BIOMAN 2011CHO-tPA Production System

Upstream Processing

Mike FinoMiraCosta College

Program Concentrations

Research• Discovery• Preclinical

studies

Development• Clinical studies• Scale-up

Production• Quality• Compliance

MiraCosta R&D MiraCosta

Bioprocessing

Program Coursework100-level courses

• Basic Techniques in Biotechnology (4-units)– Introductory experience working in

a technical environment

• Business and Regulatory Practices in Biotechnology (3-units)– Fully online– Overview of the industry from

discovery to GMPs

• Biostatistics (4-units)– Lecture and lab– UC/CSU transferrable

Program Coursework200-level courses

Hybrid Lab courses Fully online Courses• Advanced Cell Culture• Isolation and Purification of DNA• Techniques in DNA Amplification• Recombinant DNA• Principles of Separation and HPLC• Techniques in Immunochemistry and

ELISA• Qualification and Validation in

Biotechnology• Bioprocessing: Cell Culture and

Scale-up• Bioprocessing: Large Scale

Purifications• Biofuels Production and Analysis

• Data Analysis with Excel• Technical Writing for

Regulated Environments

CAREER EXPERIENCE Internship Work Co-op

Laboratory Skills

1-2 semesters

11 to 12.5-units

All BTEC classes

Bioprocess Technology

1-2 semesters

12 to 13.5-units

All BTEC classes

Research and Development

3-4 semesters

40 to 43.5-units

BTEC and GE classes

Career & Professional Development

Transfer / Long-Range

Program Certificates

Biopharmaceuticals or Biofuels

Biopharmaceutical Production

• Upstream– Product formation

• Downstream– Product purification

• Quality Control– Product safety and efficacy

A Time of Transition and Translation

• The lead candidate coming out of discovery research will now be subjected to a development process that sees it change through a prism of– Scale– Process Control– Compliance

7

From Art to ScienceScale-up

• Development is the confluence of business, engineering, manufacturing, quality assurance, quality control, and regulatory affairs

• The end goal is process understanding so that a commercial process and product are in a state of control

8

Process Control & Compliance

• Say what you do

9

Controlled process/ productMan

Machine

Materials

Methods

Environment

Do what you say Document it

The Upstream Process

• The confluence of inter-related efforts in:– Cell line development– Media development– Bioreactor process design– Bioreactor process control

11

Cell Line Engineering

12

Expression Systems

• Bacteria• Yeast• Mammalian• Insect• Transgenic Plants• Transgenic Animals

17Oct08

13

End game of cell line engineering?

• PCD = picograms per cell per day

14

15

cont’d

16

Back of the envelope calcs

• If we have a cell line that produces our product at 100 pg/cell-day and we want to produce material for a Phase I clinical trial in a single batch then…– Our product is dosed at 1-mg/kg– Our process takes about 1 week– Typical cell densities are 1e6 cells/mL– Assume 20 people in the trial– Assume average person is 75-kg

• …how big does my culture need to be?

Large scale mammalian cell culture

• We use the CHO-tPA system as a model culture system for several reasons– Seeding densities as a way to show why a step-

wise – Aseptic technique and processing to underscore

the slower growth rates vs microbes– Currently the main choice for any glycosylated

protein

18

Inherent Variability: Erythropoietin

• Contains 40% carbohydrate, only 2 disulfide bonds• 3, N-linked ASN (24, 38, 83), 1 O-link (SER126) glycosylation

sites – Glycosylation at these sites may be responsible for resistance to

denaturing conditions• O-linked site not essential for in-vitro or in-vivo activity• Sialic acid residues (avg 10 moles/mole EPO) responsible for

preserving phamacokinetic behaviour– Muteins lacking 2 or 3 N-linked sites are poorly secreted

• N-linked glycosylation and sialylation is critical to optimal secretion, structure, in-vivo potency

The Upstream Process

• The confluence of inter-related efforts in:– Cell line development– Media development– Bioreactor process design– Bioreactor process control

20

21

The Upstream Process

• The confluence of inter-related efforts in:– Cell line development– Media development– Bioreactor process design– Bioreactor process control

23

24

25

26

27

28

29

Reac

tor S

elec

tion:

Man

y fa

ctor

s to

con

side

r

30

Developing higher yields• In 1986, the industry standard from stable CHO-derived

cell lines was a specific productivity of 10 pg/cell/day, 50 mg/L titers, 2e6 cells/mL and processes lasted 7 days

• Today, we are seeing 90 pg/cell/day, 10 g/L, 10e6 cells/mL, and processes lasting up to 3 weeks1. Generation of recombinant cell lines with high specific

productivities2. Formulation of media to support high density cell

cultivation3. Understanding of bioprocess conditions for cell cultivation4. Sustained viability of cell lines in high-density batch and

fed-batch cultures

Stirred Vessel Bioreactor

• Process control of the physiologic environment– Disssolved oxygen – pH– Temperature– Medium components

(feed: glucose, glutamine; hormones, growth factors)

– Waste products (CO2, ammonium, lactate)

31

32

Batching Types (Process Modes)

