Manufacturing processes of Biotech

compounds used in Pharmaceutical and

Biopharmaceutical fields
Quy trình sản xuất các hợp chất công nghệ sinh học được
sử dụng trong lĩnh vực dược phẩm và y dược
Introduction
Two important aspects:

(a) biotechnology — as a new process technology

(b) biotechnology — as a ‘drug discovery’ research
tool.
 Overview
Recombinant production technology of therapeutic peptides
and proteins

Vaccine technology

Synthesis and manufacturing processes of monoclonal

antibodies

Biosynthesis principles and processes in:

Enzymes
Antibiotics
Anti-tumor agents
Biotech materials used in Pharmaceutical and
Biopharmaceutical fields
Recombinant
Protein
Production
Recombinant Protein Production
-Why?
• over-expression to get enough amount
• easy purification

-Application
• functional studies
• structural studies
• vaccine/antigen/antibodies
• therapeutic drug
• industrial enzymes for reaction
 Application: therapeutic proteins
• Actimmune (If g) Chronic
Granulomatous Disease (an interferon gamma)
• Activase (TPA) treat blood clots, heart
attack.
• BeneFix (F IX) helps the blood to clot
• Betaseron (If b) (interferon beta-1b) is
made from human proteins. Interferons help the body fight • Intron-A Interferons help your body's immune
viral infections
• Humulin (insulin isophane) improve blood sugar
control in adults and children with diabetes mellitus
• Novolin
• Epogen to treat anemia caused by
chemotherapy or chronic kidney disease, or anemia
caused by taking zidovudine to treat HIV (human
immunodeficiency virus)
• Regranex (PDGF) to treat diabetic
foot ulcers
• Novoseven (F VIIa)
system respond to bacteria, viruses, cancer, or other
invading substances.
• Neupogen treat neutropenia, a lack of
certain white blood cells caused by cancer, bone marrow
transplant, receiving chemotherapy

• Pegademase (AD) to replace ADA in • Pulmozyme

people with cystic fibrosis
to improve lung function in

people with severe combined immunodeficiency disease

(SCID)
• Infergen treat long-term hepatitis C infections
•Now more than 200 approved peptide and protein pharmaceuticals on the FDA
list (http://www.accessexcellence.org/RC/AB/IWT/The_Biopharmaceuticals.html)
 GENERAL PROCEDURE
Three major themes:

(1) sources of biopharmaceuticals

(2) upstream processing
(3) downstream processing
The additional element of biopharmaceutical
manufacturing, i.e. product analysis
 BIOMANUFACTURING
PROCESS

Master cell
bank
Innoculum
Chất cấy truyền Fermentation

Collection
Lyophilized
đông khô Collection Purification Concentration
 Kỹ thuật đông khô
(lyophilization)
• Trong tổng hợp các vaccine hay các polymer,v.v. hòa tan được trong nước, sau khi phản ứng hoàn tất thì các tạp
chất được loại bỏ bằng kỹ thuật (dialysis http://en.wikipedia.org/wiki/Dialysis)với nước siêu tinh khiết (đã qua
chưng cất và màng siêu lọc để loại bỏ hoàn toàn các ion tạp chất). Những hợp chất có kích thước phân tử lớn sẽ
không lọt qua được màng bán thẩm nên bên trong màn bán thẩm chỉ còn chứa dung dịch sản phẩm vacinne hay
polymer khá tinh khiết.
• Vấn đề đặt ra là làm sao loại bỏ được nước mà không làm hỏng sản phẩm tổng hợp. Ví dụ nếu dùng máy cô quay
(rotavapor hay chưng cất thì các vaccine hay polymer có hoạt tính dược học sẽ bị phá hủy hay biến dạng). Vấn
đề này có thể giải quyết một cách dễ dàng bằng kỹ thuật đông khô. Để thực hiện kỹ thuất này cần phải có máy
đông khô (Lyophilizer, có thể mua hoặc chế tạo trong điều kiện Việt Nam) và tiến hành qua các bước sau:

• Bước 1: Cho dung dịch phản ứng sau dialysis vào bình chứa thích hợp bằng thủy tinh hay nhựa.

