ppfs interim: recombinant human erythropoietin … · ppfs interim: recombinant human...

PPFS Interim: Recombinant Human

Erythropoietin Production in the Pichia

Pastoris Expression System

Monday, November 10, 2014

Professor VanAntwerp

Nicholas Giles

Zion Lee

Abby Leistra

Stephen Tubergen

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© 2014, Team eporis and Calvin College

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Executive SummaryExecutive SummaryExecutive SummaryExecutive Summary

eporis is a biopharmaceutical company that manufactures biologic proteins as active drug substances.

Erythropoietin (EPO) is a protein that stimulates red blood cell production for cancer and HIV-afflicted

patients. The current method of producing erythropoietin is a large-scale biofermentation process

using Chinese Hamster Ovary (CHO) cells. Our proposal, which bases itself on current research, is to

design an alternate process using simpler eukaryotic cells, namely yeast, of the species Pichia Pastoris.

The new process will reduce costs of manufacturing, subsequently reducing the $154/10,000 IU price

tag on this essential drug that grosses over $13 billion annually. Bioreactor design, kinetics,

chromatographic separations, thermodynamics, and controls will be used to develop a product at a

lower market price while maintaining FDA-quality purity. At eporis we strive to deliver high quality

drug substances for the innovative therapies of our customers, at affordable prices. We work to

develop the most cutting edge, cost-effective manufacturing methods in the biopharmaceutical

industry.

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Table of ContentsTable of ContentsTable of ContentsTable of Contents

Executive Summary ....................................................................................................................................... 2

Table of Figures ............................................................................................................................................. 6

Table of Tables .............................................................................................................................................. 7

1. Project Overview ....................................................................................................................................... 8

1.1 EPO Overview ...................................................................................................................................... 8

1.1.1 Medical Significance ..................................................................................................................... 8

1.1.2 Isoforms on the Market and in Development.............................................................................. 8

1.1.3 Market Trends .............................................................................................................................. 8

1.1.4 Production Methods .................................................................................................................... 9

1.1.5 Drug Substance vs Drug Product .................................................................................................. 9

1.2 Project Proposal .................................................................................................................................. 9

1.2.1 Objective ...................................................................................................................................... 9

1.2.2 Target Customers ....................................................................................................................... 10

1.2.3 Potential Competitors ................................................................................................................ 10

1.2.4 Differentiated Approach ............................................................................................................ 10

1.3 Team Organization ............................................................................................................................ 10

1.3.1 Team Profile: Student Members ................................................................................................ 10

1.3.2 Auxiliary Members ..................................................................................................................... 11

1.3.3 Team Management Method ...................................................................................................... 11

1.3.4 Work Breakdown Structure ....................................................................................................... 12

1.4 Design Norms and Criteria ................................................................................................................ 14

1.4.1 Stewardship ............................................................................................................................... 14

1.4.2 Transparency .............................................................................................................................. 14

1.4.3 Integrity ...................................................................................................................................... 14

1.4.4 Justice ......................................................................................................................................... 14

2. Constraints and Requirements ............................................................................................................... 14

2.1 Time of Reaction ............................................................................................................................... 14

2.2 Product Purity ................................................................................................................................... 14

2.3 Bioactivity and Potency ..................................................................................................................... 15

2.4 Product Yield ..................................................................................................................................... 15

2.5 Economic Feasibility .......................................................................................................................... 15

3. Deliverables ............................................................................................................................................. 16

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3.1 Poster ................................................................................................................................................ 16

3.1 PPFS ................................................................................................................................................... 16

3.3 Final Design Report ........................................................................................................................... 16

3.4 Team Website ................................................................................................................................... 16

4. General Background ................................................................................................................................ 16

4.1 EPO Structure .................................................................................................................................... 16

5. Research and Design Scope .................................................................................................................... 17

5.1 Expression System: Strain Selection ................................................................................................. 17

5.1.1 Research and Design Alternatives.............................................................................................. 18

5.1.1 Design Considerations................................................................................................................ 20

5.2 EPO Gene and Post-Expression Modifications .................................................................................. 21



5.3 Growth Media ................................................................................................................................... 23



5.4 Reactor Type ..................................................................................................................................... 25

5.4.1 Reactor Design and Alternatives ................................................................................................ 25

5.4.2 Design Criteria ............................................................................................................................ 30

5.5 Process Analytical Technology (PAT) ................................................................................................ 31

5.5.1 Dissolved Oxygen Sensors .......................................................................................................... 31

5.5.2 Cell Density Quantification ........................................................................................................ 32

5.5.3 EPO Concentration Quantification ............................................................................................. 33

5.5.3 Reactor Operating Conditions .................................................................................................... 35

5.6 Harvesting ......................................................................................................................................... 35



5.7 Isolation and Purification .................................................................................................................. 36


5.8 Recycling and Waste Management ...................................................................................................... 38


5.8.2 Design Consideration ................................................................................................................. 38

6. Proposed Laboratory Simulations ........................................................................................................... 38

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6.1 Simulations ........................................................................................................................................ 38

6.2.1 Scale – up Equations .................................................................................................................. 38

6.2.2 Simulation Software ................................................................................................................... 39

8. Business Plan ........................................................................................................................................... 39

8.1 Market Analysis ................................................................................................................................. 39

8.1.1 Target Market ............................................................................................................................ 39

8.1.2 Demographic Profile .................................................................................................................. 40

8.1.3 Market Size and Trends.............................................................................................................. 40

8.1.4 Advertising and Pricing .............................................................................................................. 40

8.2 Research and Development Costs .................................................................................................... 40

9. Financial Estimates .................................................................................................................................. 40

10. Conclusion ............................................................................................................................................. 41

11. References ............................................................................................................................................ 41

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Table of FiguresTable of FiguresTable of FiguresTable of Figures

Figure 1. Various Isoforms of EPO ................................................................................................................ 8

Figure 2. Projected growth of the biosimilar market.................................................................................... 9

Figure 3. Team Eporis .................................................................................................................................. 10

Figure 4. Range of product titer and batches per year that yield a cost competitive biosimiliar). ........... 15

Figure 5. Team Poster ................................................................................................................................. 16

Figure 6. EPO structure. .............................................................................................................................. 16

Figure 7. Representative glycosylation process in human and in glycoengineered P. Pastoris. ................ 19

Figure 8. In vivo analysis of hemocrit levels after treatment with rhEPO. ................................................. 20

Figure 9: Concentration of rHuEPO as a function of bioreactor culture time. ........................................... 23

Figure 10. Ideal Batch Reactor .................................................................................................................... 25

Figure 11. Ideal CSTR ................................................................................................................................... 26

Figure 12. Ideal PFR ..................................................................................................................................... 26

Figure 13. Semi Batch Reactor .................................................................................................................... 27

Figure 14. 2010.igem.org/File:UCL-BIOREACTSRS ...................................................................................... 28

Figure 15. Perfusion Reactor ....................................................................................................................... 29

Figure 17 Bubble Reactor Column.. ............................................................................................................ 29

Figure 17: Dissolved oxygen probes. .......................................................................................................... 32

Figure 18: Optical sensor mechanism for detecting dissolved oxygen. ...................................................... 32

Figure 19: Flow-through cuvette with internal dilution.. ........................................................................... 33

Figure 20: Basic ELISA method. ................................................................................................................... 34

Figure 21: Lab-scale, real-time monitoring of GFP co-expressed with heterologous protein. ................... 34

Figure 22. General EPO process flow diagram ............................................................................................ 36

Figure 23. Affinity Chromatography for protein purification ..................................................................... 37

Figure 24. SuperPro Designer process modeling software. ........................................................................ 39

Figure 25. Development statistics in comparison. ...................................................................................... 40

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Table of TablesTable of TablesTable of TablesTable of Tables

Table 1. Pichia Pastoris strains commonly used in recombinant work....................................................... 18

Table 2. Differences in rhEPO produced in P. Pastoris and CHO in comparison to hEPO.7 ....................... 19

Table 3. Literature references to glycoengineered P. Pastoris strains. ...................................................... 20

Table 4: EPO structure variants and post-expression modifications. Star (*) indicates a post-expression

and purification modification as opposed to a gene sequence modification. ........................................... 22

Table 5: Compositions and Concentrations of BMGY and BMMY mixed media. ....................................... 24

Table 6 Reactor Alternatives ....................................................................................................................... 30

Table 7. Signal sequences use in P. Pastoris. .............................................................................................. 35

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1. Project Overview1. Project Overview1. Project Overview1. Project Overview

1.1 EPO Overview1.1 EPO Overview1.1 EPO Overview1.1 EPO Overview

1.1.1.1.1111.1 Medical Significance.1 Medical Significance.1 Medical Significance.1 Medical Significance

EPO is a protein that can be delivered either by IV or injection that

works by causing bone marrow to increase red blood cell

production. If not enough EPO is naturally created in the kidneys,

the body will not produce enough red blood cells, leading to

anemia. EPO therapies prevent the need for blood transfusions and

the main medical use is currently for anemic patients undergoing

hemodialysis. Patients undergoing chemotherapy, HIV treatments,

or who suffer from chronic kidney failure have also benefitted from

erythropoietin stimulating agents as these therapies offset the

common side effect of anemia.

