Biological Process Development Facility Department of Chemical Engineering College of Engineering and Technology Bench to Clinic of Biotherapeutic Molecules:

Biological Process Development Facility

Department of Chemical EngineeringCollege of Engineering and Technology

Bench to Clinic of Biotherapeutic Molecules: Issues to Consider

ByBy

Dr. Michael MeagherDr. Michael MeagherDonald F. and Mildred T. Othmer Endowed ProfessorDonald F. and Mildred T. Othmer Endowed Professor

Director, Biological Process Development FacilityDirector, Biological Process Development FacilityDepartment of Chemical EngineeringDepartment of Chemical Engineering

University of Nebraska-LincolnUniversity of Nebraska-Lincoln



Objective of the Presentation

• Provide the discovery scientist with Provide the discovery scientist with information on what is required to move a information on what is required to move a “discovery molecule” to a “clinical “discovery molecule” to a “clinical candidate.”candidate.”



Starting Point• Discovery has identified a protein with Discovery has identified a protein with

therapeutic properties.therapeutic properties.

• Discovery wants to move the clinical Discovery wants to move the clinical candidate into preclinical testing.candidate into preclinical testing.



Technology Transfer to Process Development and cGMP Manufacturing

• Product characterizationProduct characterization• Identify production cell line-Identify production cell line-Pichia pastorisPichia pastoris• Analytical methodsAnalytical methods• Raw materials Raw materials • Bench-scale processBench-scale process

Fermentation (shake flask)Fermentation (shake flask) RecoveryRecovery PurificationPurification Formulation and stabilityFormulation and stability



Product Characterization• The more characterization the better.The more characterization the better.

N-terminal sequencingN-terminal sequencing Tryptic digest and peptide map (LC-MS/MS)Tryptic digest and peptide map (LC-MS/MS) Mass spectrometryMass spectrometry

Overall massOverall mass Post translational modificationsPost translational modifications

Amino acid analysisAmino acid analysis Isoelectric focusing (pI)Isoelectric focusing (pI) Bioassay(s)Bioassay(s)



Cell Line

• Discovery is accomplished through high-Discovery is accomplished through high-throughput expression systems.throughput expression systems.

• Such expression systems are not intended or Such expression systems are not intended or suitable for high-level production and suitable for high-level production and cGMP manufacturing.cGMP manufacturing.

• Therapeutic gene may not be optimized for Therapeutic gene may not be optimized for scale-up and production.scale-up and production.



Cell Line

• Production expression system is determined Production expression system is determined based on the post translations modifications based on the post translations modifications (PTM) that are required.(PTM) that are required. Bacteria to transgenic animalBacteria to transgenic animal



Cell Line

• The cell line is the most critical component The cell line is the most critical component of the production process.of the production process.

• Thorough characterization of the cell line is Thorough characterization of the cell line is strongly recommended before moving a strongly recommended before moving a process into scale-up.process into scale-up.

• Prefer that a validated Master Cell Bank be Prefer that a validated Master Cell Bank be established prior to process development.established prior to process development.



Cell Line Evaluation

• Shake flaskShake flask SDS-PAGE and Western Blot (minimum)SDS-PAGE and Western Blot (minimum)

• 5 L Bench-scale Fermentation5 L Bench-scale Fermentation SDS-PAGE and Western blot (minimum)SDS-PAGE and Western blot (minimum) Stability of supernatant (extra) or homogenate Stability of supernatant (extra) or homogenate

(intra)(intra)

• Small-scale purificationSmall-scale purification



Cell Line Evaluation-Case Study of BoNTC Hc expressed in Pichia pastoris• BoNTC Hc is expressed BoNTC Hc is expressed intracellular.intracellular.• By shake flask there was no “apparent” effect By shake flask there was no “apparent” effect

of copy number on expression based on of copy number on expression based on Western blot.Western blot.

• Evaluated 1, 2, 3 and 4 copy clones in a 5 L Evaluated 1, 2, 3 and 4 copy clones in a 5 L fermentor.fermentor. Standard basal salts media and trace minerals.Standard basal salts media and trace minerals. Methanol set point during induction was 1. 5 g/L. Methanol set point during induction was 1. 5 g/L.



USAMRIID Botulinum Toxin ProgramDirected by Dr. Leonard Smith

• Seven distinct serotypes (A-G) Seven distinct serotypes (A-G) • Current vaccine is a pentavalent toxoid of Current vaccine is a pentavalent toxoid of

serotypes A, B, C, E and F.serotypes A, B, C, E and F.

Light Chain (50 kd) Heavy Chain (100 kd)

Zn Protease Membrane binding and translocation domain

CN

Vaccine

Botulinum Toxin



Effect of Gene Copy Number on Cell Growth During MeOH Induction

Cell Growth Vs Induction Time

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 10 20 30 40 50

Induction Time (hr)

XV/XoVo (g/g)

One CopyTwo CopyThree CopyFour CopyWB (old clone)



Effect of Gene Copy Number on Methanol Consumption

Methanol Consumption Vs Induction Time

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40 50

Induction Time (hr)

g Methanol consumed/XoVo

(g/g)

One CopyTwo CopyThree CopyFour CopyWB (old clone)



Effect of Gene Copy Number on BoNTC(Hc) Production

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 10 20 30 40 50

Induction time (hr)

un

it/u

nit

of

Bo

NT

C(H

c)

293-1

299-2

300-3

301-4



Effect of Gene Copy Number on MeOH Growth Rate and Production of BoNTC Hc

Gene Copy Number

Specific MeOH Growth Rate (hr-1)

1 0.0544

2 0.0231

3 0.0054

4 Not Constant

WB (old) 0.0166

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 1 2 3 4 5

Gene copy number

max

imu

m u

nit

/un

it o

f B

oN

TC

(Hc)



BoNTC Hc Cell Line Case Study

• The effect of BoNTC Hc copy number on The effect of BoNTC Hc copy number on MeOH metabolism was unexpected.MeOH metabolism was unexpected.

