how waves of innovation in biotechnology shaped a small business venture

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 andScienceDecember 9,2016

When do multiplication and division mean the same thing?

A Riddle for You

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


Chris Kemp



Kempbio, Inc.

5119 Pegasus Ct. Suite P Frederick, MD 21704 USAwww.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

• 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– 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

100L SUB

Kempbio’s Business

Production of ProteinsFrom Genes

Kempbio is a

Gene to Protein Company

Gene to Protein Concept

Gene to Protein Reality• >Contig pCI-X-His Sequence

• ATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATAGGCTAGCGCCACCATGGGATGGTCCTGCATCATTCTGTTCCTGGTGGCAACTGCAACCGGAGCACACAGCTCCACTGAGGAACTGTTTAAGGAGTACAAACTGACCCGACCTTATATGGCCCGGTGCATCAGATGTGCTGTGGGCTCCTGTCACTCTCCAATCGCTATTGAAGCAGTGAAGTCTGACGGGCATGATGGATACGTCCGCCTGCAGACCTCTAGTCAGTATGGCCTGGACTCAAGCGGCAACCTGAAGGGGAGGACAATGCGCTACGATATGCACGGGACTATCAAAGAGATTCCTCTGCACCAGGTGAGTCTGCATACATCAAGACCATGCCACATCGTCGACGGACATGGCTATTTCCTGCTGGCAAGGTGTCCAGCAGGCGACTCTATTACCATGGAGTTTAAGAAAGATAGTGTGACACACAGCTGCTCCGTGCCCTACGAGGTCAAGTTCAACCCTGTCGGCAGGGAACTGTATACCCACCCCCCTGAGCATGGGGTGGAACAGGCTTGTCAGGTCTACGCCCACGACGCTCAGAATCGCGGAGCATATGTGGAGATGCATCTGCCTGGCTCTGAAGTGGATTCCTCTCTGGTCTCTCTGAGTGGGAGTTCAGTGACAGTCACTCCACCCGTGGGAACAAGTGCCCTGGTCGAGTGCGAATGTGGCGGGAAGAAAATCTCAGAGACTATTAATAGAACCAAGCAGTTCTCCCAGTGCACTAAGAAAGAACAGTGTCGAGCATACCGGCTGCAGAACGACAAATGGGTGTATAATAGCGATAAGCTGCCCAAAGCCGCTGGGGCTACCCTGAAGGGAAAACTGCACGTGCCCTTTCTGCTGGCAGACGGGAAGTGCACAGTCCCTCTGGCCCCCGAGCCTATGATCACTTTCGGATTTCGCTCAGTGAGCCTGAAGCTGCATCCAAAAAACCCCACTTACCTGACCACACGACAGCTGGCCGATGAGCCACACTATACCCATGAGCTGATTTCCGAACCCGCTGTGCGGAACTTCACCGTCACAGAGAAGGGCTGGGAATTCGTGTGGGGGAATCACCCTCCAAAAAGATTTTGGGCTCAGGAGACAGCACCTGGAAATCCACACGGCCTGCCACATGAAGTGATCACCCACTACTATCATCGGTACCCCATGAGCACAATTCTGGGCCTGTCCCACCATCACCATCACCATTAATGAGCGGCCGC

•• >Predicted protein sequence (397 aa, MW=44,336.58)•• MGWSCIILFLVATATGAHSSTEELFKEYKLTRPYMARCIRCAVGSCHSPIAIEAVKSDGHDGYVRLQTSSQYGLDSSGNLKGRTMRY

DMHGTIKEIPLHQVSLHTSRPCHIVDGHGYFLLARCPAGDSITMEFKKDSVTHSCSVPYEVKFNPVGRELYTHPPEHGVEQACQVYAHDAQNRGAYVEMHLPGSEVDSSLVSLSGSSVTVTPPVGTSALVECECGGKKISETINRTKQFSQCTKKEQCRAYRLQNDKWVYNSDKLPKAAGATLKGKLHVPFLLADGKCTVPLAPEPMITFGFRSVSLKLHPKNPTYLTTRQLADEPHYTHELISEPAVRNFTVTEKGWEFVWGNHPPKRFWAQETAPGNPHGLPHEVITHYYHRYPMSTILGLSHHHHHH

Promega pCi Vector

Basic Methods of Gene Transfer

Gene to Protein Reality


Chris Kemp



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

• 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

• 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

• 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

Virus Counter (ViroCyt, LLC)

Intact Virus Particles

Scatter Plot: Plaque Titer vs ViroCyt vp/mL titer

0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

1.20E+08

0.00E+00 1.00E+09 2.00E+09 3.00E+09 4.00E+09

pfu/

mL

vp/mL (ViroCyt)

r2 = 0.95Slope = 0.025 pfu/mL per vp/mLY-intercept = -3.7x106 pfu/mL

Virus Production based on Plaque and ViroCyt Titers

0.00E+00

1.00E+09

2.00E+09

3.00E+09

4.00E+09

5.00E+09

6.00E+09

7.00E+09

0 20 40 60 80 100 120

Viru

s Pa

rtic

les

per m

L

Time Post-Infection (hours)

591 A

591 B

751 A

751 B

MOI = 0.021x106 cells per mLSF900-IIIA = Plaque TiterB = ViroCyt Titer

Baculovirus in Supernatant of Stirred-tank Bioreactor

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

3.00E+09

0 20 40 60 80 100 120

Viru

s Pa

rtic

les

per m

L

Time Post-Infection (Hours)

Sf9 CellsMOI = 2.0Cell Density = 2x106 cells per mLTiters by ViroCyt 2100Each point = Mean of 3 titers

0.00E+00

5.00E+08

1.00E+09

1.50E+09

2.00E+09

2.50E+09

24 36 48 60 72

Vir

us P

artic

les p

er m

L

Elapsed Time (h.)