• Bước 2: Làm đông hoàn toàn dung dịch trên bằng hổn hợp nước đá khô (dry ice) với acetone hay methanol hay
isopropanol. Nếu dung dịch không được đông hóa một cách cẩn thận sẽ bị bắn văng khỏi bình chứa khi cho mẫu
lên máy đông khô.

• Bước 3: Cho mẫu lên máy, nước đá sẽ thăng hoa (sublime) trực tiếp ở thể rắn và thoát ra khỏi bình chứa mẫu ở
nhiệt độ phòng một cách dễ dàng. Với lượng nước khoảng 0.5L, thời gian thăng hoa sẽ khoảng 24 h với bình
chứa lớn.

• Bước 4: Kiểm tra mẫu xem đã khô một cách tuyệt đối chưa và lấy mẫu ra khỏi máy.

• Nói chung, nước được loại bỏ khỏi dung dịch một cách nhẹ nhàng để cho ra sản phẩm khô mà không cần phải
đun nóng gì cả nên mẫu thu được không bị biến tính và vẫn còn hoạt tính dược học.
Flow chart of protein production
Service
Requested

Parallel Cloning

Expression test in E. coli

additional charge
standard
Insoluble / posttranslational modification required

Soluble
Yeast system
Baculovirus system
in vitro expression systems

Protein Purification

Protease cleavage to
remove tag
Sources of biopharmaceuticals
 Sources of biopharmaceuticals

Expression systems which are/could potentially be used

for the production of recombinant biopharmaceutical
products:

E. coli (and additional prokaryotic systems, e.g. bacilli)

Yeast (particularly S. cerevisiae)
Fungi (particularly aspergilli)
Animal cell culture (particularly CHO and BHK cell lines)
Transgenic animals (focus thus far is upon sheep and
goats)
Plant-based expression systems (various)
Insect cell culture systems
 Sources of biopharmaceuticals

Bacterial Systems
Advantages Disadvantages
• Grow quickly (8 hrs to • Difficulty expressing large
produce protein) proteins (>50 kD)
• High yields (50-500 mg/L) • No glycosylation or signal
• Low cost of media peptide removal
(simple media • Eukaryotic proteins are
constituents) sometimes toxic
• Low fermentor costs • Can’t handle S-S rich
proteins
 Sources of biopharmaceuticals

Expression System Selection

• Choice depends on size and character of protein
– Large proteins (>100 kD)? Choose eukaryote
– Small proteins (<30 kD)? Choose prokaryote
– Glycosylation essential? Choose baculovirus or
mammalian cell culture
– High yields, low cost? Choose E. coli
– Post-translational modifications essential?
Choose yeast, baculovirus or other eukaryote
 Sources of biopharmaceuticals

Escherichia coli as a source of recombinant, therapeutic

proteins
Some biopharmaceuticals currently on the market which are produced by
genetic engineering in either E. coli or animal cells

Biopharmaceuti Source Biopharmaceuti Source

cal cal
tPA E. coli, CHO FSH CHO
IFN-α E. coli IFN-β CHO
IFN-γ E. coli RPO CHO
Insulin E. coli Glucocerebrosid CHO
ase
IL-2 E. coli Factor VIIa BHK
G-CSF E. coli
hGH E. coli
 Sources of biopharmaceuticals

Escherichia coli as a source of recombinant, therapeutic

proteins
Levels of expression of various biopharmaceuticals produced
in recombinant E. coli cells
Biopharmaceutical Level of expression (%
of total cellular protein
IFN-γ 25
Insulin 20
IFN-β 15
TNF 15
α1-antitrypsin 15
IL-2 10
hGH 5
 pTrxFus
1 2 3 4

Expression
1. Strong promoter
Fusion 2. Thioredoxin gene
protein 3. Nucleotide sequence coding for the
peptide sequence which serves as
cleavage site for the protease
Enterokinase (enterokinase)
4. Gene/cDNA for the protein of
interest

High-level expression of a protein of interest in E. coli in soluble

form by using the engineered ‘thiofusion’ expression system.
 Sources of biopharmaceuticals

