The document discusses upstream processing in biomanufacturing. Upstream processing involves growing cells in bioreactors to produce target proteins for pharmaceuticals. Key aspects of upstream processing include media preparation and sterilization, inoculum development, and cell culture in bioreactors. The main goal of upstream processing is to provide optimal environmental conditions for cell growth and protein production before downstream processing separates and purifies the target proteins.

1. BIOREMEDIATION AND
BIOPROCESSING
TOPIC: UPSTREAM PROCESSING
SUBMITTED BY
ALI HUSNAIN ARSHID
SUBMITTED TO
MAM POONUM RANA
BS (H) ZOOL. M.Phil. ZOOL.
GOVT. COLLEGE UNIVERSITY, FAISALABAD, PAKISTAN

2. 1.0
2.0
3.0
INTRODUCTION
OBJECTIVE OF UPSTREAM
PROCESSING
3.1 Cells and Proteins (Medium)
3.2 Upstream Processing Areas,
Equipment, and Systems
3.3 Clean in Place Systems
3.4 Media Preparation Area
3.5 Cell Culture/Fermentation
CONTENTS
UPSTREAM
BIOPROCESSING

3. 1.0 Introduction
Therapeutic cell manufacturing processes can be separated into:
1. Upstream processes
2. Downstream processes
➢Upstream Processing
The upstream process is defined as the entire process from early cell
isolation and cultivation, to cell banking and culture expansion of the cells
until final harvest.

4. ➢The upstream part of a bioprocess refers to the first step in which
microbes/cells are grown, e.g. bacterial or mammalian cell lines in
bioreactors.
➢Upstream processing involves all steps related to
• Inoculum development
• Media development
• Improvement of inoculum by genetic engineering process
• Optimization of growth kinetics so that product development can
improve tremendously.

5. 2.0 Objective of Upstream
The main objective of upstream manufacturing is to create the
environment necessary for cells to make the target protein. These proteins
serve various medicinal purposes; Bio-manufacturing is used to produce
products such as:
➢ Therapeutic Proteins
➢ Antibiotics
➢ Hormones
➢ Enzymes
➢ Amino acids
➢ Blood substitutes
➢ Alcohol
➢ Active Pharmaceutical Ingredients

6. 3.0 Processing
3.1 Cells and Proteins (Medium)
The cells that upstream technicians will care for in every step of the upstream process.
➢Cells are kept frozen in liquid nitrogen (LN2) in Dewar room/ vessels
➢ Cell required Narrow window of environmental conditions
➢Required Bioreactors (mammalian) or Fermenters (microbial)
➢It should be cheap and easily available
➢It should Maximize the growth of Micro

7. 3.2 Upstream Processing Areas, Equipment, and Systems
i. Dispensing room
• Raw materials are received and Stored
• Raw Materials/Product are weighed and measured in a traceable
• Animal-derived raw materials must be segregated to reduce risk of exposure to
adventitious viruses
• Electronic measurement more advantageous
• Dispensing booths prevent cross-contamination (Figure#1)
Figure#1

8. 3.3 Clean in Place/Steam in Place Systems
➢ Vessels piping/hoses must be free of any foreign substances (Foreign
substances include cell debris, medium and cleaning chemicals)
➢ Sterilization is critical to prevent contamination
1) Sterilization of Liquid Culture Media
➢ The constituents of culture media, water and containers contribute to the
contamination by vegetative cells and spores.
➢ The media must be free from contamination before use in fermentation.
➢ Sterilization of the media is achieved by
i. Heat sterilization
• Heat is the most widely used sterilization technique
• Around 60°C in 5-10 minutes for vegetative cells
• Around 80°C for 15-20 minutes for spores destruction

9. ii. Physical methods
➢ The physical methods such as
• Filtration
• Centrifugation
• Adsorption
• Radiation and Chemical methods are not commonly used
➢ There are following limitations of filtration technique:
• Application of high pressure in filtration is unsuitable for industries.
• Some of the media components may be lost form the media during filtration.
iii. Batch sterilization
➢ Sterilization at 121°C in batch volumes in the bioreactor
➢ By injecting the steam into the medium
➢ The steam should be Pure
There are two disadvantages of batch sterilization:
• Alteration in nutrients, change in pH and discoloration
• High energy consumption

10. iv. Continuous sterilization
➢ Carried out at 140°C for a very short period of time ranging from 30 to 120s
➢ Carried out by directly injecting the steam or by means of heat exchangers
➢ There is different stages used in Continuous sterilization (Figure#2)
Figure#2

