Introduction to

Bioprocessing

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 Introduction

Module 1
Introduction To Biological Products And
Pharmaceutical Drugs

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 Introduction

Vials used for biopharmaceutical packaging.

Biopharmaceuticals
Multiple definitions
A common one is that biopharmaceuticals are inherently biological in
nature and manufactured using living organisms.

Four major categories of biopharmaceuticals

Vaccines Cell Therapy

Gene Therapy Protein therapeutics

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 Introduction

Vaccines Cell Therapy

Biopharmaceuticals used to prevent a disease Refers to the use of living, whole cells for
by improving immunity. Applications include treatment of a disease. Examples include
flu vaccines, vaccines for measles, mumps, Kymirah and Yescarta. Kymriah and Yescarta
and rubella, and vaccines for COVID-19 are both used to treat lymphatic cancer.

Gene Therapy Protein therapeutics

Technique that uses genes, or DNA, to treat Proteins used for therapeutic application to
or prevent disease by inserting a correct copy treat an existing disease. Examples include
of a gene into patient’s cells instead of using insulin and Humira, the largest-selling drug in
drugs or surgery. Examples include Luxtuna the world. Humira treats diseases like
and Zolgensma. Luxturna treats an eye rheumatoid arthritis and Crohn’s disease.
disorder. Zolgensma treats spinal muscular
atrophy
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 Introduction

Comparing
Biopharmaceuticals to
Traditional
Pharmaceuticals
Traditional, small-molecule pharmaceuticals

Characteristics of Certain small molecules that may

pharmaceutical drugs: be produced by living cells:
● Defined chemicals. ● Antibiotics.
● Small molecules. ● Cholesterol-reducing statins.
● Often oral solid dosage. ● Amino acids.
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 Introduction

Biopharmaceuticals VS. Traditional

Pharmaceuticals
Biopharmaceuticals Traditional Pharmaceuticals
Small with well-defined structures and
Large and complex molecules/antigens
properties
Large and complex molecules/antigens
Often produced through chemical reaction
NovoRapid® is an produced using biological systems
insulin used to treat
Administered parenterally, either Administered orally (tablets, capsules, liquid),
diabetes. 
intravenously, subcutaneously, or topically, or parenterally
intramuscularly

Patient delivery
Lipitor® is atorvastatin Another big difference between biopharmaceuticals and traditional
pharmaceuticals is how they are delivered to a patient. 
calcium (a statin) that
lowers blood cholesterol. 6
 Introduction

Module 2
Proteins

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 Introduction

What are Proteins?

Special Case: Antibodies

Immunoglobulin (Ig).
Four polypeptides. Two regions
● Two heavy chains. ● Variable.
● Two light chains. ● Constant.
 Introduction

Module 3
The Four Stages of Biomanufacturing

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 Introduction

Stage 1:
Cell Growth (Production)
Stage 1 is where the biopharmaceutical product is
expressed, that is, synthesized within or using a living
organism.

The objective of this stage is to produce the product in

quantities suitable for clinical trials and/or commercial
distribution.

For the many biopharmaceuticals produced using

genetically engineered cells, this stage involves cell
growth in bioreactors.

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 Introduction

Stage 2:
Harvest

The harvest stage, also referred to as recovery,

occurs after the product has been produced in
Stage 1.

The objective of this stage is to separate product

from the production (i.e., host) system. 

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 Introduction

Stage 3: Purification
Process-related Product-related
impurities impurities
Spent cell-culture
Dimers
media
Leachables and
Aggregates
extractables
Host cell proteins (HCP) Misfolded product

Deamidated product
Endotoxin
variants
Oxidized product
Proteolytic enzymes
variants
Host cell DNA, other Enzymatically Degraded
nucleic acids products
Viruses
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 Introduction

Stage 4: Formulation and Fill

Ultrafiltration/
Diafiltration
Bulk fill 

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 Introduction

Module 4
Good Manufacturing Practice (GMP)

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 Introduction

Stage 4: Formulation and Fill

Aseptic Filling Of Drug Products

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 Introduction

Stage 4: Good Manufacturing Practice (GMP)

cGMP Control Elements Regulations that govern the
manufacture of drugs, including
both traditional pharmaceuticals
Process (small molecule) and
1
biopharmaceuticals, are called
"current Good Manufacturing
Practice." They are often referred
5 Control of 2 to by the abbreviation cGMP.
Procedures all five are Facility
required
for GMP

Personal Equipment
4 3
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 Introduction

GMP Element 1: Process

A good process is created in the process

development lab, not after it’s transferred
to the manufacturing area.  

