMathematics andScience
December 9,2016
Kempbio, Inc.
5119 Pegasus Ct. Suite P Frederick, MD 21704 USA
www.kempbioinc.com
History
• Kempbio, Inc. founded December 2008 at FITCI
– FITCI = Frederick Technology Incubator
• Graduated FITCI September 2012
• Built-out 3000 sq.ft. facility at Westview Business
Park, Frederick 2012 with 7-year lease
• Past employees of Kemp Biotechnologies, Inc.
– 1992 to 2007 (Sold in 2006)
– GeneChoice 2000 to 2008 (Sold in 2006)
• 20+ years of experience working as a CRO for the
Biopharmaceutical industry
• Private company located in Frederick, MD
Personnel
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Chris Kemp, Ph.D., President
April Birch, B.S., Director of Cell Culture
Heather Allen, B.S., Sr. Scientist Cell Culture
Pat Kemp, B.S., Vice President
Kerrie Kenefick, B.S., Purification Technician
Gary Bechanan, CPA, Comptroller
Company Information
• 72 Customers
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3 Large Pharma
4 US Government Agencies
5 Academic Institutions
60 Biopharms and Biotechnology Companies
Facility
• Kempbio, Inc. is located in Frederick, MD USA
Protein Purification Lab
• Kempbio, Inc. has 2,000 sq. ft. of laboratory space
Main Lab
• Kempbio, Inc. has 2,000 sq. ft. of laboratory space
Main Lab
• Kempbio, Inc. has 2,000 sq. ft. of laboratory space
100L SUB
Kempbio’s Business
Production of Proteins
From Genes
Kempbio is a
Gene to Protein Company
Gene to Protein Concept
Gene to Protein Reality
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>Contig pCI-X-His Sequence
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ATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACT
GGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGC
GTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTC
TTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCGCCACCATGGGATGGTCCTGCATCATTCTGTTCCTGGTGGC
AACTGCAACCGGAGCACACAGCTCCACTGAGGAACTGTTTAAGGAGTACAAACTGACCCGACCTTATATGGCCCGGTG
CATCAGATGTGCTGTGGGCTCCTGTCACTCTCCAATCGCTATTGAAGCAGTGAAGTCTGACGGGCATGATGGATACGT
CCGCCTGCAGACCTCTAGTCAGTATGGCCTGGACTCAAGCGGCAACCTGAAGGGGAGGACAATGCGCTACGATATGCA
CGGGACTATCAAAGAGATTCCTCTGCACCAGGTGAGTCTGCATACATCAAGACCATGCCACATCGTCGACGGACATGG
CTATTTCCTGCTGGCAAGGTGTCCAGCAGGCGACTCTATTACCATGGAGTTTAAGAAAGATAGTGTGACACACAGCTGC
TCCGTGCCCTACGAGGTCAAGTTCAACCCTGTCGGCAGGGAACTGTATACCCACCCCCCTGAGCATGGGGTGGAACAG
GCTTGTCAGGTCTACGCCCACGACGCTCAGAATCGCGGAGCATATGTGGAGATGCATCTGCCTGGCTCTGAAGTGGAT
TCCTCTCTGGTCTCTCTGAGTGGGAGTTCAGTGACAGTCACTCCACCCGTGGGAACAAGTGCCCTGGTCGAGTGCGAA
TGTGGCGGGAAGAAAATCTCAGAGACTATTAATAGAACCAAGCAGTTCTCCCAGTGCACTAAGAAAGAACAGTGTCGA
GCATACCGGCTGCAGAACGACAAATGGGTGTATAATAGCGATAAGCTGCCCAAAGCCGCTGGGGCTACCCTGAAGGGA
AAACTGCACGTGCCCTTTCTGCTGGCAGACGGGAAGTGCACAGTCCCTCTGGCCCCCGAGCCTATGATCACTTTCGGA
TTTCGCTCAGTGAGCCTGAAGCTGCATCCAAAAAACCCCACTTACCTGACCACACGACAGCTGGCCGATGAGCCACAC
TATACCCATGAGCTGATTTCCGAACCCGCTGTGCGGAACTTCACCGTCACAGAGAAGGGCTGGGAATTCGTGTGGGGG
AATCACCCTCCAAAAAGATTTTGGGCTCAGGAGACAGCACCTGGAAATCCACACGGCCTGCCACATGAAGTGATCACC
CACTACTATCATCGGTACCCCATGAGCACAATTCTGGGCCTGTCCCACCATCACCATCACCATTAATGAGCGGCCGC
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>Predicted protein sequence (397 aa, MW=44,336.58)
MGWSCIILFLVATATGAHSSTEELFKEYKLTRPYMARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRY
DMHGTIKEIPLHQVSLHTSRPCHIVDGHGYFLLARCPAGDSITMEFKKDSVTHSCSVPYEVKFNPVGRELYTHPPEHGVEQACQVYA
HDAQNRGAYVEMHLPGSEVDSSLVSLSGSSVTVTPPVGTSALVECECGGKKISETINRTKQFSQCTKKEQCRAYRLQNDKWVYNSD
KLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGFRSVSLKLHPKNPTYLTTRQLADEPHYTHELISEPAVRNFTVTEKGWEF
VWGNHPPKRFWAQETAPGNPHGLPHEVITHYYHRYPMSTILGLSHHHHHH
Promega pCi Vector
Basic Methods of Gene Transfer
Gene to Protein Reality
How waves of innovation in biotechnology shaped a small
business venture.
Chris Kemp
Kempbio, Inc. 5119 Pegasus Ct. Suite P. Frederick, MD 21704
PrincetonInternational SchoolofMathematics andScience
December 9,2016
INNOVATION
• The introduction of something new
• A new idea, method or device
INNOVATION
• The introduction of something new
• A new idea, method or device
Innovation Paradox: WSJ 12-9-16
TRUE INNOVATION
• 1977 Herbert Boyer clones the gene for
human insulin into E. coli and expresses the
recombinant peptide hormone
TRUE INNOVATION
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1958 BS St. Vincent College LaTrobe, PA
1963 PhD University of Pittsburgh
Post-Doc at Yale, Asst. Prof at UCSF
1976 Co-founded Genentech
Innovations That Guide Kempbio
• ViroCyt Virus Counter
• BacMam
Baculovirus Plaque Titer Method
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Perform serial 10X dilutions
Overlay with agarose
Incubate 7 Days
Stain with Neutral Red Dye
Count Plaques and correct for dilution
Endpoint is infectious titer
– Plaque Forming Unit (pfu)
Rapid Virus Titer Methods
• Plaque Titer (not a rapid method)
• Expression Systems Flow Cytometry
• Clontech BacPAK™ Rapid Titer Methods
– Immunoassay
– qPCR
• Izon particle counter
• ViroCyt virus particle counter
ViroCyt Virus Counter
Virus Counter part of a collaboration with ViroCyt
Virus Counter (ViroCyt, LLC)
• A flow cytometer-based system
developed specifically to quantify
viruses using a dual fluorescence
staining approach
• With this “Combo Dye” system, viral
genomes and surface proteins are
stained with fluorogenic dyes that emit
in the yellow and red regions of the
visible spectrum, respectively
• Fluorescence emission is detected in
two separate optical channels where
optical compensation hardware and
software elements correct for any
crosstalk between channels
Virus Counter (ViroCyt, LLC)
• When voltage pulses are
simultaneously observed in both
the nucleic acid and protein
emission channels, the
“simultaneous event” is counted
as an intact virus particle
• The number of simultaneous
events counted during the
analysis time is used in
combination with sample flow rate
to calculate the concentration of
virus particles per milliliter of
sample
Intact Virus Particles
Scatter Plot: Plaque Titer vs ViroCyt vp/mL titer
r2 = 0.95
Slope = 0.025 pfu/mL per vp/mL
Y-intercept = -3.7x106 pfu/mL
1.20E+08
1.00E+08
pfu/mL
8.00E+07
6.00E+07
4.00E+07
2.00E+07
0.00E+00
0.00E+00
1.00E+09
2.00E+09
vp/mL (ViroCyt)
3.00E+09
4.00E+09
Virus Production based on Plaque and ViroCyt Titers
MOI = 0.02
1x106 cells per mL
SF900-III
A = Plaque Titer
B = ViroCyt Titer
7.00E+09
Virus Particles per mL
6.00E+09
5.00E+09
4.00E+09
591 A
591 B
3.00E+09
751 A
751 B
2.00E+09
1.00E+09
0.00E+00
0
20
40
60
80
Time Post-Infection (hours)
100
120
Baculovirus in Supernatant of Stirred-tank Bioreactor
3.00E+09
Virus Particles per mL
2.50E+09
Sf9 Cells
MOI = 2.0
Cell Density = 2x106 cells per mL
Titers by ViroCyt 2100
Each point = Mean of 3 titers
2.00E+09
1.50E+09
1.00E+09
5.00E+08
0.00E+00
0
20
40
60
80
Time Post-Infection (Hours)
100
120
Baculovirus Particles in Culture Supernatant
2.50E+09
Virus Particles per mL
2.00E+09
1.50E+09
High Five
Sf9
1.00E+09
5.00E+08
0.00E+00
24
36
48
Elapsed Time (h.)