Escherichia coli as a source of recombinant, therapeutic

proteins
These advantages, particularly its ease of genetic manipulation,
rendered E. coli the primary biopharmaceutical production system for
many years.
 Heterologous proteins accumulate intracellularly;
 Inability to undertake post-translational modifications (particularly glycosylation) of proteins;
 The presence of lipopolysaccharide (LPS) on its surface.
 The vast bulk of proteins synthesized naturally by E. coli (i.e. its homologous proteins) are intracellular.
 Few are exported to the periplasmic space or released as true extracellular proteins. Heterologous
proteins expressed in E. coli thus invariably accumulate in the cell cytoplasm.

• Các protein dị hợp tích tụ bên trong tế bào;

• Không có khả năng thực hiện các sửa đổi sau khi chuyển đổi (đặc biệt là glycosylation) của protein;
• Sự hiện diện của Lipopolysaccharide (LPS) trên bề mặt của nó.
• Phần lớn các protein được tổng hợp tự nhiên bởi E. coli (nghĩa là các protein tương đồng của nó) là nội
bào.
• Rất ít được xuất vào không gian periplasmic hoặc được giải phóng ra như các protein ngoài tế bào đích
thực.
• Các protein dị hợp được biểu hiện bằng E. coli do đó thường xuyên tích tụ trong tế bào chất.
 Sources of biopharmaceuticals

Escherichia coli as a source of recombinant, therapeutic

proteins
 An additional complication of high-level intracellular
heterologous protein expression is inclusion body formation.
Presumably, when expressed at high levels, heterologous proteins
overload the normal cellular protein-folding mechanisms.

 Refolding protein to gain the active form

 Sources of biopharmaceuticals

Escherichia coli as a source of recombinant, therapeutic

proteins
Inclusion bodies are generally incubated with strong denaturants, such as detergents, solvents or urea.
This promotes complete solubilization of the inclusion body (i.e. complete denaturation of the proteins
therein).

The denaturant is then removed by techniques such as dialysis or diafiltration. This facilitates refolding of
the protein, a high percentage of which will generally fold into its native, biologically active, conformation.

• Đối với inclusion body, bạn có thể phá vỡ tế bào chủ

bằng mọi cách, đông tan thoải mái, dùng đệm có SDS,
siêu âm v.v... mình đảm bảo bạn không phá vỡ được
inclusion body.

Sau khi ly tâm và rửa nhiều lần để loại bỏ các protein

tan được và các mảnh vụn tế bào, bạn sẽ có 1 cục cặn
trong suốt, hơi vàng, đó là inclusion body của bạn.

Protein trong inclusion body thường rất tinh khiết và

thậm chí không cần đến sắc ký ái lực. Có điều nó
không tan. Inclusion body có thể bị làm tan bằng urea
hoặc guanidine nồng độ cao (6-8 M). Vấn đề là protein
lúc này lại bị biến tính và làm sao gấp nó lại cho đúng.

Gấp protein có thể được thực hiện bằng thẩm tích

(đệm PBS thể tích 50-100 lần thể tích protein, giảm rất
từ từ nồng độ urea/guanidine, có thể hỗ trợ protein với
L-alanine 1 M trong đệm thẩm tích).
 Sources of biopharmaceuticals

Escherichia coli as a source of recombinant, therapeutic

proteins
An alternative means of reducing/potentially eliminating inclusion body
accumulation entails the high-level co-expression of molecular chaperones
along with the protein of interest.

The inability of prokaryotes such as E. coli to carry out post-translational

modifications (particularly glycosylation) can limit their usefulness as
production systems for some therapeutically useful proteins. Many such
proteins, when produced naturally in the body, are glycosylated.

However, the lack of the carbohydrate component of some glycoproteins

has little, if any, negative influence upon their biological activity.

The unglycosylated form of IL-2, for example, displays essentially identical

biological activity to that of the native glycosylated molecule. In such cases,
E. coli can serve as a satisfactory production system.
• Một phương pháp thay thế để giảm / có khả năng loại bỏ sự tích tụ cơ thể hòa nhập
đòi hỏi sự biểu hiện đồng thời cao của chaperones phân tử cùng với protein quan
tâm.