1.1.1.1.1111.2 Isoforms on the Market and in Development.2 Isoforms on the Market and in Development.2 Isoforms on the Market and in Development.2 Isoforms on the Market and in Development

EPO is a protein with five different isoforms currently on the global

market, each with varying forms or glycosylation patterns. The five

forms tend to be prescribed interchangeably, as doses of each can

be varied to produce similar in vivo effects. Epoetin alfa, beta,

omega, delta, and darbepoetin alfa are the general structures to

which all branded erythropoietin biopharmaceuticals match (and

biosimilars resemble). Different countries have varying standards

regulating the development of erythropoietin stimulating agents,

leading to a fragmented market with over fifty global players.

1.1.1.1.1111.3 Market.3 Market.3 Market.3 Market TrendsTrendsTrendsTrends

Due to the recent breakthroughs in biopharmaceuticals, paired with the upward trends in anemic

patients and those undergoing dialysis, erythropoietin is projected to grow along with the industry.

Expiring patents of first and second generation therapeutics, along with American legislation encourages

the entry of new players in the market. This provides a healthy market climate for eporis to overcome

barriers to entry, and establish a commendable market share in the near future. The patent expiration

of Amgen’s Epogen in 2013 opens up the market from the most lucrative monopoly in modern history.

Retacrit (Hospira)

Silapo (stada)

Binocrit (Sandoz)

Procrit (Johnson and Johnson)

Recorman (Hoffmann-La Roche)

Mircera (Roche)

Dynepo (Shire plc)

Epomax (Lek)

Epoetin alfa

Epoetin beta

Epoetin delta

Epoetin zeta

Epoetin omega

Aranesp (Darbepoetin, Amgen)

Epocept (Lupin Pharma)

Epofit (Antas Pharma)

Epogen (Amgen)

Eprex (Jannsen-Cilag)

Figure 1. Various Isoforms of EPO

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1.1.1.1.1111.4 Production Methods.4 Production Methods.4 Production Methods.4 Production Methods

The current industrial production of EPO is performed in the Chinese hamster ovary cell line. These cells

are extensively researched and well understood. After cell culturing, there is great variety in the

separation and purification of EPO protein. eporis is proposing an alternate cell line, the yeast Pichia

Pastoris, to culture EPO at a faster rate. Methanol will be used to induce protein production after the

growth stage, followed by purification and polishing. This separates the EPO out from the cell mixture,

while polishing ensures the absence of viruses and other contaminants.

1.1.1.1.1111.5 Drug Substance vs Drug Product.5 Drug Substance vs Drug Product.5 Drug Substance vs Drug Product.5 Drug Substance vs Drug Product

eporis creates the active drug substance EPO. This drug substance is sold to other pharmaceutical and

biopharmaceutical companies to be used in their drug product therapies. Through this distribution

network to existing market players, eporis aims to lower the cost of EPO therapies across the entire

market by encouraging healthy price competition.

1.1.1.1.2222 Project ProposalProject ProposalProject ProposalProject Proposal

1.1.1.1.2222.1 Objective.1 Objective.1 Objective.1 Objective

At eporis we strive to deliver high quality drug substances for the innovative therapies of our customers,

at affordable prices. We work to develop the most cutting edge, cost-effective manufacturing methods

in the biopharmaceutical industry. The biosimilar drug substances produced is to be sold to all willing

market players, lowering the average price for EPO therapies in America. The feasibility of a biosimilar

production in the Pichia Pastoris cell line shall be analyzed

Figure 2. Projected growth of the biosimilar market (Sjöblom).

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1.1.1.1.2222.2 Target Customers.2 Target Customers.2 Target Customers.2 Target Customers

The target customers for our EPO drug substance are other pharmaceutical and biopharmaceutical

companies that manufacture drug products. Once the patents of current American EPO drug products

expire, biosimilars will be introduced, of which eporis will supply the key drug substance.

1.1.1.1.2222.3 Potential Competitors.3 Potential Competitors.3 Potential Competitors.3 Potential Competitors

Potential competitors are the current producers of branded EPO, Amgen, along with other potential

biopharmaceutical companies that enter the market to profit on EPO therapies.

1.1.1.1.2222.4 Differentiated Approach.4 Differentiated Approach.4 Differentiated Approach.4 Differentiated Approach

Our differentiated approach is the alternate method of producing EPO in Pichia Pastoris. The

differentiation in business practice of acting as supplier to the rest of the biosimilar EPO market allows

Eporis to operate on a larger production scale than if we enter the a potentially fragmented market.

1.1.1.1.3333 Team OrganizationTeam OrganizationTeam OrganizationTeam Organization

1.1.1.1.3333.1 Team Profile.1 Team Profile.1 Team Profile.1 Team Profile: : : : Student MembersStudent MembersStudent MembersStudent Members

Team eporis is comprised of Nick Giles, Abby Leistra, Stephen Tubergen, and Zion Lee.

• Nick Giles is majoring in international chemical engineering. During his junior year he was an

intern at Pfizer Global Supply, the world’s largest pharmaceutical team, focusing on drug

manufacturing and pharmaceutical process design and implementation. He reported to the

project manager of a large steroid capacity increase that produced the active pharmaceutical

ingredient for an anti-epileptic drug product. Nick also spent two summers at IQ Designs in the

automation engineering department working on custom machinery that filled and packaged

petri dishes in an aseptic environment.

• Zion Lee is a Chemical Engineering and Biochemistry double major. He previously researched at

the VanAndel Research Institute in Grand Rapids doing directed recombinant DNA protein

Figure 3. Team eporis (left to right) Zion Lee, Nick Giles, Stephen Tubergen, Abby

Leistra

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cloning, and he is now developing Bioluminescence Resonance Energy Transfer (BRET)

technology for molecular distance determination.

• Abby Leistra is a Chemical Engineering and Biochemistry double major. Abby researches

targeted drug delivery utilizing riboflavin as a targeting agent at Calvin College. She has been

published in the Biophysical Journal for her research.

• Stephen Tubergen is an International Chemical Engineer and Biochemistry double major. He

interned this past summer at the pharmaceutical company Boehringer Ingelheim, just outside of

Frankfurt, Germany. His job consisted of process development, which scales up lab techniques

to the industrial processes that create active pharmaceutical ingredients.

1.1.1.1.3333.2 .2 .2 .2 Auxiliary MembersAuxiliary MembersAuxiliary MembersAuxiliary Members

Team eporis is mentored by Professor Jeremy Van Antwerp. The industrial consultant for this project is

Dr. Venkatesh Natarajan from Biogen Idec in Cambridge, Massachusetts. Dr. Natarajan is a senior

chemical engineer involved in process development in the biopharmaceutical industry.

1.1.1.1.3333....3333 Team Management MethodTeam Management MethodTeam Management MethodTeam Management Method

The scrum methodology is the current management method for team eporis. This project management

tool is suited for projects with rapidly changing requirements. Scrum breaks down the design into

manageable two week sprints. After each sprint, progress is reviewed with the team mentor and the

tasks for the next sprint are established. Also, improvements to the scrum process are consistently

implemented during these times. Zion Lee serves as the scrum leader, who leads scrum meetings every

other day, which evaluate each team member’s progress on the current sprint and ensure that each

person performs to the best of his or her abilities.

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1.3.4 Work Breakdown Structure1.3.4 Work Breakdown Structure1.3.4 Work Breakdown Structure1.3.4 Work Breakdown Structure

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1.1.1.1.4444 Design NormsDesign NormsDesign NormsDesign Norms and Criteria and Criteria and Criteria and Criteria

1.1.1.1.4444.1 Stewardship.1 Stewardship.1 Stewardship.1 Stewardship

Stewardship in the biopharmaceutical industry is a critical design norm, as eporis strives to minimize

potentially harmful effects in both the production and disposal of EPO. Another important requirement

is to ensure the proper warnings are passed onto the drug product manufacturer to inform consumers

of proper EPO dosage and its potential side effects.

1.1.1.1.4444.2 .2 .2 .2 TransparencyTransparencyTransparencyTransparency

The codes outlined by the biopharmaceutical sector of Pharmaceutical Research and Manufacturers of

America (PhRMA) are to be strictly adhered to. eporis works to remain as transparent as possible in an

industry riddled with confusion and secrecy. eporis is an unbiased supplier as it will not hold the equity

in any customer’s companies.

1.1.1.1.4444.3 Integrity.3 Integrity.3 Integrity.3 Integrity

The integrity of our drug substance is expected to be verified by the approval of strict FDA regulations.

The activity and purity of eporis EPO cannot produce adverse effects that are atypical of conventional

EPO therapies.

1.1.1.1.4444.4 Justice.4 Justice.4 Justice.4 Justice

Economic justice is one of eporis’ core principles. The mission of the company is primarily to reduce the

cost of biosimilar therapies by encouraging price competition with our customers. We believe that

biosimilar therapies should be economically feasible for all Americans.

2.2.2.2. Constraints and RequirementsConstraints and RequirementsConstraints and RequirementsConstraints and Requirements

The proposed project aims to exploit faster growth times inherent to Pichia Pastoris cell lines to produce

an equivalent EPO drug substance more cost-effectively. In all aspects, then, the proposed project in

constrained by the practices of current EPO production in CHO.

2.12.12.12.1 Time of ReactionTime of ReactionTime of ReactionTime of Reaction

EPO production in CHO, in a batch or semi-batch system has a 23 day turnover period.1 Growth and

expression in Pichia Pastoris takes significantly less time. The process in P. Pastoris has a 100 hour

turnover time.2 Cells grow for 24 hours. Expression of EPO is then induced and lasts 72 hours.

