Upstream Processing

Upstream Processing Overview

Main objective – to create the environment necessary for cells to make a

protein product (recombinant protein or biologic)

§ Product of interest
• Called API’s- Active Pharmaceutical Ingredient
• Produced by mammalian cell culture (Chinese Hamster Ovary - CHO cells, mouse
myeloma NSO cells) or bacterial fermentation (E.Coli)

§ Mammalian cells excrete product into media

• can modify proteins (post translation modifications)
• harvest can be made without cell lysis
• CHO cells most commonly used

§ Bacterial cells
• do not have the machinery to secrete the desired product into the media
• cells must be lysed during harvest
Biomanufacturing Process Flow – Upstream Processing
 Upstream Processing - Cells

Cells used in upstream processing:

– contain the transfected gene that expresses the

desired API (protein)

– transfected gene is linked to a gene that imparts

special survival abilities to the cells that have it

• kept in cryovials in Dewars

» Dewar – a specialized vessel that provides
the environment to keep cell processes in
temporary frozen suspension
Upstream Processing Areas, Equipment &
Systems

§ Dispensing room
§ CIP/SIP systems
§ Media Preparation area
§ Cell Culture/Fermentation
§ Cell Banking Area
§ Bioreactors
§ Primary Recovery (harvest)
 Dispensing Room

Materials needed to make product are weighed and

measured

Environment and access are strictly monitored traceably

documented
• Containment and Access
• Air quality and flow
• Cleanliness
 Containment and Access

§ Containment booths to prevent cross contamination of one

material to another
– non animal derived and animal derived materials
segregated to reduce exposure to adventitious viruses
– only one raw material in the booth at a time

§ Weighing materials such as scoops, pipette tips, spatulas, weighing

boats, and graduated cylinders are sterile and discarded or properly
cleaned after use

§ Access to rooms monitored electronically and gowning required for

technicians
• gown suit, two layers of gloves, face masks, sleeve covers,
 Air Quality & Flow

– Air cleanliness classification – Class 100,000 (FDA Class D) clean

room

– HVAC system maintains the room at a positive pressure with

respect to surrounding rooms and corridors

– Room pressures continuously monitored by building automation

system which collects data to show that room is maintained in
controlled state

– Circulate 90% of airflow through pre-filter and HEPA filter bank

– HEPA filters certified and calibrated periodically (generally every

6 months)
Dispensing Booths
 CIP/SIP Systems

§ CIP (Clean In Place)

– automatic cleaning of processing equipment, vessels, piping and in-
line devices
– minimal manual setup or shutdown
– little or no operator intervention during cleaning
– strong base, rinse, strong acid, final rinse with WFI
– conductivity tests to monitor content of cleaning solutions and
rinse water

§ SIP (Steam In Place)

– equipment and vessels sterilized with clean steam
– After sterilization system must remain pressurized to maintain
sterility
 Cell Culture Media

Provides all the nutrition cells need within a narrow window of

environmental conditions for optimal expression of the target protein
– Major media components
• Carbohydrate energy source – glucose
• Nitrogen source such as amino acids
• Lipids – often in the form of fatty acid

– Cells also require

• Trace minerals in the form of electrolytes (salts)
• fetal bovine serum supplements – not so common due to risk of animal
viruses
• Chemically defined, serum free media commonly used – reduces threat of
adventitious animal virus contaminants
• Selective agents that cells require for optimal expression of the target
protein
 Media Preparation

§ Usually done in tanks

– Powdered media dissolved in high purity grade Water
For Injection (WFI)
– Batch Record followed with every step
– Proper mixing time essential – media must be
homogenous before introducing cells

§ Parameters monitored
– pH- measures degree of acidity or alkalinity
– Conductivity – measure of ions dissociated in the
solution; purity of solutions used in media prep
– Glucose - indicator of cell growth; main energy
source for cells in growth phase
– Osmolality - measure of osmotic pressure,
dependent on salt concentration of media
 Cell Culture/Fermentation

Cell Growth - 4 distinct phases

• lag phase – cells adapt to the environment
• log phase – exponential growth
• plateau phase – growth rate slows- rate of proliferation
equals rate of cell growth
• death phase – rate of cell death exceeds proliferation rate
and cells start to die

§ Generation – doubling of cell concentration from the original

seeding cell concentration
• Doubling time vary depending on the cell
» E.Coli - doubling time 20 minutes – culture batch ~ 24
hours
» CHO - doubling time 14-17 hours – culture batch ~ 6-
20 days
Cell Culture Growth Phases
 Master Cell Bank (MCB)