• Không có khả năng sinh con như E. coli để thực hiện các sửa đổi sau khi chuyển đổi
(đặc biệt là glycosylation) có thể giới hạn tính hữu ích của chúng như là hệ thống sản
xuất đối với một số protein hữu ích về mặt điều trị. Nhiều protein như vậy, khi được
sản xuất tự nhiên trong cơ thể, được glycosyl hoá.

• Tuy nhiên, thiếu thành phần carbohydrate của một số glycoprotein ít có, nếu có, ảnh
hưởng tiêu cực đến hoạt động sinh học của chúng.

• Ví dụ, dạng ung thư oxy hoá của IL-2 thể hiện hoạt tính sinh học giống hệt với hoạt
tính của phân tử glikozlated tự nhiên. Trong những trường hợp như vậy, E. coli có
thể phục vụ như là một hệ thống sản xuất thỏa đáng.
 Sources of biopharmaceuticals

Escherichia coli as a source of recombinant, therapeutic

proteins
 Another concern with regard to the use of E. coli is the presence on its surface of LPS
molecules.

 The pyrogenic nature of LPS renders essential its removal from the product stream.

Fortunately, several commonly employed downstream processing procedures achieve such a

separation without any great difficulty.
 Sources of biopharmaceuticals

E. coli Expression System

-challenge
• poorly expressed
• protein insoluble- inclusion bodies
• expressed and soluble: 20-30%

-improvement
• growth condition (e.g. temperature)
• codon usage
• host strain
• fusion to carrier protein
 Sources of biopharmaceuticals

E. Coli Expression System

parallel screening for soluble proteins
1. Increase the expression level and
solubility of target protein with
protein tags.

Rationale 2. Simultaneously, parallel screening

different fusion tags.

3. Has potential for automating gene

cloning.
Protein Science (2002), Shih YP
Publication
et. al., 11:1714-1719.
E. coli

Sticky-end PCR
E. coli

Parallel screening for soluble protein

 Efficient protein expression in E. coli

 Inefficient transcription No or little protein synthesized

u Promoter choice and design
 Inefficient translation No or little protein synthesized
u Codon usage
u Transcript stability
u Transcript secondary structure
 Inefficient folding (cytoplasmic or periplasmic)
Aggregation or degradation
u Improper secondary, tertiary or quaternary structure formation
u Inefficient or improper disulfide bridge formation
u Inefficient isomerization of peptidyl-prolyl bonds

 Inefficient membrane insertion/translocation

Aggregation or degradation
 Toxicity Cell death
E. coli

E. Coli Expression System Summary

• The method introduces sticky-end to target genes,

without using restriction enzymes.

• Well-induced and highly soluble recombinant proteins :

80% success
Cloning and expression of target gene:

Gene of Interest
+
Expression Vector Recombinant
Vector

Expression of Fusion Protein

 Key Parts to a Vector
• Origin of replication (ORI) – DNA sequence for DNA polymerase to
replicate the plasmid
• Selectable marker (Amp or Tet) – a gene, when expressed on
plasmid will allow host cells to survive
• Inducible promoter – Short DNA sequence which enhances
expression of adjacent gene
• Multi-cloning site (MCS) – Short DNA sequence that contains many
restriction enzyme sites

• Nguồn gốc nhân bản (ORI) - Dãy DNA cho DNA polymerase sao chép plasmid
• Marker chọn lọc (Amp hoặc Tet) - một gen, khi biểu hiện trên plasmid sẽ cho phép các tế bào chủ tồn tại
• Promoter hấp dẫn - Chuỗi ADN ngắn tăng cường biểu hiện gen liền kề
• Site đa nhân bản (MCS) - Chuỗi ADN ngắn chứa nhiều vị trí enzyme hạn chế
 Which Vector?
• Must be compatible with host cell system
(prokaryotic vectors for prokaryotic cells,
eukaryotic vectors for eukaryotic cells)
• Needs a good combination of
– strong promoters
– ribosome binding sites
– termination sequences
– affinity tag or solubilization sequences
– multi-enzyme restriction site
 Which Vector?