Subsequently cleaning and turnover requires a four hour duration. Thus the opportunity to produce EPO

faster, and thus cheaper, is great.

2.2 2.2 2.2 2.2 Product PurityProduct PurityProduct PurityProduct Purity

Drug substance and product purity is regulated by the FDA. In the case of biosimilars, product purity

must match the commercial brand name drug the biosimilar is mimicking.3 Generally, the FDA tests for

consistent product purity and potency. Specifically, “Products shall be free of extraneous material

except that which is unavoidable in the manufacturing process described in the approved biologics

license application.”4

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2.32.32.32.3 Bioactivity and Potency Bioactivity and Potency Bioactivity and Potency Bioactivity and Potency

EPO produced in P. Pastoris needs to be equivalently active in vivo as CHO derived forms. Even

though extent and type of glycosylation varies between the expression systems, equivalently active

EPO has been produced in P. Pastoris.5,6 Thus the proposed P. Pastoris strain, EPO structure, growth

conditions, and process operating conditions are constrained to those that yield equivalently active

and potent EPO.

2.42.42.42.4 Product YieldProduct YieldProduct YieldProduct Yield

Investigative work by GE into EPO biosimilar production established the fermentation yields necessary

to realistically produce EPO in the current market. Figure 4 indicates yields (titers) of 1.7 g EPO / L

reactor effluent or more as necessary to establish a competitive process. Factors including reactor type,

reaction conditions, growth media, and P. Pastoris strain can affect yield

Figure 4. Range of product titer and batches per year that yield a cost competitive biosimiliar production process (Sjöblom).

2.52.52.52.5 Economic FeasibilityEconomic FeasibilityEconomic FeasibilityEconomic Feasibility

A biosimilar is typically sold for around 70% of the price of the biologic drug it is based off of. eporis will

sell EPO drug substance to our customers ensuring that they can then go on to manufacture a profitable

drug product. This pricing strategy aims to lower the cost of the drug product by encouraging price

competition, while eporis still maintains a healthy margin. An optimized EPO process in Pichia Pastoris

will allow for the end user markup to remain lower than the current market price due to eporis’ lower

cost of goods sold.

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3. Deliverables3. Deliverables3. Deliverables3. Deliverables

3.1 Poster3.1 Poster3.1 Poster3.1 Poster

The team created an informative poster outlining the key objectives of the biopharmaceutical EPO

process. It provides an adequate background to familiarize those who may not yet have a solid base of

biochemical knowledge.

3.1 3.1 3.1 3.1 PPFSPPFSPPFSPPFS

This semester, eporis has conducted the project proposal feasibility study on the scaled production of

recombinant human EPO in Pichia Pastoris.

3.33.33.33.3 Final Design ReportFinal Design ReportFinal Design ReportFinal Design Report

The final design report is due in May 2015.

3.43.43.43.4 Team WebsiteTeam WebsiteTeam WebsiteTeam Website

The eporis website is currently being constructed.

4. 4. 4. 4. GeGeGeGeneral Backgroundneral Backgroundneral Backgroundneral Background

4.1 4.1 4.1 4.1 EPOEPOEPOEPO Structure Structure Structure Structure

EPO is an erythropoeitic agent, stimulating red blood

cell production in the bone marrow. Specifically, EPO

stimulates the proliferation and differentiation of

eythroid precursor cells via interactions with cell

membrane receptors. EPO-EPO receptor interception

initiates development of the bound cell into fully

formed red blood cells.5 Human EPO (hEPO) was first

isolated in urine of anemic patients. The sequence was

cloned, enabling development of recombinant human

Figure 5. Team Poster

Figure 6. EPO structure. Dark blue shaded areas highlight

glycan structures. (glycam.org)

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EPO (rhEPO) as a drug.

EPO’s ability to stimulate erythropoiesis is dependent upon its structure. In addition to a specific

structure mediated by folding of its amino acid sequence, complex carbohydrate structures (glycans) are

present at specific amino acid residues (Figure 6). After the EPO is translated, human adult kidney and

fetal liver cells modify the protein with carbohydrates at four residues: N24, N38, N83, and S126. While

these glycans are necessary for proper function, their precise structure is not constant in the human

body. Native EPO is present in multiple glycoforms, i.e. the kind of glycan structure present at each of

the four residues varies from molecule to molecule of EPO.6,7 Ergie and Browne showed in 2001 that the

glycosylation pattern of EPO is key to its solubility, cellular processing, secretion, and in vivo

metabolism.8

Glycans observed in hEPO have two to three, but predominately two, branching antennae, or arms, and

are typically terminated with the sialic acid carbohydrate.6 The challenge of producing rhEPO for

therapeutic use on an industrial scale is mimicking the essential glycosylation pattern of hEPO.

Mammalian cells, such as Chinese hamster ovary (CHO) and baby hamster kidney (BHK), have been used

as they are capable of post-translational glycosylation similar to that found in hEPO.5

The major commercial glycoforms of hEPO are epoeitin alfa and beta. While both of these variants are

produced in CHO cells, the glycosylation patterns are different enough to warrant alfa and beta

nomenclature.6 Briefly, epoeitin beta displays a wider variety of glycans and more glycans of negative

greater negative charge.9 Darbepoeitin differs in amino acid sequence from alfa and beta epoetin.6,10

Directed mutation was performed to yield two additional glycosylation sites, increasing the molecular

weight of the protein and the magnitude of its negative charge. The resulting rhEPO displayed increased

half-life and bioactivity, establishing a definitive correlation between EPO charge, size, and activity in

vivo.10 Darbepoeitin alfa is marketed by Amgen in the US as Aranesp, and has paved the way for further

research, development, and production of second and third generation rhEPOs.6

5555. . . . Research and Research and Research and Research and Design Design Design Design ScopeScopeScopeScope

Twelve years have passed since the FDA approved Epogen, Aranesp, and Mircera, opening the market

for biosimilars.11 Additionally patents on current EPO production processes have or will expire in the

next 10 years,12 limiting the risk of new products infringing on process patent rights. Thus a current push

is underway to develop longer-lasting, more biologically active drug products. Academic and industrial

research groups are using protein engineering, genetic engineering of host cells, and optimization of

growth and production systems to optimize efficacy of EPO drug substances in new, more efficient

expression systems.6 Similarly, eporis has researched and defined the design space of the proposed

project in light of three major areas: expression system, EPO gene and post-expression modifications,

and growth conditions.

5555.1 .1 .1 .1 Expression System: Expression System: Expression System: Expression System: Strain SelectionStrain SelectionStrain SelectionStrain Selection

eporis proposes production of EPO in the Pichia Pastoris yeast cell line. Current commercially available

EPO is produced in CHO cell lines. While mammalian cell lines like CHO are able to perform human-like

post-translation modifications on proteins, industrial CHO products are hampered by low growth rates.

Bacterial alternatives, such as E. Coli offer speed but without necessary post-translational modifications.

Yeast and protozoa cell lines offer speed with the opportunity for glycosylation. 6,13 Leishmania

18

Tarantolae is one such protozoan system.13 However this alternative has been less explored in the

literature. Thus eporis specified the yeast P. Pastoris as a platform for further research and design.

5.1.1 Research5.1.1 Research5.1.1 Research5.1.1 Research and Design Alternativesand Design Alternativesand Design Alternativesand Design Alternatives

There are a wide variety of P. Pastoris strains commercially available for recombinant protein

expression. Each of these strains allow for selection of the recombinant protein with certain growth

methods. For example, Table 1 presents several strains of P. Pastoris offered by LifeTechnologies.

However, these strains require additional glycoengineering to express EPO with human-like

glycosylations.

Table 1. Pichia Pastoris strains commonly used in recombinant work.14

P. Pastoris Strain Genotype Application

GS 115 his4 Selection of expression vectors contain HIS4

X-33 Wild type Selection of Zeocin-resistant expression vectors

KM71 his4, aox1::ARG4, arg4

Selection of expression vectors containing HIS4 to

generate strains with Muts phenotype

KM71H aox1::ARG4, arg4

Selection of Zeocin-resistant expression vectors to

generate strains with Muts phenotype

SMD1168 his4, pep4

Selection of expression vector containing HIS4 to

generates strains without protease A activity

SMD1168H pep4

Selection of Zeocin-resistant expression vectors to

generate strains without protease A activity

19

Natively, P. Pastoris heavily mannosylates its protein

products. Figure 7 illustrates how EPO, once it

passes through the endoplasmic reticulum, is

similarly glycosylated in human and P. Pastoris cells.

Further modification in the human golgi apparatus,

however, yields glycan structures capped with sialic

acid, while golgi modification in P. Pastoris gives

terminally mannosylated structures. Terminal

mannosylation poses half-life and immunogenic

response issues. 6,15 Research shows both strength

of negative charge and molecular weight affect in

vivo pharmacokinetics. Less negatively charged,

lighter EPO drugs are cleared from the human

system more quickly.5, 7, 16 Extent of branching

(termed antennae in Table 2) alters the number of

possible sialylation sites, playing a role in half-life

determination as well.

In 2006, Hamilton and colleagues worked to

“humanize” P. Pastoris through glycoengineering.