• Single Batch• Fed Batch• Continuous Batch

33

Batching Types• Single Batch

– 7-14 days– Run is over when there are no

more nutrients

34

Batching Types• Fed-Batch

– Addition of concentrated nutrients => higher product concentration

– 2-3 weeks– Intermittent common for viral vaccine– rProtein production is largely fedbatch

35

Batching Types

• Continuous– Continuous addition

of feed and collection of harvest with cell retention device => higher product concentration

– Especially for process-labile products

36

Parameters measured or controlled in bioreactors

37

38

39

40

41

The Upstream Process

• The confluence of inter-related efforts in:– Cell line development– Media development– Bioreactor process design– Bioreactor process control

Essentials to Control a Process

43

Process Variables Identify which variables are important to what you’re doing Bioreactors: pH, DO, Temperature, cell count, nutrient levels, waste levels, CO2

levelsProcess Probes

You need some instrument that can convert the chemical or physical phenomenon into an electrical one

Controller The basic function of a controller is to make a comparison between the current

reading of a Process Variable and the desired Setpoint Based on that comparison, the controller produces an output

Process Manipulation Based on the Output, you then attempt to manipulate the Process Variable to

bring it closer to the Setpoint

17Oct08

Bioreactor Control System

44

• Yellow number is the Process Variable, PV• White number is the Set Point, SP• Blue number is the Controller Output

ControllerPVSP

Output

Depending on the level of the output, something will be changed (e.g. valve opened/closed) to bring the PV closer to the SP

17Oct08

A Control System

• A simple error calculation will be made:– SP - PV = Error

• Depending on the nature of the Error an Output is calculated– Magnitude– History– Current slope

• The Output will do something to bring the PV closer to the SP

4517Oct08

4617Oct08

Our BioNet Systems

• Bioreactor– Probes– Heating blanket– Agitator and motor

• Control tower– Collects, stores, and processes information– Controls gases and pump additions– Primary interface

4717Oct08

Bioreactor Control System

48

• MANual mode– Controller not utilized, full operator instruction– Example is agitation – we set it and forget it

• AUTO mode– Controller will try to bring the PV in line with the SP– The Output of the AUTO mode will be looking to do

something, based on what’s defined in the Setup Page• CAScade mode

– These are the devices that are looking for direction from the AUTO output

– This is for all the instruments (pumps, valves, MFCs) that will be controlled in order to maintain the SP

– Their response is defined in the Setup Page

17Oct08

4917Oct08

Priming the Base50

17Oct08

Gas Controls

5117Oct08

The main controlled process variables

• pH– Cues from animal physiology dictate starting

points for this• Most normal cell cultures around 7.4• Transformed cells around 7.2

– Our cultures do not aggressively change pH so we will be using gases to maintain our pH

• The Output of the controller will change gas mixtures to affect pH

5217Oct08

• Agitation– This one, while it is technically controlled to a

setpoint, is not influenced by our cultures – we set it and forget it

– Common ranges are 80 to 120 RPM– For cell cultures we need to be concerned about

shear damage

17Oct08 54

Controlled process variables

• DO – Dissolved Oxygen– For a given temperature and barometric pressure

and solution composition, water can only “hold” so much oxygen.

– Example: if you were to keep adding sugar when you’re making Kool-aid.

• Eventually, for the given temperature of the kool-aid, there’s some critical amount of sugar it can hold. After that it just precipitates out.

5617Oct08

• The same is true for DO– Water (given pressure, temp, and composition)

can only hold so much oxygen– After that, it’s saturated– This is about 5-10 parts of O2 per 1 million part of

water

• Common setpoints for O2 are 30-50% of saturation

5817Oct08

Controlled process variables

• Temperature– Temperature takes its cues from animal physiology

for animal cells: 37oC– We keep ours at 37oC because though we’re

always cautious because cells are much more tolerant to deviations on the low side than the high.

5917Oct08

Instrumentation Calibration

• Most lab instruments contain what can be called a transducer. • It’s some component that is transducing a physical or chemical

phenomena into an electrical phenomena.

– We need to let the computer (or whatever is monitoring the probe) know what the electrical signals mean

6117Oct08

Calibration example

• For pH probes, an electrical signal is generated at about 60mV per pH unit. This is a slope.– We need to locate this line, with the intercept. – Three points is best to define a line and locate the

calibration curve; two is the minimum

6217Oct08

Dissolved Oxygen example

• For the DO we also want to establish a line (standard/calibration curve)– We don’t have nice standards though like for pH– We therefore set a zero and a span (i.e. 100%)

• Expose the probe to deoxygenated water (or a simulator)

• Expose the probe to fully saturated water

6317Oct08

Temperature example

• The same idea continues, at least two points, more is better to define the calibration curve– What can be used for temperature?

6417Oct08

Process instrumentation

• The previous examples are controlling the process environment with real-time, online measurements.– A setpoint is defined and the control system works to make sure the

process variable equals the setpoint• There is also offline instrumentation, where you need to take a

sample and use an instrument not connected to the system

6517Oct08

Process instrumentation (cont’d)

• The data from these instruments can be used to make adjustments to your culture variables, e.g.– Cell growth rate– Metabolite consumption– Toxic substance build-up

6617Oct08