Cells used for production culture batches are established from a single
clone and stored frozen in banks called Master Cell Banks

– stored at low temperatures in vapor phase of liquid nitrogen

(- 196°C)
• suspended in cryoprotecant (DMSO or glycerol) to prevent damage to
cell membrane by ice crystal formation

– Preserves characteristics of original cell line

– Prevents contamination and deterioration

– Produced in accordance with regulatory standards (21CFR 610)

 MCB- Cell Line Characterization

Cell line have to be stringently tested to

– Confirm identity of expression construct (species identity)

– Confirm purity (contamination-sterility tests for bacteria,

mycoplasma, adventitious viruses)

– Confirm genetic stability (coding region)

Quality assurance must be established from MCB to end-of-

production/post production cells (EPC/PPC)
 Upstream Processing Stages

§ Inoculum
– Frozen vials of cells from cell bank thawed and added ( “inoculated”) to spinner flasks ( 100 – 500 mL) or cell
culture bags (50-200 mL)
– Cell culture expanded to meet cell density and volume requirements to inoculate larger volume bioreactor

§ Bioreactor Seed
– Cell cultures from spinner flasks or culture bags transferred
“seeded” into larger volume bioreactors ( up to 20,000L)
– Bioreactors are either stainless steel or disposable
– Cells grow to high densities producing API in the culture media

§ Primary Recovery ( Harvest)

– main purpose is to separate the cells from the media containing
the API
• Centrifugation to separate cells from media
• Filtration to remove large debris
 Inoculum

§ Ampoule of frozen cells released from Master Cell Bank and

thawed - Out of Freeze (OOF)

§ Cells added to prepared media in spinner flasks or culture bags

and cultured in temperature controlled (35-38°C) humidified,
incubator infused with CO2 (5%)

§ Specific conditions to promote multiplication of the cells without

producing the protein

§ Monitor cell concentration, viability, and pH of culture medium

§ Replenish nutrients when culture reaches 75-100% of it’s peak

growth – ”subculture or passage”
Spinner flask containing cells in suspension in a biosafety cabinet
 Culture Parameters- pH

– Critical process parameter – fluctuations in pH can affect

growth
– Optimal pH between – 6.8 and 7.4

– Incubator
– Culture media contains bicarbonate which, when combined
with CO2 infused into the culture incubator makes a buffering
system to control pH

– Bioreactor
– pH measured by inline probe and bioreactor control system
– pH of media decreases during cell growth as cells metabolize
glucose in the presence of oxygen and produce C02
– Alkaline solutions (sodium bicarbonate, sodium hydroxide)
added to increase and control pH
 Other Culture Parameters

Temperature - optimal - 37°C

• C02 incubator-typically controlled either by a water bath that circulates
through the walls of the cabinet (water jacketed C02) or by electric coils
that give off radiant heat

• bioreactor- monitored by temperature probe and Temperature

Control Module (TCM)

Dissolved oxygen (bioreactors)

– controlled by DO probe and computer system
– based on the rate at which oxygen molecules diffuse a membrane
covering a set of electrodes
– low solubility in culture media
 Maintaining & Monitoring
Culture

Cell growth and viability monitored during culture by counting cells

methods

Cell counting:
– tallies the number of viable (living) and non-viable (dead) cells
• calculate the % viability (viable/viable + nonviable)*100
• calculate viable cell concentration
• Accurate and consistent cell counts essential to robust production
process
• Viable cell concentration & % viability used as Forward Processing
Criteria (FPC) & Critical Process Parameter (CPP)
• Can be determined offline or inline
 Offline Counting Methods

Trypan Blue Exclusion Method

• Sample of culture must be taken from the culture vessel (
offline) to count
• Live cells with intact cell membranes do not allow the trypan
blue dye into the cell (remain clear)
• Trypan blue enters dead cells; become blue
• Time sensitive – cells must be counted as quickly as possible
after staining
• Used in conjunction with hemacytometer
• Most commonly used cell counting method
– Automated cell counting – use image analysis to automate Trypan
Blue Exclusion Method
 Inline Counting Methods

Cells are counted in the bioreactor and decreases the risk of

contamination that occur during sampling
– Biomass sensors
• measures only viable cells
• Viable cells with intact plasma membranes have a different
capacitance (ability to store charge) than cells with disrupted
plasma membranes (non-viable cells)
• When an electric field is applied to the culture, biomass probes
can measure capacitance which is directly related to concentration

– Packed Cell Volume (PCV)

• Subjective and does not differentiate between viable and non-
viable calls
• Generally used to determine cell density for cell harvest
Hemocytometer showing viable & non-viable cells with trypan blue stain
 What is a Bioreactor?