60
72
Innovations That Guide Kempbio
• ViroCyt Virus Counter
• BacMam
Basic Methods of Gene Transfer
BacMam
Invitrogen 2008
• Mammalian CMV
Promoter
• Express rProteins in
a variety of
mammalian cells
using a baculovirus
as the delivery
vehicle
BacMam Transfer Vector
Second Generation Bacmam Vectors
VSV-G Coat Protein (Panels C-G) on envelope of budded virus
allows increased entry into cells
Rick Boyce 2011
GFP BacMam HEK-293
BacMam Protocol CHO and HEK
• For 1L- 100L expression
– Cells at 1.5 to 2x106 cells per mL for HEK-293
– Up to 4x106 cells per mL for CHO-S
– Generate BacMam virus in Sf9 cells
• If >10% culture volume concentrate virus
• Determine virus titer using ViroCyt 3100 to determine if need concentration
– Add cells and incubate at 37°C + 5% CO2 for 72-144h.
– Add 10 mM sodium butyrate (required for CHO, optional for HEK)
• Media formulations that work with BacMam
– LTC: Freestyle-CHO, Freestyle-293
– Expression Systems: ESF SFM, ESF 921
Effect of Cell Density & MOI on rIgG Yield
Cell Density:
A = 2x106 cells per mL
B = 3x106 cells per mL
C = 4x106 cells per mL
CHO-S/CD-CHO
MOI = 50 (25:25)
IgG B Bacmam
MOI: Various
mg Purified IgG per Liter
160
140
120
CHO-S/CD-CHO
4x106 cells per mL
100
80
60
40
20
0
0
10
20
30
40
BacMam MOI
50
60
1L Shake-Flask MOI = 50
96h. PT Harvest
Purified Yield = 150 mg/L
Humanized rIgG: 10L Bioreactor
SDS PAGE Gel Coomassie Stained
Non-Reduced
Culture Supernatant
Serum-Free Freestyle Medium
HEK-293/MOI = 50
24, 48, and 72h. Post-Transduction
10-Liter Stirred-Tank Bioreactor
Purified Yield = 140 mg/L
Total Yield: 1.4 Grams rIgG
BacMam H7N1 VLPs
Medigen 2014
BacMam H7N1 VLPs
• First Attempt
– Ratio of H7:N1:gag was 1:1:1
– Fresh Virus Tittered using the ViroCyt 2100
– MOI was 10:10:10 (30 Total)
• HEK-293 Shake-Flask 1L
– Harvested supernatant at 120h. PT
– Purified by centrifugation
– Analyzed by TEM
BacMam H7N1 VLPs
Medigen 2014
BacMam H7N1 VLPs
• Second Attempt
– Ratio of H7:N1:gag was 1:1:0.5
– Fresh Virus Tittered using the ViroCyt 2100
– MOI was 14:14:7 (35 Total)
• HEK-293 Shake-Flask 1L
– Harvested supernatant at 120h. PT
– Purified by centrifugation
– Analyzed by TEM
BacMam H7N1 VLPs
Medigen 2014
Basic Methods of Gene Transfer
Ebola Classification
Family: Filoviridae
Genus: Ebolavirus
Species:
1. Zaire ebolavirus
2. Sudan ebolavirus
3. Tai Forest ebolavirus
4. Bundibugyo ebolavirus
5. Reston ebolavirus
http://www.rcsb.org/pdb/101/motm.do?momID=178
Ebola Zaire Glycoprotein
• Mayinga Zaire Strain
– 1976
• Trimeric
transmembrane protein
• Heavily glycosylated
• Mucin domain
• Transmembrane
domain deleted = dTM
– MW ~120 kDa
http://www.rcsb.org/pdb/101/motm.do?momID=178
• Expressed as secreted
product in HEK-293
Total Ebola Cases 2014-2015
http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html
Ebola Outbreak 2014-2015
http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/index.html
Zaire-dTM Vector Comparison
PEI Vector
BacMam Vector
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pCi Transient Expression