• Promoters
– arabinose systems (pBAD), phage T7 (pET),
Trc/Tac promoters, phage lambda PL or PR
• Tags
– His6 for metal affinity chromatography (Ni)
– FLAG epitope tage DYKDDDDK
– CBP-calmodulin binding peptide (26 residues)
– E-coil/K-coil tags (poly E35 or poly K35)
– c-myc epitope tag EQKLISEEDL
– Glutathione-S-transferase (GST) tags
– Celluluose binding domain (CBD) tags
 Bacterial Transformation
• Moves the plasmid into bacterial host
• Essential to making the gene “actively” express the
protein inside the cell
• 2 routes of transformation
– CaCl2 + cold shock
– Electroporation
• Typical transformation rate is 1 in 10,000 cells (not very
efficient) for CaCl2, but 1 in 100 for electroporation
 Electroporator

25 microfarads = 2500 V
@ 200 ohms for 5 ms
 Electroporation
• Seems to cause disruption in
cell membrane
• Reconstitution of membrane
leads to large pores which
allow DNA molecules to
enter
• Works for bacteria, yeast and
animal cells
Cloning & Transforming in
Yeast Cells

Pichia pastoris
 Pichia Pastoris
• Yeast are single celled eukaryotes
• Behave like bacteria, but have key advantages of
eukaryotes
• P. pastoris is a methylotrophic yeast that can use
methanol as its sole carbon source (using alcohol
oxidase)
• Has a very strong promoter for the alcohol oxidase
(AOX) gene (~30% of protein produced when induced)
Pichia Cloning
 Pichia Pastoris Cloning
• Uses a special plasmid that works both in E. coli and
Yeast
• Once gene of interest is inserted into this plasmid, it
must be linearized (cut open so it isn’t circular)
• Double cross-over recombination event occurs to cause
the gene of interest to insert directly into P. pastoris
chromosome where the old AOX gene used to be
• Now gene of interest is under control of the powerful
AOX promoter
 Pichia Systems
Advantages More advantages
• Grow quickly (8 hrs to • Can express large proteins
produce protein) (>50 kD)
• Very high yields (50-5000 • Glycosylation & signal
mg/L) peptide removal
• Low cost of media • Has chaperonins to help fold
(simple media “tough” prtns
constituents) • Can handle S-S rich proteins
• Low fermentor costs
Baculovirus Expression

~12 days
Baculovirus (AcMNPV) Cloning Process

Transfer vector

Cloned gene
5’ 3’
x x Cloned gene
5’ 3’

Polyhedrin gene

AcMNPV DNA Recombinant

AcMNPV DNA
 Baculovirus Systems
Disadvantages Advantages
• Grow very slowly (10-12 • Can express large proteins
days for set-up) (>50 kD)
• Cell culture is only • Correct glycosylation &
sustainable for 4-5 days signal peptide removal
• Set-up is time consuming, • Has chaperonins to help fold
not as simple as yeast “tough” prtns
• Very high yields, cheap
Mammalian Expression Systems
Mammalian Cell-line Expression
• Sometimes required for difficult-to-express proteins or for
“complete authenticity” (matching glycosylation and
sequence)
• Cells are typically derived from the Chinese Hamster
Ovary (CHO) cell line
• Vectors usually use SV-40 virus, CMV or vaccinia virus
promoters and DHFR (dihydrofolate reductase) as the
selectable marker gene
 Mammalian Expression
• Gene initially cloned and plasmid propagated in bacterial
cells
• Mammalian cells transformed by electroporation (with
linear plasmid) and gene integrates (1 or more times) into
random locations within different CHO chromosomes
• Multiple rounds of growth and selection using
methotrexate to select for those cells with highest
expression & integration of DHFR and the gene of interest
 Methotrexate (MTX) Selection
Gene of interest DHFR

Transfect
dfhr- cells

Grow in Culture a Grow in Culture a

Nucleoside Colony of 0.05 uM Mtx Colony of
Free medium cells cells
Methotrexate (MTX) Selection