They genetically engineered a strain to terminate

EPO glycans with sialic acid, a carbohydrate moiety

more similar to those observed in human EPO

(hEPO). Transfecting P. Pastoris with genes enabling

sialylation coupled to elimination of genes that

produce immunogenic structures created a strain

suitable to production of human-like EPO. Table 2

compares glycosylation properties of human, CHO,

and glycoengineered P. Pastoris EPO. Results

indicate sialic acid is present as terminal residues on P. Pastoris rhEPO glycans. Predominately, one sialic

acid is attached per branched arm of the glycan, maximizing negative charge per protein molecule. An In

vivo assessment revealed dose-dependent erythropoietic activity the glycoengineered P. Pastoris rhEPO

that is consistent with biologically active forms (Figure 8). Since 2006, other groups have

glycoengineered P. Pastoris to produce human-like yeast. Table 3 presents significant advances in the

field.

Table 2. Differences in rhEPO produced in P. Pastoris and CHO in comparison to hEPO.7

Figure 7. Representative glycosylation process in human and in

glycoengineered P. Pastoris.

20

Figure 8. In vivo analysis of hemocrit levels after treatment with rhEPO in wild type P. Pastoris (blue and green bars) and rhEPO

in glycoengineered P. Pastoris (red and yellow bars). Blue and red bars correspond to 8 days and green and yellows bars

correspond to 15 days after injection.6

Table 3. Literature references to glycoengineered P. Pastoris strains.

Reference Year EPO

structure

Structure

Achievement of Note Sialylated? Branching?

Original

DNA

sequence

Hamilton 2006 rEPO Yes Biantennary rat EPO

N-glycosylation adjusted

to be human

Nett 2011 rhEPO-

PEG Yes Biantennary hEPO

Humanized yeast;

subsequent PEGYlation

Gong 2013 rhEPO Yes Biantennary hEPO

Compares bi to

tetrantenneray

glycosylation in CHO

and P. Pastoris.

5.1.1 Design Considerations5.1.1 Design Considerations5.1.1 Design Considerations5.1.1 Design Considerations

The current progress in the field of glycoengineered P. Pastoris represents the design alternatives we

are considering (Table 3). The design decision in this arena is twofold. First a base strain must be chosen

(Table 1) and a subsequent series of glycoengineering modifications be selected (Table 3). Reliability of

growth and product yield, reproducibility of glycosylation patterns, and waste fermenter effluent

composition, shall be considered in selecting a P. Pastoris strain.

The design norms of justice and integrity are inherent in strain selection. P. Pastoris strain selection will

effect purity, potency, and cost of production of the rhEPO product. Consistently high product purity

and potency and reliably low prices is justice to American consumers. Integrity is seen in producing an

rhEPO product consistent with the eporis vision for quality, availability, and transparency. Strain

selection plays a role in maintaining company integrity.

21

5555.2 .2 .2 .2 EPO Gene and EPO Gene and EPO Gene and EPO Gene and PostPostPostPost----Expression ModificationsExpression ModificationsExpression ModificationsExpression Modifications

5.2.15.2.15.2.15.2.1 Research and Research and Research and Research and Design AlternativesDesign AlternativesDesign AlternativesDesign Alternatives

Several variants on the traditional EPO structure have been pursued as alternative drug substances. The

overarching goal is to optimize half-life and bioactivity in vivo. Thus modifications that increase the

EPO’s molecular weight and overall negative charge are being explored.

At the post-expression level, MacDougall and others explored conjugating polyethylene glycol (PEG) to

EPO. PEG is a large, negatively charged molecule. Interestingly, PEGylated EPO variants have in vivo

effects similar to darbepoietin alfa. The amino acid sequence of darbepoetin alfa is modified in five

locations to produce two additional N-linked glycosylation sites. The five-glycan EPO structure and

PEGylated 3-glycan EPO structure were observed to have similar half-lives and bioactivity.7 Post-

expression modifications come with intense capital investment. While PEGylation chemistry is straight

forward, capital and operating costs of an additional reactor and separation units are drawbacks.

Typically an additional chromatography column and filtration step is required. 7, 17 Sytkowski, in the late

1990s, modified rhEPO sulfhydryl groups to induce dimerization and trimerization. The increased

molecular weight served to improve half-life and thus bioactivity. However, the bench top modification

process lends itself poorly to industrialization.18

At the DNA level, recombinant technology enables structural changes to EPO before downstream

processing. Dimerization, conjugation to other proteins, and amino acid sequence changes that enable

hyperglycosylation have been investigated. In each case the structural modification was made to

increase protein stability, molecular weight, or potential for negative charge. The clear advantage of

genetically engineering size and charge changes to EPO through recombinant cDNA work is less

downstream processing. However, expressing more complicated recombinant sequences can negatively

affect transcription, translation, modification, and secretion efficiencies, thus diminishing yields.

Table 4 additionally includes a “Biosimilar Potential” column. This qualitatively asses how similar the

activity of the reference’s rhEPO is to an existing commercial rhEPO product. Score is presented on a

one to ten scale, with one indicating a high biosimilarity to the commercial product listed.

The clear advantage of genetically engineering size and charge changes to EPO through recombinant

cDNA work is less downstream processing. However, expressing more complicated recombinant

sequences can negatively affect transcription, translation, modification, and secretion efficiencies, thus

diminishing yields.

22

Table 4: EPO structure variants and post-expression modifications. Star (*) indicates a post-expression and purification

modification as opposed to a gene sequence modification.


Biosimilar potential shall be of primary consideration in selecting an EPO sequence. The commitment

made by eporis to a reliable, cost effective product rhEPO shall likely be best realized if the product is a

biosimilar. Research and development costs, clinical trials necessary for FDA approval, and time to

market are considerably less for a biosimilar product than for a new biologic. In light of the information

presented in Table 4, post-expression or gene sequence modification will be necessary to produce a

Reference Structural

Modification

Biosimilar

Potential

Commercial

Comparison Result

Nett (2011) PEGylated rhEPO* 8 Aranesp Research study, about comparable

pharmacokinetics

Maleki

(2011) PEGYlated rhEPO* - - -

Ergie and

Brown

(2001)

Hyperglycosylated

rhEPO - - -

Elliot et al

(2003)

Hyper

glycosylated

rhEPO

- - -

Macdougall

(1999) hEPO 1 Epoetin alfa

Clinical trial; three time longer

circulation time; better efficacy

Macdougall

(2010)

PEGylated hEPO

beta 5

Darbepoetin

alfa

Clinical trial; close to same efficacy

in first 26 weeks; better efficacy in

second 26 weeks

Way (2005)

hEPO with an

optimized

disulfide linkage

in an antibody

fusion protein

2 Procrit,

Aranesp

Similar in vitro and

pharmacokinetics to Procrit, but

not as good as Aranesp; Better in

vivo act than Procrit

Fares

(2007)

hEPO fused to the

carboxyl terminus

of human

chorionic

Gonadotropin

1

Commercial

hEPO (not

uniquely

specified)

One dose of 660 IU hEPO-CTP was

approximately the same as three

doses of 220 IU commercial hEPO

Sytkowski

1998

Dimerized and

trimerized EPO via

modified

sulfhydryls*

- - -

Sytkowski

(1999)

Double EPO cDNA

construct with a

flexible amino

acid linker

- - -

23

rhEPO in P. Pastoris that is comparable bioactivity to CHO derived commercial products. Specifically,

PEGylated varieties are indicated to have comparable activity to darbepoetin/Aranesp.

Stewardship is applicable to EPO structure design. Post-expression modification approaches require

additional process units and their associated energy duties, capital costs, and waste stream treatments.

However, PEGYlation as a post-expression modification may yield the most biosimilar rhEPO. Producing

a biosimilar lends itself well to implementation of integrity and justice. Thus design norms shall be

considered in the EPO structure design process.

5555.3 Growth Media.3 Growth Media.3 Growth Media.3 Growth Media

5.3.1 Research and Design Alternatives5.3.1 Research and Design Alternatives5.3.1 Research and Design Alternatives5.3.1 Research and Design Alternatives

The typical growth strategy for P. Pastoris is a methanol-limited fed batch process.2 Here, the yeast grow

and enumerate on a repressing carbon source such as glycerol for 24 hours. Repressing indicates that

expression of recombinant protein is inhibited by glycerol. Then, a glycerol-limited methanol fed phase

initiates the transcription of the AOX1 promoter and the following recombinant genes. This stage lasts 4

hours, and is a crucial transition to the next stage. Finally, a methanol-only feed source induces

expression of recombinant protein fully for 72 hours. The reactor contents are harvested at this point.

These guidelines need to be customized for each system in order to induce the yeast at just the right

phase of growth. The time for expression should also be adjusted, as harvesting should occur when

recombinant protein levels are at a maximum. If too much time elapses, proteases can begin to degrade

the amount of protein in the media, as shown in Figure 9.

Figure 9: Concentration of rHuEPO as a function of bioreactor culture time.19

The standard media to culture P. Pastoris are buffered complex glycerol medium (BMGY) and buffered

complex methanol medium (BMMY). These media go beyond “minimal media”, which is defined as a

carbon source and essential nutrients, and are better classified as “mixed media,” because they also

contain yeast extract and peptone for amino acid supplementation, as shown in Table 5. The added

yeast extract and peptone improve growth, stabilize proteins, and reduce degradation of secreted

24

proteins by proteases.20 These are also buffered with phosphates, which allow for pH stability as well as

choice in terms of operating pH.20

Table 5: Compositions and Concentrations of BMGY and BMMY mixed media.