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 Introduction

GMP Element 2: Facility

Maximum concentration limits (particles/m3 of air)
FED STD For particles equal to and larger than sizes listed below
Class 209E
Equivalent 0.1 micron 0.2 micron 0.3 micron 0.5 micron 1 micron 5 micron

ISO 1 10 2
ISO 2 100 24 10 4
ISO 3 1 1,000 237 102 35 8
ISO 4 10 10,000 2,370 1,020 352 83
ISO 5 100 100,000 23,700 10,200 3,520 832 29
ISO 6 1,000 1,000,000 237,000 102,000 35,200 8,320 293
ISO 7 10,000 35,2000 83,2000 2,930
ISO 8 100,000 3,520,000 832,000 29,300
ISO 9 35,200,000 8,320,000 293,000
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 Introduction

GMP Element 3: Equipment

Important considerations:
● Design.
● Controls (identification and equipment status).
● Operation.
● Cleaning.
● Maintenance.

Equipment Qualification: IQ, OQ & PQ 00:19

During and after equipment is commissioned and installed, it
must be qualified. There are three types, usually performed in
sequential order.
● Installation Qualification (IQ).
● Operational Qualification (OQ).
● Performance Qualification (PQ).
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 Introduction

GMP Element 4: Personnel

Important considerations
Personnel that ensure quality must reside in a part of
the organization totally independent of
manufacturing.

● Independent quality oversight.

● Qualified and trained operators.
● Personal hygiene.
● Attention to detail.

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 Cell Growth

Module 5
Expression systems & biotechnology

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 Cell Growth

Module 5: Expression systems & biotechnology)

Cells or unused Impurities like host cell proteins,
media/cell debris endotoxin, DNA, Virus

Stage 1: Stage 2: Stage 3:

Cell Growth
Harvest Purification
(Production)

Stage 4:
Genetically
modified cells
Formulation
Drug and Fill
Substance

Drug Product
Unwanted buffer
components
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 Cell Growth

Module 6
Nutritional Strategies and Cell Culture Media

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 Cell Growth

Basal Medium Components

Carbohydrate Buffer Amino acids

Glutamine Proline

Primary. Sodium bicarbonate Protein synthesis.

● Glucose. (NaHCO3). Energy source.
Others. ● Works with carbon Two important amino acids for CHO
dioxide sparged into growth:
● Galactose.
bioreactor.
● Fructose.  ● Glutamine.
● Proline. 24
 Cell Growth

Basal Medium Components

Fatty Acids And Lipids Growth Factors
Recombinant insulin or insulin-like growth factor
(IGF-1, LongR3®).
● Improves growth.
● Improves productivity.
● Facilitates glucose uptake and lipid metabolism.
● Recombinant transferrin.
● Iron carrier.
● Can be replaced with various forms of iron.

Cholesterol

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 Cell Growth

Basal Medium Components

Surfactants
Cells can be damaged when bubbles burst

Straight Pipe Frit

Surfactants are used to reduce sensitivity to shear in bioreactors.

Pluronic F68 is most common.

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 Cell Growth

Module 7
Cell Growth

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 Cell Growth

Module 7: Cell Growth

Design parameters
Cells or unused Impurities like host cell proteins,
media/cell debris endotoxin, DNA, Virus

Stage 1: Stage 2: Stage 3:

BIOPROGRESS
Cell Growth
Harvest Purification
(Production)
Inputs Outputs

Stage 4:
Genetically
modified cells
Formulation
Drug and Fill
Substance

Drug Product
Unwanted buffer
components
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 Cell Growth

Process Output

Cells
(Cell therapy)
Viral
vectors Therapeutic
(gene Proteins
therapy)

VLPs Monoclonal
(Vaccines) Bioprocess antibodies

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 Cell Growth

Modes of Bioreactor Operation

Batch Fed Batch Perfusion (continuous

● No nutrients are added. ● Nutrients are added when ● Nutrients added.

● Run time < week. needed. ● Spent medium removed.
● Run time ~14 days. ● Cells returned.
●
●
Periodic removal of cells.
Run time = month(s). 30
 Cell Growth

Anchorage-dependent Cells

T-flasks. Roller Bottles. Stacked Systems.