Grow in Culture a Grow in Culture a

0.25 uM Mtx Colony of 5.0 uM Mtx Colony of
cells cells

Foreign gene
expressed in
high level in
CHO cells
 Mammalian Systems
Disadvantages
• Selection takes time (weeks • Can express large proteins
for set-up)
• Cell culture is only
sustainable for limited period
of time
• Set-up is very time
consuming, costly, modest
yields
Advantages
(>50 kD)
• Correct glycosylation &
signal peptide removal,
generates authentic proteins
• Has chaperonins to help fold
“tough” prtns
 Conclusion
• Isolation of gene of interest
• Introduction of gene to expression vector
• Transformation into host cells
• Growth of cells through fermentation
• Isolation & purification of protein
Expression systems

• E. coli
• Baculovirus
• Yeast
• Cell-free
• Mammalian cell
( not open for service)
 Upstream processing

Biopharm production can be divided into ‘upstream’ and ‘downstream’

processing.

Upstream processing refers to the initial fermentation process that

results in the initial generation of product, i.e. the product biosynthesis
phase.

Downstream processing refers to the actual purification of the protein

product and generation of finished product format (i.e. filling into its fi
nal product containers, freeze-drying if a dried product format is required),
followed by sealing of the fi nal product containers.

Upstream processing is deemed to commence when a single vial of the

working cell bank system (see later) is taken from storage and the cells
therein cultured in order to initiate the biosynthesis of a batch of product.
 Upstream processing

Overview of the production process for a biopharmaceutical product.

 Upstream processing

Cell banking systems

uRecombinant biopharmaceutical production cell lines are most often initially

constructed by the introduction into these cells of a plasmid housing a
nucleotide sequence coding for the protein of interest

u After culture, the resultant product-producing cell line is generally aliquoted

into small amounts, which are placed in ampoules and subsequently immersed
in liquid nitrogen.

 The content of all the ampoules is identical, and the cells are effectively
preserved for indefinite periods when stored under liquid nitrogen. This batch
of cryopreserved ampoules forms a ‘cell bank’ system, whereby one ampoule
is thawed and the cell therein cultured in order to seed, for example, a single
production run. This concept is applied to both prokaryotic and eukaryotic
biopharmaceutical-producing cells.
 Upstream processing

Cell banking systems

 The cell bank’s construction design is normally two tiered, consisting

of
a ‘master cell bank’ and a ‘working cell bank’.

 The master cell bank is constructed first, directly from a culture of the
newly constructed production cell line. It can consist of several hundred
individually stored ampoules.
 Upstream processing

Figure 6 The master cell bank/working cell bank system. For simplicity, each
bank shown above contains only five ampoules. In reality, each bank would
likely consist of several hundred ampoules. Working cell bank number 2 will be
generated from master cell bank vial number 2 only when working cell bank
number 1 is exhausted and so on
 Upstream processing

Cell banking systems

The ampoules are not used directly to seed a production batch. Instead, they
are used, as required, to generate a working cell bank.

The generation of a single working cell bank normally entails thawing a

single master cell bank ampoule, culturing of the cells therein and their
subsequent aliquoting into multiple ampoules.

The ampoules are then cryopreserved and form the working cell bank.

When a single batch of new product is required, one ampoule from the
working cell bank is thawed and used to seed that batch.

When all the vials that compose the first working cell bank are
exhausted, a second vial of the master cell bank is used to generate a
second working cell bank, and so on.
 Upstream processing

Cell banking systems

The rationale behind this master cell bank/working cell bank system is to
ensure an essentially indefinite supply of the originally developed
production cells for manufacturing purposes.
 Upstream processing

Cell banking systems

The upstream processing element of the manufacture of a batch of

biopharmaceutical product begins with the removal of a single ampoule of
the working cell bank. This vial is used to inoculate a small volume of
sterile media, with subsequent incubation under appropriate conditions.