BMGY BMMY

1% yeast extract 1% yeast extract

2% peptone 2% peptone

100 mM potassium phosphate, pH 6.0 100 mM potassium phosphate, pH 6.0

3.4 g/L Yeast Nitrogen Base 3.4 g/L Yeast Nitrogen Base

10 g/L Ammonium Sulfate 10 g/L Ammonium Sulfate

4 × 10-5% biotin 4 × 10-5% biotin

1% glycerol 0.5% methanol

One alternative for the standard culturing process is to use sorbitol as a co-substrate during the final

production phase of growth.21 Sorbitol, being a non-repressive source of carbon, allows the cells to have

excess feed while still being induced with methanol. This solves the problem of limited growth due to

methanol being at low concentrations because of toxicity at high concentrations.

Another alternative involves using mannitol as a co-substrate to methanol during the final production

phase of growth. Eskitoris et al. showed that pulse-feeding mannitol to a concentration of 50g/L in six-

hour increments led to higher rHuEPO expression than feeding sorbitol in shorter times.19 The proposed

mechanism for this effect is the ease in assimilating mannitol into the glycolytic metabolic pathway over

sorbitol.


In selecting optimum incubation and expression times, a number of criteria exist. Most important is the

combination of growth times that will yield the most protein in the shortest time. However, in the event

that recombinant protein continues to increase in concentration, a maximum time as prescribed by the

maximum culturing time will be adhered. Another criteria is the incremental cost of feed compared to

the incremental gain in recombinant protein. A level of expression may be reached where more protein

is produced, but that may not be worth the extra feed cost required to get there. Thus, expression time

may be less than the time corresponding to maximum recombinant protein concentration.

In selecting alternative feeding components, the main criteria is obviously improved protein expression.

However, other criteria exist, such as the relative costs of the feed, and the stewardship design norm,

which considers the sources of the components. Harvesting of these sugars in countries with poor

working conditions may be unfavorable compared to the synthetic production of these sugars in

humane ways. It is not unfeasible that a slightly more expensive component may be used because it is

produced in just ways.

25

5555.4.4.4.4 Reactor Reactor Reactor Reactor TypeTypeTypeType

5.4.1 5.4.1 5.4.1 5.4.1 Reactor Design and AlternativesReactor Design and AlternativesReactor Design and AlternativesReactor Design and Alternatives

There are many different types of reactors, each having their own advantages and disadvantages. The

most general reactor types include batch reactors, continuous stirred reactors (CSTR), and plug flow

reactors (PFR).

Batch Reactors: These reactors are constructed so that there is no flow in or out for the extent of the

reaction. Batch reactors are mainly used for liquid phase reactions and are considered a closed system

of thermodynamics. There will be some form of agitation in order to ensure complete mixing of the

solution. Generally the reaction is marked from when the chemicals are loaded and brought up to

reaction temperature to when the chemistry stops. There is usually a higher percent conversion than

with other types of reactors as conversion is a function of time and not a function of reactor volume.

Figure 10. Ideal Batch Reactor22

CSTR Reactors: These reactors are generally much larger than batch reactors and have a constant flow in

and out of the reactor. CSTRs can be used for both liquid and gas type reactions and have an open

system of thermodynamics. The basic concept of the CTSR is that the feed concentration and

temperature change instantaneously to the reactor concentrations and temperature. There is generally

not as high of conversion with this system compared to the batch process as the conversion is a function

of reactor volume and not time. Thus, in order to achieve a higher conversion the reactor volume has to

increase.

26

Figure 11. Ideal CSTR22

PFR Reactors: These reactors resemble large cylinders and have flow that passes through the tubular

construct. PFRs can handle both liquid and gas phase reactions and have a flow both in and out of the

reactor. These are modeled in an open system of thermodynamics. Key assumptions for an ideal reactor

include no mixing in the direction of the flow along with no variation of concentrations, temperature,

and flow rate in the radial direction. Similar to CSTRs, the conversion of these reactions are a function of

volume.

Figure 12. Ideal PFR22

27

Different variations of these ideal reactors allow for more complex systems. For example one

modification to the batch reactor is the semi batch reactor. A semi batch reactor is similar to a batch

reactor in that conversion is a function of time and that there is no flow out of the reactor. However, it

differs by charging one of the reactants at a continuous rate over the extent of the reaction. Thus, a low

concentration of the charged reactant is maintained until the reaction is run to the equilibrium

conversion.

Figure 13. Semi Batch Reactor22

For reactions where a high conversion is essential, a variant of the ideal batch reactor or semi batch

system is generally used because conversion is dependent solely on time as opposed to volume.

Specifically, for growing up cell cultures, the reactor is called a fermenter. Pictured below is a typical

fermenter.

28

Figure 14. 2010.igem.org/File:UCL-BIOREACTORS23

A fermenter is the most common type of reactor in which cell cultures are grown. The cell culture is kept

in a growth media containing a nutrient source essential to rapid reproduction. Cultures can range from

animal cells to bacterial. Similar to a semi batch reactor, the fermenter does not have a liquid outflow.

One distinguishing characteristic of a fermenter is the continual gas feed. Processes operating under

aerobic conditions will need a constant supply of oxygen; this is achieved via the gas inlet and exit

streams. Modifications can be made to the fermenter by changing the agitation equipment, the heat

transfer methodology, and the air sparger.

One adaptation to the fermenter involves modifying the process to become similar to a CSTR. Known as

a perfusion reactor, there is flow into and out of the reactor. The reactor is continuously supplied with

fresh growth media while the spent media is siphoned off. The cells are separated out of the spent

media and returned to the bioreactor to ensure constant cell density. This method is the optimal

process when the stability of the protein is low.24

29

Figure 15. Perfusion Reactor25

Another variation of the fermenter is a bubble bioreactor column. These bioreactors are vessels with

large aspect ratios (height to diameter ratio).26 While maintaining many of the core concepts of the

fermenter, the bubble bioreactor column differs by the agitation method. Instead of using a mechanical

shaft the mixing occurs by forcing compressed gas into the reactor and then the gas bubbles up through

the liquid. These reactors have relatively low capital cost, require only simple mechanics and incur

reduced operating costs based on the lower energy requirements.

Figure 16 Bubble Reactor Column.26 Schematic diagram showing the framework of a model for a bubble column reactor with a

recycle. The right hand side details a section of the column’s phase material balances.

Below is a compilation of the various design options for each reactor needed.

30

Table 6 Reactor Alternatives

Reactor Alternatives

Fermentation

Fermenter

Perfusion Reactor

Bubble Bioreacter Column

PEGylation

Semi Batch

PFR

CSTR

5.4.5.4.5.4.5.4.2222 Design CriteriaDesign CriteriaDesign CriteriaDesign Criteria

The production of eporis will require two different reactors: one reactor will be used for growing the

yeast and the other will be used for the pegylating the protein product. Below are listed the criteria that

will be used to determine the optimal reactor for each process.

Considerations for the Fermenter:

Temperature Control:

The yeast culture is sensitive and as so maintaining a key temperature range must be possible. The

reactor must have a heating and cooling system that can readily manage the required temperature. As

such either a jacketed reactor or a reactor with heating and cooling coils should be used in order to

ensure a consistent temperature throughout the vessel.

Oxygen Injector:

Getting oxygen dispersed throughout the growth media is crucial for the survival of the yeast. An

excellent oxygen distribution system is needed in order to accommodate the substantial oxygen

requirements needed by the yeast. A typical air sparger is used to generate the needed oxygen feed to

the growth media. If possible, coupling the sparger with a disk impeller increases bubble dispersion is

greatly enhanced and the aeration system’s efficiency increases as a whole.

Agitation Method:

Two different types of agitation will be considered: mechanical agitation using a shaft with various

impellers attachments or agitation through compressed gas flow (much like the bubble column reactor).

For mechanical agitation, the type of impeller attachments should have a lower dissipative energy

transfer in order to prevent cell rupture. These impellers include the three-blade-segmented impeller

and the marine-type impeller. However, in bioreactors with an H/D ratio above 1:1.4 multiple impellers

are used to ensure efficient mixing throughout the solution. For example, a reactor may have a disk

impeller right above the air sparger to promote good air distribution and a marine-type impeller to

ensure thorough mixing of the media above the disk impeller. For agitation without a mechanical shaft,

compressed air would be used to agitate the media. The compressed air would have duo-functionality as

it would not only serve to mix the fluid, but also to provide sufficient oxygen to the solution.

Amount of Solution:

31

Depending on the volume of the media that the reactor will need to hold, different reactors will be

better suited for the production process. For example, less volume will allow for different agitation

methods and for variations to the temperature maintenance of the system.

PEGylation Reactor:

The PEGylation (PEG) reactor design criteria is similar to the criteria of the fermenter except no cell

culture is grown within the PEG reactor. Also, unlike the fermenter where additional time leads to extra

conversion, if the protein remains in contact with PEG, over polymerization will occur resulting in

undesired product.

Temperature Control:

The reactor will need to stay within the appropriate range in order to maintain the structural integrity of

the protein.

Agitation Method:

Similar to the fermenter, the agitation will be crucial to ensuring a full extent of reaction. Less delicate

mixing techniques can be considered as cell rupture is no longer a worry.

5555....5555 Process Analytical Technology (PATProcess Analytical Technology (PATProcess Analytical Technology (PATProcess Analytical Technology (PAT))))

Tight control of the bioreactor environment must be held in order to consistently produce maximal

desired protein. Process Analytical Technology provide information on the state of the reactor.