11. 2) Sterilization of Air
➢The air should be completely sterile, and free from all microorganisms
➢Air can be filter by means of
i. Air sterilization by heat
➢ Air is passed over electrically heated elements and sterilized
➢ But this is quite expense, hence not in use these days
ii. Air sterilization by filtration
➢ Filtration of air is the most commonly used
➢ There are different filters used for Air Sterilization:
A. Depth filters
➢ Air is passed through a glass wool containing depth filters the particles are
trapped and removed (Figure#3)
B. Membrane cartridge filters
➢ Removable pleated membrane filters made up of cellulose ester, nylon or
polysulfone
➢ Membrane cartridge filters are smaller in size, simpler for replacement

12. Figure#3: Depth Filter in Air Sterilization

13. 3.4 Media Preparation Area
➢ Technicians working in media preparation area
➢ As a human body requires a certain amount of carbohydrates, fats (lipids),
and proteins in a diet to remain healthy, cells must receive proper nutrition
to produce the protein product.
➢ Major components of media include:
• a carbohydrate energy source such as glucose
• a nitrogen source such as amino acids
• lipids often in the form of the subunit fatty acid
➢ Some cell lines require supplemental feeding of cholesterol
➢ Cells also require trace minerals in the form of salts, just as a human tissues
and organs require specific minerals.
➢ In most manufacturing plants, media components are usually in powder
form

14. ➢ For each step of scale-up, media contain all the nutrition and selective agents that the
cells require for optimal expression of the target protein.
➢ Following parameters are measured included:
I. pH (degree of acidity or alkalinity)
• pH is defined as the inverse log of the hydronium ion activity
• Figure 4 illustrates a typical pH scale.
II. Conductivity
• Concentration of charged particles called ions
III. Glucose
• Glucose is a water-soluble sugar added to all cell culture media
• The amount of sugar ranges from 1 g/L to 10 g/L
III. Osmolality (a measure of particle concentration)
• An osmole is the number of osmotically active particles
exerting an osmotic pressure of one atmosphere in 22.4 L
of solvent at zero degrees Celsius.
Figure#4

15. 3.5 Cell Culture/Fermentation
The stages within the upstream biomanufacturing area are generally referred
i. Inoculum
ii. Bioreactor stage (seed and production)
iii. Primary recovery (Production of upstream)
I. Inoculum
• The inoculum stage involves the thawing of a frozen vial of cells (Figure#5)
• And its introduction into a bioreactor
• Cells are given specific conditions that promote the multiplication
• Cells can be grown in inoculum using a variety of equipment
• Suspension culture is grown in culture flasks such as a spinner flask (Figure#6) in a
temperature-controlled incubator at approximately 35–38 degrees Celsius
• CO2 gas can be applied in a passive manner to incubator
• Biological Safety Cabinets (BSC) are used during critical steps in fermentation

16. Figure#5: A 2mL cryovial is sub-cultured into larger and
larger volumes until there is enough culture to inoculate a
seed reactor.
Fig#6: Spinner bottle containing cells and media on
spinner plate within a Biosafety Cabinet.

17. II. Bioreactors
• Bioreactors are classified as
A. Disposable
• Generally used in cell culture for mammalian processing
• High-titer development processes
B. Stainless Steel
• Double walled vessel Bioreactor
• There are four layers to the stainless steel
bioreactor vessel:
i. Interior wall
ii. Jacket
iii. Insulation
iv. Outer sheathing Figure# 7: Single-use disposable
Bioreactor

18. IV. Primary recovery (harvest)
 The main objective is to separate the cells from the media containing the target Active
Pharmaceutical Ingredient.
 It is the first step in recovery of the protein product from the culture
 Generally based on the quality and quantity of product
 Harvest steps
• The first step in mammalian harvest is usually centrifugation
• Microbial harvest is similar to mammalian harvest; however, a lysing step must occur prior
• The next step in cell harvest is the filtration step to remove large debris
❖ Membrane filter is that of mechanical straining, or size exclusion In sterile
❖ Biomanufacturing, vent filters are also used to filter gases
• The first filtration step in harvest is depth filtration
• The next and often last step in harvesting is to perform sterile grade membrane filtration
 Filter integrity testing
• Ensure the integrity of the filter element
• The two main types of integrity tests are:
A. Bubble point test: measures the pressure point at which a continuous stream of
B. Forward flow test: measures the rate of flow of a gas through a wetted filter

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