This describes the growth of laboratory-scale starter cultures of the

producer cell line. This starter culture is, in turn, used to inoculate a
production-scale starter culture that is used to inoculate the production-
scale bioreactor.
 Upstream processing

Cell banking systems

The media composition and fermentation conditions required to: Promote

optimal cell growth/product production.
Bioreactors are generally manufactured from high-grade stainless steel
and can vary in size from a few tens of litres to several tens of thousands of
litres.

At the end of the production-scale fermentation process, the crude

product is harvested, which signals commencement of downstream
processing.
 Upstream processing

Outline of the upstream processing stages involved in the production of a

single batch of product.
 Upstream processing

The manufacture of a batch of biopharmaceutical product entails filling

the production vessel with the appropriate quantity of purified water.

Heat-stable nutrients required for producer cell growth are then added
and the resultant medium is sterilized in situ  heat, and many
fermenters have in built heating elements or, alternatively, outer jackets
through which steam can be passed in order to heat the vessel contents.
Heat-labile ingredients can be sterilized by filtration and added to the
fermenter after the heat step.
 Downstream processing application
for pharmaceutical biotechnology
Introduction

Downstream processing serves to

(a) recover the therapeutic protein from its producer cell

source upon completion of the upstream processing phase

(b) purify the protein

(c) formulate the protein into final product format

 Fermentation

Recovery of producer Removal of cells from media

cells (centrifugation or (centrifugation or filtration)
filtration)

Cellular disruption
(homogenization)
Removal of
cellular debris
(centrifugation
or filtration)
Concentration of product-containing
extracellular media (ultrafiltration or
Initial purification/concentration precipitation)
(ultrafiltration/ion exchange or
precipitation)

Chromatographic
purification; usually
2-4 chromatographic
steps
 Adjustment of
potency and addition
of excipients

Sterile filtration
and aseptic filling

Freeze drying

Powder preparation Liquid preparation

Sealing of final product

container, labelling and packing

Figure 1. Overview of a generalized downstream processing procedure employed

to produce a finished product (protein) biopharmaceutical. QC also plays a
prominent role in downstream processing. Quality control personnel collect
product samples during/after each stage of processing. These samples are
analysed to ensure that various in-process specifications are met. In this way, the
production process is tightly controlled at each stage
 Cell disruption

 Sonication or treatment with the enzyme lysozyme

 Confined to laboratory-scale
 Equipment limitations or on economic grounds.

 Disruption of microbial cells (and, indeed, some

animal/plant tissue types) : mechanical methods, such as
homogenization or by vigorous agitation with abrasives.
 Cell disruption

Some chemical, physical and enzyme-based techniques that

may be employed to achieve microbial cell disruption.

 Treatment with chemicals:

- detergents
- antibiotics
- solvents (e.g. toluene, acetone)
- chaotropic agents (e.g. urea, guanidine)
 Exposure to alkaline conditions
 Sonication
 Homogenization
 Agitation in the presence of abrasives (usually glass beads)
 Treatment with lysozyme
Removal of nucleic acid

.
 Treatment with nucleases.

 Precipitation : polyethylenimine, a long-chain cationic

polymer .
Initial product concentration

Concentration

Ion-exchange chromatography (in which proteins

bind to charged beads immobilized in a column)

Ultrafiltration
 Diafiltration

Reduce or eliminate low molecular mass molecules from a

solution.

The level of reservoir is maintained at a constant volume.

 Chromatographic purification

 Column chromatography refers to the separation of

different protein types from each other according to their
differential partitioning between two phases: a solid
stationary phase (the chromatographic beads, usually packed
into a cylindrical column) and a mobile phase (usually a
buffer).

 With the exception of gel filtration, all forms of

chromatography used in protein purification protocols are
adsorptive in nature.
Series of chromatographic purification
Initial product characterization
The physicochemical and other properties of any newly identifi ed
drug must be extensively characterized prior to its entry into
clinical trials.
As the vast bulk of biopharmaceuticals are proteins, a summary
overview of the approach taken to initial characterization of these
biomolecules is presented.
Initial purification of the protein - High-resolution chromatographic
steps.
Moreover, once these characteristics have been defined, they form
the basis of many of the QC identity tests routinely performed on the
product during its subsequent commercial manufacture.
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