5.5.1 Dissolved Oxygen Sensors5.5.1 Dissolved Oxygen Sensors5.5.1 Dissolved Oxygen Sensors5.5.1 Dissolved Oxygen Sensors

As mentioned in the previous section, maintaining high oxygen levels is critical to maximum yeast

growth. Three types of on-line probes are available for measurement of dissolved oxygen in the cell

culture media. These are galvanic, polarographic, or optical probes (see Figure 17). In galvanic or

polarographic probes, the partial pressure of oxygen is measured inside the probe with a membrane

selectively permeable to oxygen. Inside these probes, oxygen is reduced by water to form hydroxide

ions under catalysis of a platinum cathode, pulling electrons from the anode. In galvanic probes, the

anode in turn oxidizes lead to its ionic form, supplying electrons to the cathode. In this way current is

generated, and the voltage can be measured and correlated to the oxygen partial pressure at the

cathode. In polarographic probes, a voltage is applied between the cathode and anode, causing silver

metal at the anode to oxidize with chloride ions in the probe into silver chloride, supplying electrons to

the cathodic reaction. The measured current correlates to the oxygen partial pressure at the cathode. In

both cases, the major drawbacks are accumulation and depletion of probe ions such as hydroxide and

chloride, the potential degradation of the semi-permeable membrane, and the elimination of oxygen in

the process of measurement.

32

Figure 17: Polarographic (left) and optical (right) dissolved oxygen probes made by Mettler-Toledo.

The more recent, optical probes avoid the drawbacks of the other two by using optical technology. A

fluorescent dye at the tip of the probe responds to blue light by re-emitting red light (see Figure 18). The

phase and intensity of this red light changes depending on the amount of oxygen dissolving and

interacting with the fluorescent dye. Thus, measuring both of these parameters allows for calculation of

the total dissolved oxygen in the media continuously. While this technology is newer and more costly, it

avoids the disadvantages of earlier probes. The final design will weigh the need for the improved

functionality with the added cost of the optical sensors.

Figure 18: Optical sensor mechanism for detecting dissolved oxygen.27

5.5.2 Cell Density Quantification5.5.2 Cell Density Quantification5.5.2 Cell Density Quantification5.5.2 Cell Density Quantification

To determine timing of feeds and control growth of P. Pastoris, a number of design options are available

to monitor cell density. First, samples from the bioreactor can be removed (typically 1 mL) and placed in

a cuvette to measure absorbance, also known as optical density, at 600nm in a UV-Vis

spectrophotometer. According to Invitrogen, a supplier of Pichia Pastoris yeast, an optical density of 1

corresponds to about 5×107 cells/mL.20 While this would be the simplest option, it has inherent

drawbacks, such as decreasing accuracy in optical density to cell density correlations at high cell

densities and requiring personnel to take samples at given intervals and quantify cell concentrations.

33

Consequently, another feasible option would be to build in a loop to cycle bioreactor contents through a

spectrophotometer to continuously measure optical density. Careful engineering of the loop, also called

a flow-through cuvette with internal dilution, can decrease the path length measured by the

spectrophotometer and allow for lower optical density measurements that would correspond to higher

density cell cultures when dilution is factored in (see Figure 19). The final design will consider the labor

cost of a technician monitoring optical density manually versus the capital cost of the automated

system.

Figure 19: Flow-through cuvette with internal dilution. Path length is reduced by inserting a tube of deionized water into the

cuvette.

5.5.3 EPO Concentration Quantification5.5.3 EPO Concentration Quantification5.5.3 EPO Concentration Quantification5.5.3 EPO Concentration Quantification

In addition to monitoring the cell density, monitoring the concentration of recombinant protein is also

necessary. This information gives insight into production rate, optimum harvest time, and potential

expression problems. Classical methods involve periodically taking samples from the bioreactor. They

include the Bradford assay, which measures the absorbance of a dye that binds to any protein in the

sample. The Bradford assay quantifies total protein, so it may not reflect the concentration of the

protein of interest. Contrastingly, an Enzyme-Linked Immuno-Sorbent Assay (ELISA) can provide a

specific measure of protein. ELISA first captures all proteins in a sample onto a surface, then uses

antibodies to detect a specific protein on the surface, then introduces a colored substrate that reacts

because of an enzyme attached to the antibodies (see Figure 20). The color change because of

34

metabolized substrate indicates the concentration of the protein of interest. ELISA is especially useful in

that it can analyze a sample for many proteins for which antibodies can be generated, such as proteases

and cell-surface markers.26 Alternatively, SDS-PAGE, which separates proteins on a gel based on size and

quantifies the intensity of the protein bands, can also quantify multiple proteins in a sample.28 However,

as mentioned above, these have drawbacks in requiring labor and time.

Figure 20: Basic ELISA method, where analyte (Ag) is detected by an antibody with conjugated enzyme for substrate reaction.

A number of methods seek to accomplish this by co-expressing a fluorescent protein along with the

desired recombinant protein in the same genetic sequence. For example, Infrared Fluorescent Protein

(IFP) responds to light at 684nm with an emission of 708nm.29 Alternatively, Green Fluorescent Protein

(GFP) responds to light at 395nm with an emission of 508nm.26 With a spectrophotometer on-line as

shown in Figure 21, the magnitude of the emitted light can be quantified and correlated with well-

established calibration curves to derive the protein concentration in solution. Drawbacks to this method

are that co-expression of other proteins could reduce overall expression or secretion of the

heterologous protein and that the other proteins would have to be cleaved in an additional process step

downstream.

Figure 21: Lab-scale, real-time monitoring of GFP co-expressed with heterologous protein using excitation and spectroscopy.2

35

Design considerations for recombinant protein monitoring include the total costs of the systems.

Sampling methods have intrinsic costs of reagents, equipment, and labor, while on-line fluorescence

methods have opportunity costs of otherwise greater expression of EPO instead of the recombinant

protein, intrinsic equipment costs, and further processing costs.

5.5.3 Reactor Operating Conditions5.5.3 Reactor Operating Conditions5.5.3 Reactor Operating Conditions5.5.3 Reactor Operating Conditions

As mentioned in previous sections, pH and temperature have large effects on the efficiency of

recombinant protein production, the reduction of undesired organisms, and the inhibition of proteases.

The pH can be monitored using pH probes that measure the electrochemical potential between the

solution and an intrinsic standard.26 The temperature can be measured by thermocouples.26 Many

suppliers exist for these very common instruments, and the different options will be weighed on cost,

longevity, and ability to be sterilized easily.

5555....6666 HarvestingHarvestingHarvestingHarvesting

5.6.1 Research5.6.1 Research5.6.1 Research5.6.1 Research and Design Alternativesand Design Alternativesand Design Alternativesand Design Alternatives

Pichia Pastoris is able to secrete EPO post translation and glycosylation if the EPO protein is properly

labeled.33 Signaling sequences are added to cDNA constructs are translated with the protein of interest

and serve to direct proteins towards secretion.30,31 Product secretion is advantageous in that its limits

the magnitude of the initial separation steps. Cell broth will generally contain a low level of cell parts

from apoptosis. These can be sufficiently removed by centrifugation and filtration. 32 Table 7 lists

secretion signals commonly used in the P. Pastoris system. Secretion signals need to be removed,

however, to meet purity and potency standards. This can be done intracellularly or extracellularly as

part of a downstream process unit.34, 35

An advantage to intracellular secretion label cleavage is that sometimes, paradoxically, secretion signals

can hinder final entry into secretary vesicles. This has been observed with the alpha-factor sequence.33 A

group included an endoprotease, kex2, in the expression vector to resolve this. Overexpression of kex2

enabled more efficient cleavage of the signal sequence in the golgi apparatus, serving to increase

secretion.33 A subsequent drawback follows, though. Expressing multiple proteins in a vector limits

expression of the desired product.28 The dominant effect varies between systems.

Table 7. Signal sequences use in P. Pastoris.

Secretion Signal Original Organism

Alpha factor S. cerevisiae

Alpha Amylase Asp. Niger

Glucoamylase Asp. Awarmori

Inulilnase k. maxianus

Serum albumin H. sapien

Killer Protein S. cerevisiae

Invertase S. cerevisiae

Lysozyme G. gallus34, 35

36

Alternatively, a secretion factor can be excluded from the cDNA construct. EPO will be deposited

intracellularly and wholescale cell lysis will be necessary. Strain is then placed on downstream

separation process units. The approach, however, can give greater EPO yields if the protein displays

inconsistent or inhibited secretion. It can also be useful if cleaving the signal sequence presents

biochemical challenges.32


Efficiency of secretion, ease of separation, and addition of downstream process units all need to be

considered in harvest design. EPO yield and cost of subsequent process units (capital and operational)

shall be particularly considered in evaluating secretion and wholescale lysis approaches. Stewardship is

key to these design considerations. Wholescale lysis and secretion yield distinctly different waste

streams with different treatment requirements and opportunities

for recycling.

5555....7777 Isolation and PurificationIsolation and PurificationIsolation and PurificationIsolation and Purification

In biopharmaceutical production, separation processes are often

categorized into three stages: clarification, purification, and final

polishing. Broadly, clarifying removes extra cell parts, purifying

utilizes chromatography columns to isolate the protein of interest,

and final polishing additionally filters the isolated protein, ensuring

removal of viruses.36

Clarification is the first sequence of separatory steps in industrial

biopharmaceutical production. It serves to roughly separate cell

parts from the reactor effluent. Clarification plays an emphasized

role in expression systems that do not secrete the protein of

interest but instead deposit it internally. In these cases cell lysis

and drastic separation must occur.