Vessels or containers designed to support the optimal growth and

metabolic activity of cells producing a product of interest

Can be classified in several ways including:

– Type of mixing
• Stirred Tank
• Airlift
– Mode of operation
• Batch culture
• Fed batch
• Perfusion
– Type of vessel
• Stainless steel
• Single use/ disposable
 Bioreactor- Mixing Types

§ Stirred Tank
• required gases (e.g. 02) nutrient media, cells are continuously stirred by
agitator impellor (stirrer) at the bottom or top of the vessels
• Baffles in the center of vessels ensures proper mixing and prevents
formation of vortexes that might shear cells

§ Airlift
• gas is pumped from below through a sparge tube within the bioreactor
creating bubbles which mixes the contents of the vessels
• contains baffle that guides gas up through bioreactor on one side of the
baffle and then over and down the other side
 Bioreactor –Modes of Production

Batch culture
• culture grows without additions until harvest
• approximately 5-8 days culture

Fed batch
• depleted and/or limited nutrients are added back to the reactor
• 10-20 days

Perfusion (Continuous)
• equal volumes of fresh media are added and culture fluid is removed
from the bioreactor
• can last for months
 Bioreactor Types – Stainless
Steel
§ Made of durable material that can accommodate high volumes (up to 20,000L) of
culture

§ Double walled, glycol jacketed with 4 layers that provide insulation/temperature

control and sterile contact for cell cultures

§ Very large industrial sized reactors bioreactors have

fixed vessel configurations with predefined port
assemblies that can not be easily reconfigured

§ Expensive and time consuming cleaning procedures

– high costs to produce purified water and steam for
cleaning (CIP/SIP)
Types of Bioreactors- Glass and Stainless Steel
Bioreactor Types – Single Use

– Disposable bioreactors- intended for one time use

– Components are typically made of plastic and are disposed of after

use

– Generally used for mammalian cultures

– Cultivation chamber is inflated plastic bag

– Single use technology in biomanufacturing is becoming widespread

- has advantages and disadvantages
 Advantages of Single Use Technology

Reduction or elimination of cleaning, sanitization, and sterilization steps.

This reduces:

– consumption of water – pure water is extraordinarily expensive to

produce

– energy used to produce purified water

– consumption of cleaning and sanitizing chemicals (CIP)

– eliminates the need to sterilize bioreactors (the vendor has done this)

– eliminates the need to generate clean steam

– need for cleaning and sterilization validation (and moves it to the vendor)
– eases regulatory compliance
 Single Use Advantages Continued

§ lower upfront capital costs – this becomes a major

advantage for a small company or a new company
wanting to start production quickly

§ faster cycle times and faster, less expensive

changeover between campaigns – less fear of cross-
contamination (by eliminating the need for cleaning)

§ lower risk – lower probability of cross-contamination

with another product or microbial contamination
 Single Use Disadvantages

§ Scale Limitations (only up to 2000 liter cell culture)

• increasing product titers and cell densities are making this less important

§ Limited to mammalian cell culture - low oxygen transfer

coefficient rates excludes use of bacteria

§ Increased reliance on outside vendors – potential supply chain

problems

§ Concerns over leachables/extractables from the plastics

§ Additional consumables cost

§ Environmental impact – increase in solid waste that currently

goes to landfill
Disposable wave bioreactor and its mechanics
Single Use Bioreactors
 Cell Harvest

§ Main objective is to separate the cells from the media

containing the target API

§ During culture samples taken at pre-determined intervals to

monitor progress

§ Time to end culture and begin harvest depends on cell line

and is pre-determined
• based on the quality and quantity of the product accumulated in
the bioreactor
• generally highest amount of quality product is reached when cell
number drops
 Cell Harvest Continued

2 steps:
1. Centrifugation
• separate cells from culture media
• Rapid spinning of the culture from bioreactor; cells
sink to bottom of centrifuge
• For microbial harvest lysing step prior to
centrifugation to release protein product contained
inside cell wall

2. Filtration – remove large debris

– Depth filtration – API passes through as the filtrate
along with other proteins and cell particles
– Sterile grade membrane filtration – remove smaller
particles and potential microbial contamination
» filters with 0.22 micron pore size
Cell Harvest Centrifuge
Types of Harvest Filters