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 Cell Growth

Module 8
Bioreactor Design, Operation and Control

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 Cell Growth

Main Objectives Of Bioreactor Design:

● Maintain sterility throughout operation.
Bioreactor Design ● Provide agitation to maintain a homogeneous
Objectives environment throughout the bioreactor.
● Deliver adequate aeration to meet the
metabolic requirements of cells.
● Stainless steel bioreactors are fabricated using
a variety of materials. In general, materials
should not be additive, adsorptive, or
reactive. Common metals are: 
● 304 stainless steel and 316 stainless steel. 316
stainless steel contains 2% molybdenum, which
helps prevent corrosion. 
● Polymers such as Teflon®, Viton® or silicone
are used for the gaskets and O-rings that
ensure leak-free connections. 
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 Cell Growth

Agitation

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 Cell Growth

Baffles

Bioreactor With No Bioreactor with

Baffles. baffles.
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 Cell Growth

Aeration

Sample Sparger
Designs.
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 Cell Growth

Single-use bioreactor (SUB) 

50 L SUB 200 L AND 2,000 L SUBS

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 Harvest

Module 9
Cell Separation: Centrifugation and Depth Filtration

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 Harvest

Stage 2: Harvest, part of downstream

operations. Cells or unused Impurities like host cell proteins,
media/cell debris endotoxin, DNA, Virus

Stage 1: Stage 2: Stage 3:

Cell Growth
Harvest Purification
(Production)

Stage 4:
Genetically
modified cells
Formulation
Drug and Fill
Substance
Harvesting a Bioreactor
Drug Product
Unwanted buffer
components
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 Harvest

Centrifugation

For most biopharmaceutical

applications, centrifuges are used
to separate liquid containing solids.
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 Harvest

Depth Filtration
Filter

Depth filtration
Cell equipment
Target Protein
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 Harvest

Comparaison: Centrifugation vs. Depth Filtration 

Capabilities Centrifugation Depth Filtration

Particle-free stream No No

Collection of solid possible Yes NO

Solid Capacity High High

Capital Costs High Low

Consumable cost Low Medium

Single-use options Limited Yes

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 Purification

Module 10
Chromatography

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 Purification

Chromatography is
part of Stage 3. Cells or unused Impurities like host cell proteins,
media/cell debris endotoxin, DNA, Virus

Stage 1: Stage 2: Stage 3:

Cell Growth
Harvest Purification
(Production)

Stage 4:
Genetically
modified cells
Formulation
Drug and Fill
Substance

Drug Product
Unwanted buffer
components
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 Purification

Purification by Chromatography
Achieve final high-
level purity
Remove
Isolate, impurities
Concentrate
and stabilize. Increasing
Intermediate Polishing Purity
Purification Step
Capture
Step Step

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 Purification

Module 11 - Viral Clearance

Cells or unused Impurities like host cell proteins,
media/cell debris endotoxin, DNA, Virus

Stage 1: Stage 2: Stage 3:

Cell Growth
Harvest Purification
(Production)

Stage 4:
Genetically
modified cells
Formulation
Drug and Fill
Substance

Drug Product
Unwanted buffer
components
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 Purification

Sources of Viral
Contamination
Viruses can be:
● Endogenous (exist naturally within the cell).
● Latent in infected cells.
● Introduced during cell construction.
● Added during the production process (media,
materials, cross-contamination).
● Spread by operating personnel.

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 Purification

Below are several examples of viral contamination that

have had an impact on biomanufacturing and, in some
cases, human health.
Virus
Contamination – A Recombinant:
● 1996: minute virus of mice (MVM) contaminated
Reality bioreactors.
● 2009: Vesivirus 2117 contaminated bioreactors.

Vaccine Contamination:
● 1950s and 60s: SV40 in polio vaccine. 
● 2010: Porcine circovirus in Rotavirus Vaccine. 

Plasma Product Contamination:

● 1980s: HIV contaminated Factor VIII.
● 1994: Hepatitis C contaminated IgG. 

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 Purification

Virus inactivation. Virus removal by filtration.

Ways to inactivate virus: Ways to remove virus:

● Low pH. ● Virus filtration. 
● Solvent/Detergent. ● Precipitation.
● Heat. ● Chromatography.
● Irradiation/Ultraviolet light.

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 Purification

Module 11 - Viral
Clearance

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 Purification

Module 12
Ultrafiltration and Diafiltration

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 Purification

How Does
Ultrafiltration
Work?

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 Purification

Tangential Flow
Filtration (TFF)

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 Purification

Module 12
Bulk Filling

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 Formation & Fill

Bulk Filling Goals Bulk filling facilities

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 Formation & Fill

Viable Counts

Passive Air Sampling Limit

Class Active Air Sampling Limit (cfu/m3)
(cfu/4 hours/90mm plate)

ISO 5 <1 <1

ISO 6 7 3

ISO 7 10 5

ISO 8 100 50

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 Formation & Fill

Single-use and
Closed Systems

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Thank You
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