Common clarification approaches centrifuge and filter reactor

effluent. Centrifugation removes dense cell parts. It is a technique used to separate the proteins based

on mass and size based on the drag and inertial forces experienced from the outward forces due to

rotation. Massive particles with low drag will fall to the bottom, while non-compacted particles remain

in the liquid as the supernatant. Typically continuous models are used, such as the disk stack centrifuge.

Subsequent filtration further purifies effluent. Depth and membrane filtration are commonly used at

this stage.36 Depth filtration uses a packed bed to filter flow through, offering the advantage of

increased surface area for absorption.36 Traditional membrane filtration makes use of two dimensional

membranes for purification. Microfiltration is performed first and is often followed by ultrafiltration.

They differ in size of particles excluded. Microfiltration filters at the micro level, whereas ultrafiltration

filters at a more fine scale. Both use membranes and pressure driven flow to drive separation.

Ultrafiltration membranes can separate based on size, charge, molecular structure and other factors. 37

Purification/column chromatography is used to separate out the EPO from the other proteins left over

after clarification. This can occur by several types of separation based on size, charge, biological activity,

and bonding affinity.

Figure 22. General EPO process flow

diagram

37

Hydroxyapatite is a naturally occurring mineral form of calcium, used in affinity chromatography that

can be used several times without regeneration. It involves interactions between positively charged

calcium ions and negatively charged phosphate ions on the stationary phase with negatively charged

carboxyl groups and positively

charged amino groups of the target

protein (Figure 23).

Ion exchange chromatography is a

separation technique that isolates

molecules (or proteins) based on

charge. The charged molecules bind

to the stationary phase of the

column, followed by an elution by a

wash of differing pH (or charge),

therefore isolating the target.

Membrane Ion electrophoresis is a

separation technique based on the

mass to charge ratio of proteins.38

His-tag chromatography is very similar to affinity chromatography, and can be used even when there are

no readily available antibodies for the target protein, but rather histidine groups attach to the nickel

column and eluted with standard chromatography techniques.

HPLC stands for high pressure liquid chromatography where a solvent is pumped through an adsorptive

stationary material in a column, and can be used in tandem with several of the other column

chromatography techniques.39

Blue-dye (sepharose 6) binds to many proteins, including EPO. It is currently used in several routine

commercial production processes.17 It can also be used to see the proteins separate into different bands

during electrophoresis.

Final polishing separates the target structure of EPO with the proper glycosylation and other post

translational modifications. This ensures the uniformity in activity, and allows for proper dosage to be

established. Viruses and other potential contaminants are removed in this critical aseptic stage.

A second tier of centrifugation and filtration follows to prepare the drug substance for final

formulation.5 Formulation buffer is key considersation.43 Techniques applicable at this step include

centrifugation, depth filtration and ultrafiltration.


Biopharmaceutical processes are often determined to be not economically feasible in the separation

phases. Considerations are made in respect to cost, scalability, and yield of each individual separation

step. Column chromatography makes use of a mobile solvent phase to elute the separated protein, but

we must pay close attention to the amount, price, and safety of the materials and chemicals used in

each process when determining the best option. Increasing the number of columns used will produce

more waste streams where we must consider the impact on sustainability of the process.

Figure 23. Affinity Chromatography for protein purification

38

5555....8888 Recycling and Recycling and Recycling and Recycling and Waste ManagementWaste ManagementWaste ManagementWaste Management

5.8.1 Research and Design Alternatives5.8.1 Research and Design Alternatives5.8.1 Research and Design Alternatives5.8.1 Research and Design Alternatives

Any part of the outlet fermentation broth that is not EPO is considered waste. Thus much waste is

generated in the reactor and along the downstream separation process units in both solid and liquid

forms.40 Solid waste treatment and process water treatment present an important design consideration

in the proposed EPO process.40, 41 In order to optimize the fermentation process as a whole, waste

streams from downstream units need to be considered both in separation and fermenter reactor

design.40

Waste water streams from fermentation and pharmaceutical plants contain higher than average

amounts of dissolved oxygen, higher carbon to nitrogen ratios, low pH, and traces of other organic

compounds (typically from the fermentation media) that must be separated out prior to standard waste

water processing. Waste water streams are commonly passed through a bed of activated sludge to

accomplish this.41 Waste cellular material can be recycled and sold as animal feed if properly deactivated

first. eporis proposes shipping waste yeast P. Pastoris material to a designated facility for deactivation

and future resale as feedstock.42 Waste streams emerging from chromatography columns, centrifuges,

and filters have other treatment needs. Solid waste from filter cakes, centrifuges, and column effluent

(once crystalized) can be treated as disposed as waste salts.40

Recycle streams are not typically employed in biopharmaceutical processes. Contamination issues and

control issues typically make the practice unfavorable.43 However, certain media-derived elements of

fermenter waste may be easily recycled, such as salts and minerals.40

5.8.2 Design Consideration5.8.2 Design Consideration5.8.2 Design Consideration5.8.2 Design Consideration

Waste and recycle design alternatives are inherently linked to each process step. Design of each unique

process unit shall take waste streams and their treatment into consideration. Stewardship is key when

considering waste generated and how it will be treated. The opportunity to deactivate and recycle

cellular fermenter waste into animal feed fulfills this design norm well.

6666. Proposed Laboratory Simulations. Proposed Laboratory Simulations. Proposed Laboratory Simulations. Proposed Laboratory Simulations

6666.1 Simulations.1 Simulations.1 Simulations.1 Simulations

6666.2.1 Scale .2.1 Scale .2.1 Scale .2.1 Scale –––– up Equations up Equations up Equations up Equations

Generally, bioreactors are readily scalable from the bench top up to large processes. The vessels must

have a geometrically similar design is considered important and straightforward scale up process. In

general the power input per volume is used as scale-up criterion. Tip speed or other shear related

parameters are used especially if shear sensitive cells are in the media.

The height-to-vessel-diameter ratio (aspect ratio) should be within a range of 1:1 to 3:1 for stirred

reactors44. A lower value results in a larger headspace surface to filling volume which enables an

improved gas exchange at the gas-liquid interface. Contrastingly, a larger aspect ratio offers a better

direct sparging due to the longer residence time of the gas bubbles in the liquid. This yields a better

oxygen transfer rate. Animal cell cultivations often us a value of 2:1.44

39

Impeller diameter to vessel diameter: 0.33 – 0.5 for animal cells. This influences the mixing efficiency

and the generated shear forces. Typically three-blade-segment impellers or marine-type impellers are

commonly used for animal cell cultures. They generate large circulation loops due to the axial flow

patterns.

Disk impellers generate radial flow leading to a higher power input per volume and enhanced gas-

bubble dispersion. It is common to install multiple impellers in bioreactors with an H/D ratio above 1:1.4

to ensure efficient mixing throughout the entire cultivation chamber.44

6666.2.2 Simulation Software.2.2 Simulation Software.2.2 Simulation Software.2.2 Simulation Software

Honeywell UNISIM software does not have the capability of modeling biochemical or pharmaceutical

processes. The software that eporis will use is called SuperPro Designer, developed by Intelligen Inc. This

process modeling software is currently used in the biochemical, pharmaceutical, specialty chemical, and

food processing industries. SuperPro Designer will allow us to model EPO production, estimate its cost,

as well as evaluate the environmental impact of each stage of the process.

8888. . . . Business PlanBusiness PlanBusiness PlanBusiness Plan

8888.1 .1 .1 .1 Market AnalysisMarket AnalysisMarket AnalysisMarket Analysis

8.1.1 Target Market8.1.1 Target Market8.1.1 Target Market8.1.1 Target Market

The target market of eporis is other pharmaceutical companies that wish to enter the market of

biosimilar drug products. Other pharmaceutical companies are able to use cheaper drug substance for

Figure 24. SuperPro Designer process modeling software.

40

their drug products than they would be able to produce themselves. They can diversify and enter the

market of biosimilars without developing new drugs to do so, bypassing the drug substance

manufacturing process altogether.

8.1.8.1.8.1.8.1.2222 Demographic ProfileDemographic ProfileDemographic ProfileDemographic Profile

The different demographics of the companies that eporis will do business will be mainly large

pharmaceutical companies who are not involved in the research and development of biologic and

biosimilar drugs. Companies that have optimized drug product manufacturing processes will also buy

from us. We are focusing on the American market, so we will deal primarily with companies that sell

drugs within the country.

8.1.3 Market Size and Trends8.1.3 Market Size and Trends8.1.3 Market Size and Trends8.1.3 Market Size and Trends

The current market for biosimilars in America is fairly small at $1.9 billion - $2.6 billion in 2015, and is

expected to grow to around $20 billion by 2020 due to biologic patent expiration. EPO is the largest

biologic based drug in the market.

8.1.4 Advertisi8.1.4 Advertisi8.1.4 Advertisi8.1.4 Advertising and Pricingng and Pricingng and Pricingng and Pricing

eporis has no need for advertising or promotion as we do not produce a drug product. Representatives

approach potential customers to sign contracts where drug substance is supplied to be used in the

customers’ drug products.

