Drug Manufacturing - Faculty Pages

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Drug Manufacturing

BIT 230

Walsh Chapter 3

Drug Manufacturing


Most regulated of all manufacturing industries


Highest safety and quality standards


Parameters include:


Design and layout of facility


Raw materials


Process itself


Personnel


Regulatory framework

Pharmacopeias


Discussed before in other units and classes


Martindale
-

not a standards book


Gives information about drugs


Physiochemical properties


Pharmacokinetics


Uses and modes of administration


Side effects


Appropriate doses

GMP guidelines


Different publications world wide, but
generally have similar information


Go over everything from raw materials to the
facility


US guidelines issues publications called
“Points to Consider” for additional guidelines
for newer biotech products (will go over
these later in semester)

Manufacturing facility


Most manufacturing facilities have
requirements, but some specifics to biotech
products, especially



Clean room



Water

Clean Rooms


Clean room views


Environmentally controlled areas


Critical steps for bio/injectable drugs are
produced in clean rooms


Contain high efficiency particulate air (HEPA)
filters in the ceiling


Figure 3.1 page 98

of chapter



Classification of Clean Rooms

for Pharma industry

Class


# microrganisms/m
3
of air

A



<1

B




5

C



100

D



500


See
table 3.5 page

100 of chapter



Other considerations


Exposed surfaces


smooth, sealed, non
-
penetrable surface


Chemically
-
resistant floors and walls


Fixtures (lights, chairs, etc.) minimum and
easily cleaned


Proper entry of materials and personnel into
clean room to reduce risk of contamination in
clean room

Gowned person in Clean room

Clean Room clothing


Covers most of operators body


Change in a separate room and enter clean
room via an air lock


Clothing made from non
-
shredding material


Number of people in a clean room at once
limited to only necessary personnel (helps
with automated processes)

CDS


Cleaning, decontamination and sanitization


C
-

removal or organic and inorganic material
that may accumulate


D
-
inactivation and removal of undesired
materials


S
-

destroying and removing viable
microorganisms

CDS cont’d


Done on surfaces that either are direct or
indirect contact with the product




Examples of surfaces in both categories?


CDS of process equipment


Of course trickier because comes in contact
with the final product


Clean equipment, then rid equipment of
cleaning solution


Last step involves exhaustive rinsing of
equipment with pure water


WFI


Followed by autoclaving if possible


If possible use CIP (cleaning in place)

Examples of CIP agents used to clean
chromatography columns


0.5
-
2.0 M NaCl


Non
-
ionic detergents


0.1
-
1.0 M NaOH


Acetic Acid


Ethanol


EDTA


Protease

Water


WFI
-

talked about this extensively before


30,000 liters of WFI needed for 1kg of a recombinant
protein


Use tap water just for non
-
critical tasks


Purified water


not as pure as WFI, but used for
limited purposes (in cough medicines, etc.)


WFI used exclusively in downstream processing


Will not cover pages 105
-
112
-

water and
documentation pages

Sources of Biopharmaceuticals


Genetic engineering of recombinant
expression systems


Your talks will be about types of systems and
how they are used
-

mammalian cells, yeast,
bacteria etc.


Most approved products so far produced in
E. coli

or mammalian cell lines


E. coli


Cultured in large quantities


Inexpensive (relatively speaking)


Generation of quantities in a short time


Production facilities easy to construct
anywhere in the world


Standard methods (fermentation) used

Current products from

E. Coli


tPA (Ekokinase)


Insulin


Interferon



Interleukin
-
2


Human growth hormone


Tumor necrosis factor

Heterologous systems


Expression of recombinant proteins in cells
where the proteins do not naturally occur


Insulin first in
E. coli



Remember the drawbacks of expression in
E. coli
?


Other problems with
E. coli



Most proteins in
E. coli

expressed
intracellularly


Therefore, recombinant proteins expressed
in
E. coli

accumulate in the cytoplasm


Requires extra primary processing steps
(e.g. cellular homogenization) and more
purification (chromatography)

Other problems with
E. coli,
cont’d


Inclusion bodies


Insoluble aggregates of partially folded product


Heterologous expressed proteins overload the
normal protein
-
folding machinery


Advantage
-

inclusion bodies are very dense, so
centrifugation can separate them from desired
material

Preventing inclusion bodies


Lower growth temperature (from 37

C to
30

C)



Use a fusion protein (thioredoxin)
-

native in
E. coli



protein expressed at high levels and
remains soluble



Expression in animal cells


Major advantage
-

correct PT modifications


Naturally glycosylated proteins produced in:


CHO
-

Chinese hamster ovary


BHK
-

baby hamster kidney


HEK


human embryonic kidney

Current products from animal cells


tPA


FSH


Interferon
-



Erythropoietin


FSH


Factor VIIa

Disadvantages of animal cells

(compared to
E. coli
)