Vector
CMV Promoter
HA-Tag
Synthetic Gene
Human Signal Sequence
Transmembrane domain
deleted
Codon Optimized for Human
Cells
Gateway Entry Vector
– Synthesized in standard cloning
vector with ATT sites flanking
ORF
– Bacmid transfected into Sf9 for
BacMam generation
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CMV Promoter
Transmembrane domain
deleted
Human Signal Sequence
Codon Optimized for Human
Cells
1-Liter Z-dTM PEI and BacMam
• HEK-293 Cells
• 1x1000 mL flasks
– Duplicate loadings
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1.5x106 cells per mL
Freestyle, serum-free
PEI: 4:1 ratio with DNA
PEI: 1 mg DNA per L
PEI: Harvest 96h. PT
BacMam: MOI = 50
BacMam: Harvest 72h. PT
Load concentrated
Q-capture pool
onto S-200
Sephacryl column
Buffer = PBS
Flow = 0.5 mL per
min.
Run SDS PAGE
Pool ZdTM
fractions
Concentration and QC
10mL QSepaharose/L
Batch Bind 16h.
Pack Column
Wash
Elute using linear
gradient 50-250
mM NaCl at pH 7.0
Size-Exclusion
Q Capture Step
Z-dTM Purification Scheme: 1-40-liters
Concentrate using
Centricon 10kDa
MWCO
Sterile Filtration
Vialing
Coomassie-stained
SDS PAGE and
Western Blot (antiZdTM
Z-dTM PEI: Q-Sepharose
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Q-Sepharose FF
20 mM Tris pH 7
Linear Gradient
– 50-250 mM NaCl
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5 mL Fractions
SDS PAGE
– Coomassie-stained
Z-dTM BacMam: Q-Sepharose
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Q-Sepharose FF
20 mM Tris pH 7
Linear Gradient
– 50-250 mM NaCl
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5 mL Fractions
SDS PAGE
– Coomassie-stained
Z-dTM PEI: S-200 SEC
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Sephacryl S-200
– 16/60
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PBS pH 7.4
0.5 mL per min.
3 mL Fractions
SDS PAGE
– Coomassie-stained
Z-dTM BacMam: S-200 SEC
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Sephacryl S-200
– 16/60
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PBS pH 7.4
0.5 mL per min.
3 mL Fractions
SDS PAGE
– Coomassie-stained
Z-dTM BacMam and PEI Final
SDS PAGE
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PEI Yield = < 0.5 mg/L
BacMam Yield = 4 mg/L
SDS PAGE
– Coomassie-stained
– Reduced
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Western Blot
– Anti-ZdTM
– BCIP/NBT Color
Development
Z-dTM BacMam and PEI Final
Western Blot
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PEI Yield = < 0.5 mg/L
BacMam Yield = 4 mg/L
SDS PAGE
– Coomassie-stained
– Reduced
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Western Blot
– Anti-ZdTM
– BCIP/NBT Color
Development
Some Parting Thoughts
Some Parting Thoughts
Some Parting Thoughts
Thanks
• To Adam for the Invitation
• To Everyone for Your Attention
• Kempbio, Inc.
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April Birch
Heather Allen
Kerrie Kenefick
Pat Kemp
• Medigen (Infuenza VLP)
– Peter Pushko
– Irina Tretyakova