High Five

Sf9

Baculovirus Particles in Culture Supernatant

Innovations That Guide Kempbio

• ViroCyt Virus Counter• BacMam

BacMam

• Mammalian CMV Promoter

• Express rProteins in a variety of mammalian cells using a baculovirus as the delivery vehicle

Invitrogen 2008

BacMam Transfer Vector

Second Generation Bacmam VectorsVSV-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 mLC = 4x106 cells per mL

CHO-S/CD-CHOMOI = 50 (25:25)

0

20

40

60

80

100

120

140

160

0 10 20 30 40 50 60

mg

Purif

ied

IgG

per

Lite

r

BacMam MOI

IgG B BacmamMOI: VariousCHO-S/CD-CHO4x106 cells per mL

1L Shake-Flask MOI = 5096h. PT HarvestPurified Yield = 150 mg/L

Humanized rIgG: 10L BioreactorSDS PAGE Gel Coomassie StainedNon-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

Ebola Classification

http://www.rcsb.org/pdb/101/motm.do?momID=178

Family: FiloviridaeGenus: EbolavirusSpecies:

1. Zaire ebolavirus2. Sudan ebolavirus3. Tai Forest ebolavirus4. Bundibugyo ebolavirus5. Reston ebolavirus

Ebola Zaire Glycoprotein• Mayinga Zaire Strain

– 1976

• Trimerictransmembrane protein

• Heavily glycosylated• Mucin domain• Transmembrane

domain deleted = dTM– MW ~120 kDa

• Expressed as secreted product in HEK-293http://www.rcsb.org/pdb/101/motm.do?momID=178

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 ComparisonPEI Vector

• pCi Transient Expression Vector

• CMV Promoter• HA-Tag• Synthetic Gene• Human Signal Sequence• Transmembrane domain

deleted• Codon Optimized for Human

Cells

BacMam Vector

• Gateway Entry Vector– Synthesized in standard cloning

vector with ATT sites flanking ORF

– Bacmid transfected into Sf9 for BacMam generation

• 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

• 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

Z-dTM Purification Scheme: 1-40-litersQ

Cap

ture

Ste

p 10mL Q-Sepaharose/LBatch Bind 16h.Pack ColumnWashElute using linear gradient 50-250 mM NaCl at pH 7.0

Size

-Exc

lusi

on Load concentrated Q-capture pool onto S-200 Sephacryl columnBuffer = PBSFlow = 0.5 mL per min.Run SDS PAGEPool ZdTMfractions C

once

ntra

tion

and

QC Concentrate using

Centricon 10kDa MWCOSterile FiltrationVialingCoomassie-stained SDS PAGE and Western Blot (anti-ZdTM

Z-dTM PEI: Q-Sepharose

• Q-Sepharose FF• 20 mM Tris pH 7 • Linear Gradient

– 50-250 mM NaCl• 5 mL Fractions• SDS PAGE

– Coomassie-stained

Z-dTM BacMam: Q-Sepharose

• Q-Sepharose FF• 20 mM Tris pH 7 • Linear Gradient

– 50-250 mM NaCl• 5 mL Fractions• SDS PAGE


Z-dTM PEI: S-200 SEC

• Sephacryl S-200 – 16/60

• PBS pH 7.4 • 0.5 mL per min.• 3 mL Fractions• SDS PAGE


Z-dTM BacMam: S-200 SEC

• Sephacryl S-200 – 16/60

• PBS pH 7.4 • 0.5 mL per min.• 3 mL Fractions• SDS PAGE


Z-dTM BacMam and PEI Final

• PEI Yield = < 0.5 mg/L • BacMam Yield = 4 mg/L • SDS PAGE

– Coomassie-stained– Reduced

• Western Blot– Anti-ZdTM– BCIP/NBT Color

Development

SDS PAGE

Z-dTM BacMam and PEI Final

• PEI Yield = < 0.5 mg/L • BacMam Yield = 4 mg/L • SDS PAGE

– Coomassie-stained– Reduced

• Western Blot– Anti-ZdTM– BCIP/NBT Color

Development

Western Blot

Some Parting Thoughts

Thanks• To Adam for the Invitation • To Everyone for Your Attention• Kempbio, Inc.

– April Birch– Heather Allen– Kerrie Kenefick– Pat Kemp

• Medigen (Infuenza VLP)– Peter Pushko– Irina Tretyakova