8888....2222 Research Research Research Research andandandand Development CostsDevelopment CostsDevelopment CostsDevelopment Costs

Bringing a biosimilar to the market is more difficult than a small molecule generic in terms of FDA

approval. There is a development cost of about fifty times that of a generic pharmaceutical. With the

difficulty of bringing a biosimilar to market, comes a reduction in competition. For example, there will

likely be no competition from Target or Walgreens brand EPO therapies. With the increased risk of

failure compared to generics, comes an increased rate of return with a successful biosimilar.

9999. . . . Financial EstimatesFinancial EstimatesFinancial EstimatesFinancial Estimates

The financial estimates to consider throughout the design process consist of both business capital costs

as well as operational costs of the production process. We estimate the price that our biosimilar to be

seventy percent of the price of the drug our EPO variant is similar to. We evaluating various business

strategies and their associated risk based on the project’s financial estimates. We will also consider

various process design alternatives for economic feasibility to meet the target production cost.

Figure 25. Development statistics in comparison.

41

10101010. Conclusion . Conclusion . Conclusion . Conclusion

The main lesson learned through the analysis within the scope of this design process is that eporis must

work to verify major assumptions for the success of the project. The research conducted, along with

insight from an industrial consultant has made us aware of the several pitfalls in a biopharmaceutical

process. Future work consists of designing each unit operation in the process, leading to an accurate and

empirical economic analysis of the project.

11111111. . . . ReferencesReferencesReferencesReferences

1 Surabattula et al. Research in Biotechnology, 2(3): 58-74, 2011

2 M. Sjöblom, Pichia pastoris as a Platform for the Production of Therapeutic Glycoproteins, Luleå University of

Technology. 27 (2008).

3 The Patient Protection and Affordable Care Act (2010). U.S. Department of Labor. Washington, DC.

4 FDA documentation. Accessed 11/09/14.

http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=610.13

5 J. S. Lee, T. K. Ha, S. J. Lee, G. S. Lee, Current state and perspectives on erythropoietin production, Appl.

Microbiol. Biotechnol. 95 (2012) 1405-1416.

6 S. R. Hamilton, R. C. Davidson, N. Sethuraman, J. H. Nett, et. al., Humanization of yeast to produce complex

terminally sialylated glycoproteins. Science. 313 (2006) 1441-1443.

7 J. H. Nett, S. Gomathinayagam, S. R. Hamilton, et. al., Optimization of erythropoietin production with controlled

glycosylation-PEGYlated erythropoietin produced in glycoengineered Pichia Pastoris, J. Biotechnol. 157 (2012) 198-

205.

8 J. C. Ergie, J. K. Browne, Development and characterization of novel erythropoiesis stimulating protein (NESP),

British J. Cancer Res. 84 (2001) 3-10.

9 P. L. Storring, R. J. Tiplady, R. E. Gaines Das, et. al., Epoetin alfa and beta differ in their erythropoietin isoform

compositions and biological properties, British J. Haematol. 10 (1997) 79-89.

10S. Elliot, T. Lorenzini, S. Asher, et. al., Enhancement of threapetuic protein in vivo activities through

glycoengineering, Nat. Biotech. 21 (2003) 414-421.

11 U.S. Department of Health and Human Services, Food and Drug Administraiton, et. al., Guidance for Industry.

Biosimilars: Questions and answers regarding implementation of the biologics price competition and innovation

act of 2009. (2012).

12 US $54 Billion worth of biosimilar patents expiring before 2020. Accessed 11/09/14. Retrived from Generatics

and Biosimilars Initiative Onlie: http://www.gabionline.net/Biosimilars/Research/US-54-billion-worth-of-biosimilar-

patents-expiring-before-2020

13 N. Davoudi, A. Hemmati, Z. Khodayari, et. al., Cloning and expression of human IFN-gamma in Leishmania

tarentolae, World J. Microbiol. Biotechnol. 27 (2011) 1893-1899.

14 X-33, Pichia Pastoris Yeast Strain, 9 Novemeber 2014. [Online]. Available:

http://www.lifetechnologies.com/order/catalog/product/C18000.

15 B. Byrne, G. G. Donohoe, R. O'Kennedy, Sialic acids: carbohydrate moieties that influence the biological and

physical properties of biopharmaceutical proteins and living cells, Drug Discov. Today. 12 (2007) 319-325.

42

16 A. Maleki, F. Roohvand, H. Tajerzadeh, High Expression of Methylotrophic Yeast-Derived Recombinant Human

Erythropoietin in a pH-Controlled Batch System, Avicenna Journal of Medical Biotechnology. 2 (2010) 197-205.

17 W. Hinderer and S. Arnold.United State of America Patent US 2012/0264688A1, 2012.

18 A. J. Sytkowski, E. D. Lunn, K. L. Davis, Human erythropoietin dimers with markedly enhanced in vivo activity,

Proc. Natl. Acad. Sci. USA. 95 (1997) 1184-118.

19 M. S. Eskitoros, P. Calik, Co-substrate mannitol feeding strategy design in semi-batch production of recombinant

human erythropoietin production by Pichia pastoris, Chem Technol. And Biotechnol., 89 (2013) 644-651.

20 Pichia Expression Kit User Guide, Life Technologies, 2014.

21 E. Celik, P. Calik, S. G. Oliver, Fed-batch methanol feeding strategy for recombinant protein production by Pichia

pastoris in the presence of co-substrate sorbitol, Yeast. 26 (2009) 473-484.

22 H. S. Fogler, Elements of Chemical Engineering, Westford, Massachusetts: Pearson Education Inc., 2006.

23 T. U. London, "IGEM 2010 UCL," UCL, 2010. [Online]. Available: http://2010.igem.org/File:UCL-BIOREACTSRS.png.

[Accessed 9 Novemeber 2014]

24 http://www.novasep.com?technologies/Upstream-perfusion-process.asp

25 J. Bonham-Carter, A Brief History of Perfusion Biomanufacturing, BioProcess International 9(9), October 2011.

26 J. E. Bailey and D. F. Ollis, Biochemical Engineering Fundamentals, New York: McGraw-Hill Inc., 1986.

27 S. Bell, F. Dunand, A comparison of amperometric and optical dissolvedoxygen sensors in power and industrial

water applicationsat low oxygen levels (<5μg·kg-1), Power Plant Chemistry, 12 (2010) 296-303.

28 F. Valero, Bioprocess Engineering of Pichia pastoris, an Exciting Host Eukaryotic Cell Expression System, in: T.

Ogawa (Ed.), Protein Engineering - Technology and Application, InTech, 2013, pp. 3-32.

29 H. Dortay, U.M. Akula, C. Westphal, M. Sittig, B. Mueller-Roeber, High-Throughput Protein Expression Using a

Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection, PLoS ONE. 6

(2011) e18900.

30 B. Gaun, F. Chen, J. Lei, Y. Li, X. Dunn, et. al., Constitutive expression of a rhIL-2-HAS fusion protein in Pichia

Pastoris using glucose as carbon source, Appl. Biochem. Biotechnol. 171 (2013) 1792-1804.

31 N. Gavindoppa, M. Hanumanthappa, K. Venkataranhaiah, et. al., A new signal for recombinant protein secretion

in Pichis Pastoris, J.Microbiol. Biotechnol. 24 (2014) 337-345.

32 G. Yao, X. Qin, J. Chu, X. Wu, J. Qian, Expression, purification, and characterization of a recombinant methionine

Adenosyltransferase pDS16 in Pichia Pastoris, Appl. Biochem. Biotechnol. 172 (2014) 1241-1253.

33 S. Yang, Y, Kuang, L. Hongbo, Y. Liu, Enhanced Production of Recombinant Secretory Proteins in Pichia Pastoris by

optimizing Kex2 P1’ site, PLOSone. 8 (2013) 75347.

34 EasySelect™ Pichia Expression Kit. Accessed 11/09/14.

www.lifetechnologies.com/order/catalog/product/K174001

35 Yeast Expression Vectors. Accessed 11/09/14. https://www.dna20.com/products/expression-vectors/yeast.

36F. Capito, J. Bauer, Feasibility Study of Semi-Selective Protein Presipitation With Salt-Tolerant Copolymers for

Industrial Purification of Therapeutic Antibodies, Biotechnol. and Bioeng. 110 (2013) 2915-2927.

37 C. J. Geankoplis, Transport Processes and Unit Operations, Englewood Cliffs: Prentice - Hall International Inc.,

1993.

43

38 Santos (2012) Separation of Recombinant Human Erythropoietin (rhEPO) using the European Pharmacopoeia

Method. Global Tactical Marketing, Beckman Coulter Life Sciences, Brea, CA USA.

39 Duong, BioPharma. Fast Separation of Recombinant Human Erythropoietin Using Reversed Phased Agilent.

40 P. Van Hoek, A. Aristos, J. J. Hahn, A. Patist, Fermentation goes large scale. Chem. Eng. Prog. 99 (2003) 37s.

41 Grismer (2000) Water Environment Federation. Water Environment Research, Volume 72, Number 5.

42 DiMarco. (2014, August 13). Abbvie Laboratory Tour. (S. Tubergen, Interviewer).

43 Natarajan, V. (2014, October 28). Industrial Consultant Meeting. (T. 15, Interviewer).

44 D. De Wilde and T. Dreher, "Superior Scalability of Single-Use Bioreactors," BIOProcess International, pp. 14-19,

2014

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