Complex nutritional requirements


Slower growth


More susceptible to damage


Increased costs



WILL NOT cover bottom of page 116 to page
124 (up to biopharmaceuticals)
-

you will
cover these in your presentations


Final Product Production


Focus on
E. coli

and mammalian systems


Process starts with a single aliquot of the
Master Cell Bank


Ends when final products is in labeled
containers ready to be shipped to the
customer

Production: Upstream and
Downstream


Upstream: initial fermentation process; yields
initial generation of product



Downstream: purification of initial product
and generation of finished product, followed
by sealing of final containers



biomanufacturing process overview

Upstream processing


Remove aliquot from MCB


Inoculate sterile medium and grow (starter
culture)


Starter culture used to inoculate larger scale
production culture


Production culture inoculates bioreactor


Bioreactors few to several thousand liters


See
figure 3.13

of chapter (page 129)

Upstream cont’d


Pages 129
-
133 go over specific details for microbial
fermentation


Pages 133
-
134 go over specific details for animal cell culture


Properties of animal cells


Anchorage dependent


Grow as a monolayer


Contact inhibited


Finite lifespan


Longer doubling times


Complex media requirements


Downstream processing


Diagram page 135 of chapter 3



Detailed steps considered confidential



Clean room conditions for downstream


Downstream cont’d


Steps involved (intracellular products


E. coli.)


mammalian products secreted in media, so easier to
isolate)


Centrifugation or filtration


Homogenization


Removal of cellular debris


Concentration of crude material (by precipitation or ultra
filtration)


High resolution chromatography (HPLC)


Formulation into the final product

Downstream cont’d


Final product formulation


Chromatography yields 98
-
99% pure product


Add excipients (non active ingredients), which
may stabilize the final product


Filtration of final product, to generate sterile
product


Freeze drying (lyophilization) if product if to be
sold as a powder (dictated by product stability)

Separation methods


Page 142,tables 3.18 and 3.19


Familiar with:


Ion
-
exchange


Gel
-
filtration


Affinity chromatography


Protein A chromatography


Immunoaffinity chromatography

Factors that influence biological
activity


Denature or modify proteins


Results in loss of/reduced protein activity


Need to minimize loss in downstream work


Problems can be chemical (e.g., oxidizing,
detergents); physical (e.g., pH, temperature);
or biological (e.g., proteolytic degradation)


Table 3.20 page 143

Proteolytic degradation


Hydrolysis of one or more peptide bonds


Results in loss of biological activity


Trace quantities of proteolytic enzymes or chemical
influences


Several classes of proteases:


Serine


Cysteine


Aspartic


Metalloproteases (also in other ppt)

Protease inhibitors


PMSF


serine and cysteine proteases


Benzamidine


serine proteases


Pepstatin A


aspartic proteases


EDTA


metalloproteases



a.a residue known to be present at active site
of protein, so disruption of it causes loss of
activity

Others (mentioned before)


Deamidation


hydrolysis of side chain of
asparagine and glutamine


Happens at high temp and extreme pH


Oxidation and disulphide exchange


Oxidation by air (met and cys in particular)


Alterations of glycosylation patterns in
glycoproteins (more than one sugar)


Affect activity or immunological properties

Excipients


Substances added to final product to
stabilize it



Serum albumin


Withstands low pH or elevated temps


Keeps final product from sticking to walls of
container


Stabilize native conformation of protein


Excipients cont’d




Amino acids



Glycine


stabilizes interferon, factor VIII, stabilizes against heat



Alcohols (and other polyols)


Stabilize proteins in solution



Surfactants


Reduces surface tension; proteins don’t aggregate, so don’t denature

Final product fill


See
figure 3.27

page 153


Bulk product gets QC testing


Passage through 0.22

m filter for final
sterility


Aceptically filled into final product containers


Uses automated liquid handling systems

Final product fill cont’d


Freeze drying (lyophilization)


Yields a powdered product


Reduces chemical and biological
degradation of final product


Longer shelf life than products in solution


Storage for parenteral products (those
administered intravenously or injected)

Freeze drying cont’d


Need to add cryoprotectors


Glucose or sucrose


Serum albumin


Amino acids


Polyols



Freeze drying can be done in many steps

Labeling and Packing


After sealed in final container, product
quarantined


Samples are QC’d


Check potency, sterility and final volume


Detection and quantitation of excipients


Highly automated procedures


Labeling function critical
-

biggest error where
many products are made

Label


Name and strength of product


Specific batch number


Date of manufacture and expiry date


Required storage conditions


Name of manufacturer


Excipients included


Correct mode of usage

Other final product items


Biopharmaceutical products undergo more
testing than traditional pharma products


Products made in recombinant systems have
more potential to be contaminated than
synthetic chemical drugs


Larger, more complex molecules