• Essential to evaluate clones under Essential to evaluate clones under production fermentation (and purification) production fermentation (and purification) conditions before deciding on a clone.conditions before deciding on a clone.



Establishing a Seed Bank• PurityPurity

• Determine the cell line is mono-cultureDetermine the cell line is mono-culture• IdentityIdentity

• PhenotypicPhenotypic Growth morphology Growth morphology Carbon Carbon Auxotrophic markerAuxotrophic marker

• GenotypicGenotypic Confirm and sequence gene insertConfirm and sequence gene insert Restriction mapRestriction map Ribosomal typingRibosomal typing



Establishing a Seed Bank• StabilityStability

• Generational studies in shake flask and Generational studies in shake flask and fermentorfermentor

Analyze productAnalyze product Copy numberCopy number mRNAmRNA

All aspects of establishing a seed bank must be All aspects of establishing a seed bank must be documented. Information is required for the documented. Information is required for the Master Cell Bank.Master Cell Bank.



Analytical Methods

• Product characterization assaysProduct characterization assays• Purpose is to sufficiently characterize the Purpose is to sufficiently characterize the

product so as to create a reference standard.product so as to create a reference standard.

• Bioanalytical reagentsBioanalytical reagents• Antibodies, cell lines for bioassay, enzymes, Antibodies, cell lines for bioassay, enzymes,

etc.etc.• Essential to insure sources of these reagents.Essential to insure sources of these reagents.



Analytical Methods

• In-Process Test(s)In-Process Test(s)• FastFast• ReliableReliable• RobustRobust• Quantitative for productQuantitative for product• Able to handle all types of samples Able to handle all types of samples • Provide an indication that process is operating Provide an indication that process is operating

within specifications within specifications



Analytical Methods• Lot release assays for productLot release assays for product

• Amino acid analysisAmino acid analysis• Tryptic digest and peptide mapTryptic digest and peptide map• N-terminal sequencingN-terminal sequencing• 2 HPLC methods and size exclusion2 HPLC methods and size exclusion• EndotoxinsEndotoxins• SDS-PAGE and Western BlotSDS-PAGE and Western Blot

Reducing and non-reducingReducing and non-reducing• Host protein and nucleic acidsHost protein and nucleic acids• Bioassay(s)Bioassay(s)



Process Description

• Raw MaterialRaw Material• Anything used to produce or analyze the Anything used to produce or analyze the

product.product.• Important to specify several vendors.Important to specify several vendors.• Establish methods to I.D. raw materials.Establish methods to I.D. raw materials.• Understand “shelf life” of raw materials.Understand “shelf life” of raw materials.



Process Description-Fermentation• Monitor Critical Parameters-Metabolic ActivityMonitor Critical Parameters-Metabolic Activity

• pHpH• Dissolved oxygenDissolved oxygen• On-line sensorsOn-line sensors• Off gasOff gas

• Calculate RQ and OURCalculate RQ and OUR• Consumption of nutrients, acid or baseConsumption of nutrients, acid or base• Generate a fermentation historyGenerate a fermentation history• Move towards greater computer controlMove towards greater computer control



Process Description-Purification• RecoveryRecovery

• The most difficult step in process development.The most difficult step in process development.

• PurificationPurification• Identify critical parameters.Identify critical parameters.

pH, conductivity, temperature, protein pH, conductivity, temperature, protein concentration, resin, membrane, etc… concentration, resin, membrane, etc…

• Determine scalability of each stepDetermine scalability of each step



BoNTE Hc Purification



Conclusions

• The most critical “raw material” is the cell The most critical “raw material” is the cell line.line.

• Essential to evaluate cell lines under Essential to evaluate cell lines under process development conditions.process development conditions.

• The greater the interaction of the discovery The greater the interaction of the discovery scientists with the process development scientists with the process development scientists and engineers the faster and more scientists and engineers the faster and more effective the transfer into the clinic.effective the transfer into the clinic.



Credit Goes To

• Mehmet Inan (Molecular Biology)Mehmet Inan (Molecular Biology)• Vijay Jain (Molecular Biology/Fermentation)Vijay Jain (Molecular Biology/Fermentation)• Wenhui Zhang (Fermentation)Wenhui Zhang (Fermentation)• Mark Gouthro (Fermentation)Mark Gouthro (Fermentation)• Rick Barent (Purification)Rick Barent (Purification)• Joey Wu (Purification)Joey Wu (Purification)



Acknowledgements

• BoNT Hc work was funded by the United BoNT Hc work was funded by the United States Army Medical Research and Materiel States Army Medical Research and Materiel Command. Command.

Contract No.: DAMD17-02-C-0107Contract No.: DAMD17-02-C-0107

Documents

Biological Process Development Facility Department of Chemical Engineering College of Engineering and Technology Bench to Clinic of Biotherapeutic Molecules: