PROTEIN BACKGROUNDER FOR GQAC (2-1/4 ... - bobseltzerms.com

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6 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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1




PROTEIN BACKGROUNDER
by Robert Seltzer

(Training given in 2004 to Global Quality Audits and Compliance at former
Schering
-
Plough Corporation)



X
-
ray Crystallography

of the Transport Protein
Myoglobin

2

PROTEIN BACKGROUNDER

I.

Definitions & Descriptions of Various Biochemicals (15 min)


II.

Categories, Structures, and Facts about Amino Acids & Proteins (15 min)


III.

Biosynthesis of Proteins, “The Genetic Code” Basis for Molecular
Genetics (8 min)


IV.

How Biologics Differ from Pharmaceuticals (7 min)


V.

Biopharmaceutical Manufacturing of Proteins (70 min)


A. Fermentation versus Mammalian Cell Culture


B. Aide
-
Memoire for Culturing, Fermentation & Harvesting


C. Purification


D. Aide
-
Memoire for Purification


E. PEGylation

F. Aide
-
Memoire for PEGylation


VI.
Analytical Techniques Used w/ Protein Mf’g (25 min)


VII.
Stability Testing (3 min)

VIII.
Comparability Protocol (2 min)

IX.
Training Effectiveness Worksheet (10 min)

X.
Discussion of Select Brinny FDA Audit Responses (25 min)

3

How Prokaryotes Dif
fer from Eukaryotes



4

How Prokaryotes

Differ
from Eukaryotes

1. No nucleus: all DNA on a single
circular, free
-
floating chromosome

A Nucleus holds multiple linear
chromosomes where DNA resides

2. No organelles except ribosomes

Various membrane
-
bound
organelles

3. Never carry viruses harmful to
animals

Mammalian
-
pathogenic viral DNA
can infect eukaryotic cell DNA

4. One kingdom of prokaryotes, Gram
negative bacteria, have lipopoly
-
saccharide outer cell walls
pyrogenic to humans (after bacterial
cell dies)

Eukaryotic cells do not disintegrate
into human
-
toxic components.

5. Bacterial cell diameters = 0.3
-

3 um;
mycoplasma = 0.1


0.2 um

Eukaryotic cell diameters

= 2 to 100 um

6. Can splice only enough DNA for a
small to medium
-
sized protein

Much larger genes encoding large
proteins easily spliced into a
eukaryotic cell

7. Prokaryotes enable minimal post
-
translational modification of protein

Cells’ enzymes enable all post
-
translational modification of protein

5

Biochemistry

Lipids, Carbohydrates, Nucleic Acids, & Proteins


Biochemistry
: The study of chemical substances and vital processes
occurring in
living

things


Biochemicals
: Chemicals unique to
living

things, which usually
refers to lipids, carbohydrates, nucleic acids, & proteins.


Life
: That characteristic of a member of a population whose
members can exhibit all the following phenomena at least once
during their existence:

1. Growth, full development, maturity

2. Metabolism, consuming, transforming, and storing energy/mass;


growing by absorbing and reorganizing mass; excreting waste

3. Motion, either moving itself, or having internal motion

4. Reproduction: the ability to create entitities that are similar to, yet


separate from, itself

5. Response to stimuli (homeostatis): the ability to measure


properties of its surrounding environment, & act upon certain


conditions



Lipids
: Biochemicals insoluble in water but soluble in nonpolar
organic solvents, and include fats, oils, waxes, sterols, triglycerides





6

Polypeptides


Polypeptides
: Biochemicals consisting of two or
more amino acids joined linearly by a form of amide
bond known as peptide bonds.





7

Amino Acid Facts

1.
There exist twenty natural amino acids encoded by the DNA of all
organisms.

2.
Eight amino acids are generally regarded as essential for humans (i.e,
must be supplied in the diet)

Ile, Leu, Lys, Met, Phe, Thr, Trp, Val.

Two others, His and Arg are essential only in children.

3.
Amino acids vary between extremely hydrophilic (e.g., the acidic and
basic amino acids) to extremely hydrophobic (e.g., the aromatic and the
long nonpolar aliphatic amino acids), with degrees in between.

4.
Amino acids found within protein molecules as opposed to stand
-
alone
are called
amino acid residues
.

5.
Most amino acid residues can undergo chemical reactions such as
protonation, deprotonation, oxidation, reduction, hydrolysis, as well as
covalent modification by simple or exotic, wavelength
-
absorbing or
fluorescent, short or long molecules (e.g., PEG).

6.
The aromatic amino acid residues Trp, Tyr, and Phe confer a 280 nm
absorbance maximum to a protein.

7.
Pairs of cysteine residues (can be far away from each other along the
chain) within a protein can form disulfide (
-
S
-
S
-
) bonds.


8

Polypeptide and Protein Facts

1.
Polypeptides, when they are pure and homogeneous, form crystalline solids. The
peptide bonds confer a UV absorption from 180 to 220 nm.

2.
Proteins

are a subset of polypeptides that are both a) naturally occurring and b)
serving a biological function.

3.
There believed to be only 5,000 to 12,000 natural proteins in the world.

4.
Proteins range in MW between 3,000 and 1 x 10
6

Daltons.

5.
Proteins always possess a net + or


charge because of the ionization state of the
acidic and the basic amino acids as well as the terminal amino group and the
terminal carboxyl group at both ends of the molecule.

6.
At one pH unique to each protein, the isoelectric point (p
I
), there are an equal
number of + and
-

charges, resulting in a zero net charge. Most proteins begin to
precipitate out of solution at their p
I
.

7.
When the solution pH < its p
I
, a protein has a net + charge.

8.
When the solution pH > its p
I
, a protein has a net


charge.

9.

The sequence of amino acids in a protein


its
primary structure

(click link
amino
acid, peptide bonds, and protein structure

)

10.

Proteins do not branch but are linear molecules with an amino terminus

(


N
-
terminus) and a carboxy terminus (


C
-
terminus).







9

Polypeptide and Protein Facts

11.
H
-
bonds can form between one peptide bond carbonyl oxygen and
another peptide bond amide hydrogen. This allows some local sections
of a protein to assume an “alpha helix” or a “beta sheet.” The existence
of these shaped local segments of a protein


secondary structure
.

12.
Protein molecules assume a unique three
-
dimensional conformation in
space


tertiary structure
. A combination of disulfide bonds, hydrogen
bonds, hydrophobic groups “hiding” from water, van der Waals forces,
etc. are responsible for the tertiary structure.

13.
The reduction of disulfide bonds to individual sulfhydral groups on
cysteine residues followed by oxidation and “mismatching” or
“scrambling” of disulfide bonds (when a protein has 3 or more cysteine
residues) may lose a protein its inherent biological activity (bioactivity)

14.
When disulfide bonds form between (instead of within) two or more of
the same protein molecules, aggregates/oligomers are formed (can be
dimers, trimers, tetramers, etc.).

15.
A non
-
amino
-
acid organic or metallic group held tightly but non
-
covalently to a protein is called a
prosthetic group
.

16.
Proteins with a physiological, catalytic function are called
enzymes
.

17.
Enzymes that mediate the hydrolysis of proteins are called
proteases

or
have
proteolytic

activity).





10


Protein Categories


a) Structural Proteins (e.g., collagen, elastin, chitin, lignin)

b)
Contractile Proteins (e.g., actin, myosin)

c) Storage Proteins (e.g., casein, ovalbumin)

d) Protein Toxins (e.g., diptheria,
Clostridial botulinum
)

e) Cell Receptors (e.g., opiate receptors, cholesterol receptors)

f) Enzymes (e.g., trypsin, elastase, HMG
-
CoA reductase)

g) Transport Proteins (e.g., hemoglobin, serum albumin)

h) Protective Proteins (e.g., antibodies, thrombin)

i) Hormones* (e.g., insulin, adrenocorticotrophic hormone )

j) Growth Factors (e.g., interferons, interleukins, erythropoietin)


*Hormones differ from growth factors by the fact that a hormone
modulates (promotes or retards) production or modification of
another protein(s), while a growth factor modulates
cellular

proliferation and/or differentiation.

11


Carbohydrates
:
Biochemicals

with the formula C
n
H
2n
O
n,

that
include sugars, starches, celluloses, & gums.

The monomers
known as
monosaccharides

(sometimes also called sugars) exist
primarily as ringed
hemiacetals

or
hemiketals

(the self
-
condensation from the intermediates
polyhydroxyaldehydes

or
polyhydroxyketones
, respectively.
Monosaccharides

can combine
via dehydration to form polymers

polysaccharides
.





12



Nucleic Acids
: Biochemicals composed of a
phosphate group connected to a 5
-
membered ring
sugar (ribose or deoxyribose) connected to a
heterocyclic compound called a nucleotide base



DNA (deoxiribonucleic acid)
: A long chain of nucleic
acids with deoxiribose as the sugar and guanine,
cytosine, adenine, and thymine as the nucleotide
bases.


Note: DNA in organisms normally exists as two strands
arranged alongside each other in a helical shape, thus
the term double helix. The two strands of nucleic acid
chains are held together by H
-
bonds formed between
specific pairs of nucleotide bases.





Biochemistry

Lipids, Carbohydrates, Nucleic Acids, & Proteins

13

H
-
bonds between the Single
-
Ring Pyrimidine
Nucleotide Bases and the Two
-
Ring Purine
Nucleotide Bases Comprising DNA

(imagine dotted lines below from O’s and N’s to share H’s, there being
3 between Cytosine & Guanine and 2 between Thymine & Adenine)

Thymine

Adenine

Cytosine

Guanine

14









15

Protein Biosynthesis Sequence of Events

1)
A cell’s DNA double helix enzymatically unravels then separates into
two strands along the length where the gene to be decoded exists.

2)
A messenger RNA (= mRNA) is enzymatically assembled directly off the
portion of a DNA single strand (the gene) serving as its template.

3)
This mRNA moves away from the DNA to a place in the cytoplasm
--
organelles called ribosomes
--
where the mRNA sequence of nucleotide
bases is translated into a sequence of amino acids. Every 3 nucleotide
bases (a triplet) encodes a unique amino acid. The first triplet
transcribed and later translated is always for the amino acid methionine
in eukaryotes and initially formylmethionine in prokaryotes.

4)
Peptide bonds form between the amino acids during each elongation
step of the growing protein molecule.

5)
During translation or at the end of translation as a protein molecule
moves away from the ribosomes, enzyme
-
mediated post
-
translational
modification usually affects specific amino acids in the protein (either a
covalent attachment or a proteolysis).

http://en.wikipedia.org/wiki/Genetic_code

16

Polypeptides (pre
-
proteins) Biosynthesized from the DNA

17

Protein Biosynthesis Sequence of Events

6) Examples of posttranslational modifications are:


proteolytic removal of 1 or more amino acids @ N
-

or C
-
terminus
deformylation (usually of the N
-
terminal methionine)

acetylation

phosphorylation

pyruvation

hydroxylation (occurs only in eukaryotes)

sulfonation

glycosylation



NOTE
: Whereas disulfide bond formation is not found in natural bacterial
proteins, many mammalian proteins contain disulfide bonds crucial for the
folding and maintenance of the 3D structure (and therefore bioactivity) of the
protein.




18

How Biologics Differ from Traditional
Pharmaceuticals

Definition of a
biologic

(Public Health Service Act Sec 262i)
:



A virus, therapeutic serum, toxin, antitoxin, vaccine,
blood, blood component or derivative, allergenic
product, or analogous product, or arsphenamine or
derivative of arsphenamine (or any other trivalent
organic arsenic compound), applicable to the
prevention, treatment, or cure of a disease or
condition of human beings.



Note: Proteins that are biosynthesized by and isolated from
living cells are also biologics. However, polypeptides
synthesized by a method discovered by Nobel prize
winner Bruce Merrifield are technically not biologics.





19

Traditional Pharmaceutics

Versus Biologics

1. Molecular wt. usually in 100s
(maybe low 1000s) of Daltons

Molecular wt. varies from the thousands to
millions of Da

2. Can be described by a fixed
molecular formula

Typically can’t be described by a molecular
formula (e.g., a virus or a protein, the latter w/
many different states of ionization)

3. Chemically synthesized by
workers per a “cookbook”

Biosynthesized by organisms (e.g., bacteria,
mammalian cells) or viruses

4. API mf’g process doesn’t
define the product: structure
and chemical assays define it

Perfectly characterizing the API every lot is far
more difficult than ensuring a consistent
process

5. Contamination during mf’g is
more easily detected,
removed, and validated

Contamination w/ viruses & other adventitious
agents (e.g., prions) very possible & not 100%
detectable; clearance validation often complex

6. Simple physicochemical
characterization methods

Complex physicochemical characterization
methods

7. Potency determined via a
mass quantitation assay
(usually chromatographic)

Potency usually determined using
both

an
activity assay and a mass quantitation assay

20

Facts about Interferons (IFNs)

1)
IFNs a category of proteins known as cytokines, a
subset of the growth factors produced by immune
system cells.

2)
IFNs fall into any of three categories:



慬灨a
-

⡳灥汬敤⁡汦愩 ⁰牯摵捥搠批 汥畫潣y瑥猻t



扥瑡
-
Ⱐ灲潤畣敤⁢e 晩扲潢污獴f㬠慮搠



条浭m
-
Ⱐ灲潤畣敤e批 T
-
捥汬猠☠湡瑵牡氠歩汬敲 捥汬献s


周攠慬晡⁡湤 扥瑡t䥆乳⁩湤畣攠慮瑩捡湣敲Ⱐ慮瑩癩t慬Ⱐ
慮瑩灡牡獩瑩挠慣瑩癩瑹⸠ 䝡浭愠䥆丠桡猠s敡步爠慮瑩癩t慬a
慣瑩癩ty⸠⁉琠楳t畮摥牧潩湧⁰n慳攠䥉䤠捬楮楣慬c瑲楡氠i条楮獴i
multi
-
drug resistant tuberculosis.






21

Facts about IFN
-
alfa

1)
Alfa
-
Interferons (IFN
-
alfa) are stable down to just below pH 2.0

2)
Disulfide bonds are formed between IFN
-
alfa cysteines 1 and 98 and
between cysteines 29 and 138. The 29/138 disulfide bond is essential
for bioactivity, while 1/98 can be reduced w/o effect.

3)
IFN alfa
-
2b (Intron A) contains 165 amino acids, has a molecular weight
of 19,271 Daltons. It differs from Hoffmann
-
La Roche’s Interferon alfa
-
2a (Roferon A) by only one amino acid residue at position 23: Intron A
has an arginine residue and Roferon A has a lysine residue.

4)
Intron A is indicated for:

i) Hairy cell leukemia (2% of all leukemias),

ii) AIDS
-
related Kaposi’s sarcoma,

iii) Condylomata acuminata (genital or veneral warts),

iv) chronic hepatitis B,

v) Chronic hepatitis C,

vi) Malignant melanoma, and

vii) Follicular [non
-
Hodgkin’s] lymphoma.

5)
PEG
-
Intron is a covalent conjugate of Intron A with monomethoxy
succinimidyl carbonate polyethylene glycol (12,000 Daltons) at

histidine 134 (note: the abbreviation for this specific form of PEG is
mSC
-
PEG)











22

rDNA & Other Relevant Definitions
(in learning order)

Subsets of Therapeutic Protein Impurities


Protein isoforms
: Versions of a protein with some small differences; theoretically,
Roferon A is an isoform of Intron A


Protein Variants (or just variants)
: Protein products formed during manufacture (including
biosynthesis) and/or storage which deviate only slightly in covalent groups or atoms from
its standard form; variants are divided into two subcategories

product
-
related
substances

and
degradation products



Note: There are few real differences between isoforms & variants,


except that isoforms are generated inside the host cell and


variants are a more
-
encompassing term that includes


isoforms, product
-
related substances, and degradation products.


Product
-
related substances
: Variants of the desired product formed during manufacture
and/or storage which are active and have no deleterious effect on the safety and efficacy
of the drug product, e.g., a protein having a PEG molecule covalently attached at other
than the target amino acid


Contaminants
: A subset of impurities which can consist of adventitious agents, host cell
proteins, host cell nucleic acids, host cell endotoxins, leachables (from process
equipment or process disposables), or culture media components (e.g., polysaccharides,
lipids, protein digests, antifoam agents, etc.)




23

rDNA & Other Relevant Definitions


Degradation products (or just degradants)
: Variants resulting from relatively
minor, yet undesired changes in the desired product or product
-
related
substances. Changes are brought about over time and/or by the action of any
single or multiple forces (e.g., light, temperature, pH, water, reaction with
excipients, reaction with immediate container/closure, etc.)

Note: Degradants that can arise in rDNA protein mf’g can be the products of:

-

Aggregation/oligomerization

-

Denaturation

-

Deamidation

-

Proteolytic digestion

-

Oxidation of the methionine sulfur

-

Reduction of a disulfide to two sulfhydrals

-

Scrambling of disulfide bonds


Adventitious agents
: Bacteria, yeast, mold, mycoplasma, viruses, or prions,
that can potentially contaminate prokaryote or eukaryote cells used in
production; potential sources include the media raw materials (esp. if of animal
origin), infected cells, or the environment


Impurities
: All undesired components of intermediate or final bulks of a drug or
biologic; note: impurities can be contaminants, degradants, or product
-
related
substances; also, impurities are not necessarily always inferior (as in very
similar product
-
related substances with identical bioactivity)







24

rDNA & Other Relevant Definitions


Biotechnology
: The application of biological systems and organisms to
technical and industrial processes


Recombinant

(r): Of or related to DNA (called rDNA) that contains
genes from different sources that have been combined by methods of
genetic engineering as opposed to traditional breeding experiments.


Cell Line
: Cells that acquire the ability to multiply indefinitely in
-
vitro


Clone
: A cell line stemming from a single ancestral cell and normally
expressing
all

the same genes; ICH Q5D calls this a “cell substrate”


Genetic construct (sometimes also called “expression construct”)
:

An artificially prepared segment of nucleic acid that is to be
“transplanted” into a target tissue or cell



Plasmid
: An extrachromosomal, self
-
replicating, circular segment of
double
-
stranded DNA (found naturally in bacteria and some yeasts);
they can be used by scientists to introduce up to 10,000 nucleotide
base
-
pairs of foreign DNA into a recipient (host) cell; Genetic
engineering plasmids usually contain a
relevant genotypic and
phenotypic marker

http://en.wikipedia.org/wiki/plasmid







25

rDNA & Other Relevant Definitions


Host
: A cell whose metabolism is used for the growth and reproduction
of a foreign DNA


Relevant Genotypic Marker
: Specific

gene that permits the
identification (and possibly isolation) of the strain of cell line
possessing the expression construct for the protein of interest


Relevant Phenotypic Marker
: Specific outward expression of a gene
that permits identification (and possibly the isolation) of the strain of
cell line possessing the expression construct for the protein of interest;
Example: the resistance to the antimicrobial compound tetracycline
which can be selected for on a culture plate containing tetracycline


Expression vector (or simply vector)
: A plasmid or phage (bacterial
virus) into which and by which foreign DNA may be inserted into a

host cell


Master cell bank (MCB)
: A homogeneous suspension of the original
cells already transformed by the expression vector containing the
desired gene, aliquoted into individual containers for storage.


Working cell bank (WCB)
: A homogeneous suspension of cells derived
from the MCB(s) by a finite passage level, aliquoted into individual
containers for storage








26

Required by EU for Marketing Applications and Mf’g of
rDNA Derived Proteins

1)
Origin of cell line & nature of any known relevant condition of donor, e.g.,
Burkitt’s lymphoma, active mononucleosis, virus or mycoplasma infection

2)
Growth characteristics of cell line & stability throughout the cultivation used

3)
If serum or plasma is included in the cell cultivation, evidence that it is free
from bacteria, fungi, mycoplasma, or viruses

4)
A description of host cell & expression vector (e.g., plasmid) used in prod’n

5)
Method of introducing vector into host cell, & condition of vector inside host

6)
Full details of the nucleotide sequence of the gene of interest in the vector,
including adjacent control regions of the vector

7)
The quantity (if >1) of that gene in the vector (defined as the “copy number”)

8)
Description of any inducer (cell culture additive to stimulate prod’n)

9)
Description of any enhancer (substance added to an induced cell culture to
improve final yield)

10)
The DNA sequence of the cloned gene confirmed at the seed lot stage and at
least once after a full
-
scale cultivation (done at least once for each MCB)

11)
The limit for in vitro cell age, which is defined as the time between thaw of
MCB vial(s) to harvest of the production vessel in terms of a) chronological
time, b) population doubling level of the cells, or c) passage level of the cells
when subcultivated by a defined procedure for dilution of the culture).






27

FDA
-
Required Characterization of (modified
E
.
coli
) Cell Line

1)
The location, identity and inventory of individual ampoules of
cells in the working cell bank (WCB) should be thoroughly
documented.

2)
The MCB and WCB should each be stored in two or more
widely separate areas within the prod’n facility as well as at a
distant site.

3)
Limited identity testing (genotypic or phenotypic) is performed
on each WCB.

4)
The WCB needs to be spot checked for contaminants possibly
introduced from culture medium.

5)
When a proprietary medium or medium supplement is used,
the supplier supplies required data to CBER via a Master File
Application.


NB: The majority of the “Draft of Points to Consider in the
Characterization of Cell Lines Used to Produce Biologicals
(1993)” deals with controlling the risks associated with
mammalian cell cultures (such as viruses and TSE).










28


Fermentation vs.

Mammalian Cell Culture





1. Fermentation is large
-
scale
cultivation of microorganisms

Cultivation of cells derived from
multicellular organisms such as
mammals

2. Prokaryotic cells and yeast
enable only a subset of
mammalian eukaryotic cell
mediated post
-
translational
modification of proteins

Mammalian cells enable all the full post
-
translational modification of mammalian
proteins

3. Protein usually biosynthesized
inside the cell, necessitating a
cell disruption during harvesting

Protein
is

secreted outside the cell,
saving a cell disruption step


4. Bacterial and yeast cells contain
a cell wall, making them sturdier
(allows mixing rates >350 rpm
maybe even near 800 rpm)

Mammalian cells lack cell wall, making
them more fragile and destroyed by
process shear (mixing rate usu <250 rpm)

5. Bacterial cultures almost always
can grow in suspension (free
floating)

Mammalian cell cultures are generally
adherent (i.e., require anchorage to a
surface), although some can be
suspended

29

Fermentation vs.

Mammalian Cell Culture

6. Usually contain no
adventitious agents but, if
Gram negative, do yield
endotoxins

Can yield potentially adventitious agents
such as viruses and mycoplasma,
requiring purification validated for their
removal; yield no endotoxin

7. Requires a simple growth
medium containing sugar(s)
and amino acids or protein
digests

Requires a more complex and expensive
growth medium such as mammalian serum
or plasma (which, again risk carrying
viruses or mycoplasma)

8. The cell populations double on
the order of minutes; thus
only 5 days usually required
for usable protein levels at
harvest

The cell populations double on the order
of a day; thus 4
-
5 months often required
for usable protein levels

9. Wider temp and pH tolerated
by microbial cells (e.g., temp
need be controlled +/
-

1
o
C) in
fermentation; titrant can be
NaOH or HCl w/o significant
localized cell death

Relatively narrow pH and temperature
range (and temp needs +/
-

0.1
o
C control)
in mammalian cell culture; also acid
add’n must be gentler, e.g., via CO
2


30

Audit Questions on Fermentation

1.
Ask for the MCB and WCB QC specifications, then compare
them to trended results (w/ some raw data review as well).

2.
How is the frozen storage of cell banks monitored (must be
24/7)? Is there a disaster recovery plan w/ multiple freezers
and off
-
site storage? Is cell bank access limited to authorized
personnel?

3.
Ask for printed inventory of cell banks, then ask for the
culturing/passage history of one or more cell banks chosen.

4.
Ask to see the C.O.A. & QC testing on the media used for a
sampled fermentation batch.

5.
Ask to see the elapsed time between thaw of the WCB vial to
harvest of the prod’n vessel (

in vitro cell age). How is this
trending?

6.
Is the data logging/monitoring of fermentation process
variables computerized; if yes, is it validated?


31

Audit Questions on Fermentation

7.
What tests and criteria are used to monitor for
contaminating organisms (e.g., growth rate, culture purity,
bacteriophage assay, fatty acid profile)?

8.
What are the trends of identified contaminating organisms
over the last 5 yr?

9.
How often does site standardize and clean the pH probe?

10.
What indicates end of fermentation/time for harvesting? Do
batch records show this to be a consistent endpoint?


32

Preparative Chromatography of Protein

Adsorption/desorption category of column chromatography

--
ion exchange, affinity, or hydrophobic interaction
--
can employ either
isocratic or gradient elution to desorb the protein of interest.


Isocratic elution
: step change (or no change at all) in the buffer ionic
strength or pH, introduced to a column in order to desorb a protein or
proteins of interest


Gradient Elution
: a linear or other gradual change in the buffer (usually in
its ionic strength) introduced to a column in order to desorb a protein
or proteins of interest



If the less common strategy had the protein of interest passing through
the gel w/o binding, accompanied by binding of the impurity(ies), then
a very strong isocratic (one
-
step) elution would remove to waste the
impurity(ies).


Gel filtration chromatography, having no adsorption, would not benefit
from a gradient of any kind; and thus always uses isocratic elution.


33

Ion Exchange Chromatography


This form of adsorption/desorption (on/off) chromatography relies on:

a) adsorbents with fixed negative charges (e.g., carboxymethyl,
sulfonates) that will bind net positively charged biomolecules (this is
called cation exchange); or on

b) adsorbents with fixed positive charges (e.g., diethylamino ethyl,
quaternary ammonium groups) that will bind net negatively charged
biomolecules (this is anion exchange).


The protein of interest with a known pI is diafiltered into a low ionic
strength buffer at a pH below the protein’s isoelectric point (for cation
exchange) or above its isoelectric point (for anion exchange).


The column is washed with load buffer to desorb unbound
contaminating proteins, then the protein is eluted via a pH step or via a
high salt step or increasing salt gradient.


The first and third chromatography steps for Intron A, are cation and
anion exchange, respectively. These and the Blue Sepharose Affinity
Chromatography gel specifications and packing and testing
instructions are contained in an attachment taken from the web site of
Amersham Biosciences.
(see “Chromatography Gel” Handout).

34

Gel Filtration Chromatography of Protein
(analogous to HP
-
SEC)



35

Affinity Chromatography of Protein


This form of adsorption/desorption (on/off) chromatography
relies on selective interactions between a protein of interest and
a gel containing immobilized ligands that form (reversible)
bonds with it. These ligands can be the protein’s specific
monoclonal antibody, a substrate (if the protein is an enzyme),
or it can be a dye to which the protein of interest has particular
affinity, e.g., Cibacron Blue dye, used as the middle of three
chromatography steps in Intron
-
A purification.

36

Hydrophobic Interaction Chromatography (HIC)


This adsorption/desorption (on/off) chromatography relies on
the presence of exposed hydrophobic groups on the surface of a
protein. They interact with immobilized nonpolar functions on
the column gel such as short alkyl chains and phenyl rings.


Adsorption of a protein is promoted by high ionic strength, while
elution often employs a salt gradient of
decreasing

concentration.


The method can be used directly after a high salt elution step (as
in ion exchange) or a protein salting
-
out step (e.g., ammonium
sulfate precipitation).


HIC is analogous to RP
-
HPLC except that it is much less
denaturing to a protein. HIC solid supports have a density of C
2



C
8

aliphatic or simple aromatic compounds of 10
-

50
umoles/ml gel versus the C
4



C
18

aliphatics at several hundred
umoles/ml gel in RP
-
HPLC.

37

Audit Questions on Purification

1.
For any of the pre
-

and post
-
column IFN
-
containing pools, ask for
maximum holding time support data.

2.
How are column gels preserved between purification campaigns?

3.
Ask to see the preservative effectiveness
-
like validation. Was
bacteriostasis/ fungistasis testing performed as part of it

4.
How are preservatives removed and QCed for complete removal
before production use?

5.
What are the trends of identified contaminating organisms in
column gels or product pools over the last 5 yr?

6.

How is progressive endotoxin reduction through purification
monitored and trended? Are the % reduction at measured steps
proving consistent batch
-
to
-
batch?

7.
What bacterial claims and controls versus hold times placed on
chromatography buffers? Verify a sampling of those test results.

8.
Ask to see references to dates when extractables/leachables studies
were conducted on all filters, ultrafiltration media, column gels, and
bulk containers.



38

Audit Questions on Purification

8.
Review a sampling of historic tables for batch
-
to
-
batch and
measured purification step
-
by
-
step consistency in specific
activity increase over the purification train.

9.
If blending of the product pools from multiple runs on the same
column, is expiration date or hold time based on the age of the
oldest component of the blend?

10.
Which column chromatography validation reports have FDA and
IMB reviewed? Whichever ones not so reviewed should be
audited, looking for the following:

a) column & media cleaning validation

b) column & media validated number of reuses

c) pre
-
run suitability test(s)

d) proven clearance of host cell endotoxins, DNA, and proteins

11.
Have all disposable process devices or materials that are
routinely reused been validated for their max. no. of reuses, e.g.,
filters, tubing, chromatography gel?

12.
Whatever valves are used in the column purification train, are
they on a PM system for complete internal examination and
possible cleaning (e.g., solenoid valves that entrap mat’l)?



39

In
-
Vitro Reactions Proteins are Prone to

1.
Aggregation/ oligomerization (covalent and non
-
covalent)

2.
Acid hydrolysis of peptide bonds

3.
Base hydrolysis of peptide bonds

4.
Proteolytic hydrolysis of peptide bonds (from host cell
protease enzymes)

5.
Deamidation of glutamine and asparagine to glutamic acid
and aspartic acid, respectively

6.
Reduction of disulfide bonds with or without disulfide
rearrangement (latter could cause oligomerization)

7.
Oxidation of methionine sulfur atom to a sulfoxide

8.
Overall protein denaturation that is either reversible or
irreversible (see next page)

40





Protein Denaturation:
Defined as any
noncovalent change in a protein altering its 2
o

or 3
o

structure



Structural (denaturing) changes are often not directly observable
but the effects on some measurable protein properties
are

observable. For example:


a) insolubility,

b) increased susceptibility to proteolysis, and/or

c) loss of bioactivity


The causes of protein denaturation include:


a) Temperatures in excess of 38
o
C;

b) pH extremes;

c) Changes in solvent dielectric constant;

d) Changes in solvent ionic strength;

e) Shear forces; and

f) Exposure to either liquid
-
air or liquid
-
liquid interfaces



41

Where in Intron A Process Could Protein
Degradations Occur?


1.
Proteolysis can occur at early or intermediate
stages of protein purification when it is possible
for co
-
purified host cell proteases to act upon
other proteins in concentrated protein pools
(column loads or column eluates). Concentration
and time can be early “enemies” of IFN.

2.
Excessive heat and shear from mixing, flow,
filtration, and homogenization result in both some
reversible and irreversible denaturation of IFN
molecules. Irreversible denaturation leads to lower
specific activity of IFN.




42

Protein PEGylation


Polyethylene glycol (PEG) has the general formula

HO
-
(CH
2
CH
2
O)n
-
CH
2
CH
2
OH


It is used for the covalent modification of biochemicals, especially
therapeutic proteins. The reasons for using PEG to modify a
therapeutic protein are the following:


1.
Increases blood
-
circulation half
-
life via reduced renal clearance
rate and reducing (destructive) uptake by the reticuloendothelial
system (immune system, including spleen)

2.
Enhances solubility (for highly hydrophobic proteins)

3.
Improves in vitro stability (greater shelf life)

4.
Reduces immunogenicity/ antigenicity

5.
Reduces proteolysis by blood
-
circulating proteases

6.
Is nontoxic and doesn’t harm active proteins or cells




43

Protein PEGylation

PEG technology as a therapeutic protein delivery system
began in the Rutgers lab of Prof. Frank Davis and his post
-
doctoral candidate Abe Abuchowski. They founded Enzon,
Inc. in South Plainfield, NJ, where various therapeutic
enzymes were found more stable in the bloodstream
following PEGylation.

Today’s trainer did post
-
Masters work at Enzon,
characterizing mSC
-
PEG which, today, Schering
-
Plough
licenses from Enzon for percentage of PEG
-
Intron sales.


See two PEG articles among the handouts.



44

Audit Questions on PEGylation

1.
Ask to see the supplier quality management that’s occurred (audits,
certification, etc.) for the mSC
-
PEG manufacturer (contractor for
Enzon), including performance trends in meeting specifications.

2.
Ask for SOP on acyl group titration (QC to quantitate the active
group on mSC
-
PEG); then ask to be shown records for the most
recent test.

3.
How has acyl group content (on mSC
-
PEG) trended over time &
versus specification?

4.
Up to what shelf life is mSC
-
PEG stable during storage, and what
data supports mSC
-
PEG expiration dating?

5.
Ask for the PEGylation process validation report, then compare its
conclusions on CPPs (e.g., temperature, speed of mixing, timing of
rxn before quenching w/ glycine, etc.) against a current batch record.

6.
Review trend data on stability testing of PEG content.

7.
Read batch records for variability of time between moment of glycine
addition to (quench) PEGylation rxn and time to pass entire batch
through subsequent ion exchange column.




45

Analytical Techniques Used in Intron A / PEG
-
Intron QC and
in Labs Outside Brinny

















METHOD

ATTRIBUTE
(plus specific purpose, or example)

Online or offline pH

Solution pH

Appearance

General quality

A
280

Protein concentration

SDS
-
PAGE

Identity, purity, stability (reducing and non
-
reducing, latter
for detecting disulfide bond oligomerization)

RP
-
HPLC, HP
-
SEC


Identity, purity, stability;

also separate RP
-
HPLC quantitative method for [protein]

Online or offline
Conductivity

Solution conductivity (e.g., column buffer QC)

MTT
-
CPE Antiviral
Bioassay

Potency

E. Coli Protein Elisa

Purity

N
-
terminal Methionine

Purity (would detect a variant impurity IFN)

Peptide Mapping

Identity, purity

PCR

DNA impurities

46

Analytical Techniques Used in the QC of Intron A,

PEG
-
Intron, Non
-
Schering IFNs, and Proteins other than IFN

















METHOD

ATTRIBUTE
(plus specific purpose,
example)

Flat
-
bed Isoelectric Focusing (IEF)

Purity (deamidated asn or gln residues)

Capillary IEF

ID, purity (and measure of isoelectric point)

N
-
terminal Sequencing

ID, purity

MALDI
-
TOF (Matrix
-
Assisted Laser
-

Desorption Time
-
of
-
Flight) Mass Spec

ID, purity (estimated MW
--
intact primary
structure)

BioRad Protein Assay

Protein concentration

Circular Dichroism (not at Brinny)

Conformation (intact 2
o
or 3
o

structure)

Polymerase Chain Reaction (PCR)

Purity (detects nucleic acid impurities)

Active Acyl Assay (not at Brinny)

Quantitative measure of active mSC
-
PEG

Klett

IPC (fermentation cell density)

LAL

Quantitative endotoxin impurity

Bioburden

Quantitative total aerobic bacteria assay

Amino Acid Analysis (not @ Brinny)

Provides relative proportion every amino
acid in a homogeneous protein sample

47

Therapeutic Protein Stability Studies & Shelf Life

1.
There is no
single

stability
-
indicating assay or parameter that profiles the
stability characteristics of a therapeutic protein. One must select a
stability
-
indicating profile (of orthogonal methods, see below) that
assures detecting changes in product identity, purity, and potency.

2.
Orthogonal Analytical Methods
: concurrently run tests that collectively
provide a stability
-
indicating profile that no single test can. An example
would be the use of RP
-
HPLC and HPSEC separate proteins based on
differences in polarity and molecular size, respectively.

3.
Expiration dating is based on real
-
time/real
-
temp. data.

4.
Degradants arising during a developmental stability study and exceeding
a pre
-
determined threshold results in either

a) an end
-
of
-
shelf
-
life or

b) the need for a thorough understanding of the degradation pathway


in terms of physicochemical mechanism(s) involved and the


adverse impact of that degradation pathway.

5.
Protein degradants, at worst, can create new antigenic sites (


epitopes)
and more rapid destruction by the immune system or even systemic
allergic reaction (anaphylactic shock) in a patient.



48

Comparability of Biotechnological/Biological
Products Subject to Mf’g Process Changes

1.
Principle of Comparability
: “When a mf’g process change has
been made that has the potential to have an impact on quality
attributes, a complete or limited (but rationalized) repetition of
the characterization activity conducted for the market application
[e.g., that which accompanied pivotal clinical trials] is generally
warranted to directly compare the prechange and postchange
product.”

2.
Comparability Protocol



a well
-
defined, detailed, written plan
for assessing the effect of a specific change on the identity,
strength, quality, purity, or potency of a specific drug product as
these factors relate to the safety and effectiveness of the product

3.
If a mf’g change is sufficiently radical, a comparability protocol
might need more than analytical tests & bioassays, but possibly
an animal toxicity (GLP) study and a [human] clinical (GCP)
study.






49

Reducing & Non
-
Reducing SDS PAGE


The sample (containing proteins usu. totaling less than some
capacity/resolving maximum, e.g., 50ug) is incubated with SDS. The
proteins all bind SDS via hydrophobic interactions, such that numerous
SDS negative sulfate groups surround the proteins. The proteins all have
their net surface charge “swamped” by the sample prep SDS’s negative
charge, making charge one less distinguishing variable among any
proteins. This leaves MW and molecule shape as the remaining variables.
The polyacrylamide gel acting like a sieve, retards larger MW proteins
relative to smaller MW proteins as all the negatively charged SDS
-
bound
protein molecules migrate toward the positive pole (the anode). Multiple
lanes in the gel will carry sample as well as MW markers (usu. in one or
both outermost lanes).


If there are disulfide
-
bonding oligomers such as dimers, trimers, and/or
other higher order aggregates of a protein, they will migrate more slowly as
linear function of their higher MW.



If the initial SDS/sample incubation also contains a reducing agent such as
mercaptoethanol or dithiothreitol, then any

S
-
S
-

oligomers will reduce to
all monomers. This is called a reducing SDS
-
PAGE, while the former is a
non
-
reducing SDS
-
PAGE. After staining (with Coomassie Blue or silver
stain, and then destaining with a methanol/acetic acid solution, subsequent
densitometry or a digital imaging system provides relative proportions of
the bands within a lane for purity calculations.


Non
-
reducing SDS PAGE as well as HPSEC would indicate if protein
oligomers had formed.

50

Flatbed Isoelectric Focusing


The sample (containing proteins usu. totaling less than some capacity ug) is
loaded in wells near the center of a flatbed of a specially formulated
polyacrylamide or agarose gel, again with two opposite electrodes energized.
The special formulation of polymer contains what are known as “ampholytes,”
polyionic molecules that generate a linear pH gradient when the voltage is
applied to the gel.


The protein species within the sample migrate initially toward the opposite pole
of their net charge and then stop when they reach a portion of the gel pH
gradient at which the protein’s pI equals that of the ampholyte right there and
the protein, with now a net charge of zero moves no more in the electric field.


Commercial marker proteins of known and spread
-
out pI’s are used in
neighboring well(s). The gel is later stained and destained so the protein bands
stand out against a clear background.


IEF can discriminate a single charge difference between very similar protein
molecules, e.g., one that’s been deamidated, sulfated, phosphorylated, as to
change the charge behavior of the transformed amino acid residue.

51

Peptide Mapping


It is a powerful technique for confirming the primary structure of a
therapeutic protein. This also confirms whether the genetic construct
has been properly preserved in the host organism (of the WCB).
Peptide mapping can qualitatively detect single amino acid
substitutions (caused by DNA mutation or a mistake during
transcription).


A protein of interest is digested (chemically or with a protease) into
discrete peptide fragments that are subsequently separated/mapped
(e.g., via chromatography or electrophoresis). A comparison is made
to a control peptide digest of a (1
o

or 2
o
) standard of that protein.


Before a protein sample undergoes digestion, its disulfide bonds
must be reduced and the sulfhydrals blocked. This helps eliminate
protein folding/tertiary structure to expose all peptide bonds to the
action of the chemical or protease that targets a specific side of one
or more specific amino acid residue(s) for hydrolytic digestion.


The choice of digestion agent depends on the particular protein with
its known native amino acid composition and sequence. See the next
page for “Commonly Used Digestion Agents.”


Most typically, a peptide digest mixture is loaded on a RP
-
HPLC and
eluted w/ a gradient of diminishing TFA in acetonitrile with 214 nm
detection.

52

Peptide Mapping
(cont’d)

Commonly Used Digestion Agents


Digestion Agent

Specificity

Chemicals

2
-
nitro
-
5
-
thiocyanobenzoic acid

Cys (N
-
terminal side)

O
-
Iodosobenzoic acid

Trp, Tyr (C
-
terminal side)

Cyanogen bromide

Met (C
-
terminal side)

Enzymes

Trypsin

Arg, Lys (C
-
terminal side)

Chymotrypsin

Phe, Trp, Tyr (C
-
terminal side)

Lysyl
-
C
-
endopeptidase

Lys (C
-
terminal side)

Glutamyl endopeptidase from
Stapholococus

aureus

strain V8

Glu, Asp (C
-
terminal side)

53

Circular Dichroism (CD)


CD measures the difference in absorption between left
-

and right
-

circularly
polarized light as a function of wavelength. When light passes through an
optically active chromophore, differential absorption results in light with an
electronic vector that is eliptical rather than circular. The secondary structure
of a protein causes an asymmetric environment, and CD can distinguish
between proteins with differing percentages of alpha
-
helix, beta
-
sheet, and
random coil secondary structure.


Also, a protein with tertiary structure (i.e., a high degree of three
-
dimensional
folding) has a CD spectrum.


When comparing a protein standard to a protein that may have had deleterious
conformational changes (as reflected in diminished bioactivity), CD can confirm
whether denaturation caused the lost activity. Amino acid covalent
modifications, degradations, or even substitutions caused by a mutation or by a
transcription error can rob a protein of its bioactivity while not affecting its
secondary or tertiary structure (and thus its CD spectrum).

54

MALDITOF Mass Spectrometry

(Matrix
-
Assisted Laser
-

Desorption Time
-
of
-
Flight)


A sample is heated in a vacuum, and the gaseous
breakdown products are then analyzed using mass
spectrometry. The test spectrum generated is compared
to a database of spectra.








55


Restriction mapping involves digesting DNA with a series of restriction
enzymes (

endonucleases that cleave specifically recognized sequences of
DNA) and then separating the resultant DNA fragments by agarose gel
electrophoresis. The distance between restriction enzyme sites can be
determined by the patterns of fragments that are produced by the restriction
enzyme digestion.


In the Intron A process, restriction mapping sequences the human IFN gene and
50 basepair flanking regions using plasmid DNA. Every three years, MCB and
EOF cells (latter from a single fermentation batch) are DNA sequenced. During
WCB requalification, this restriction mapping is repeated.

-------------------------------------------------------------------------------------------------------------





It’s a qualitative or quantitative test of whole or fragmented DNA in a
sample, making use of the enzyme DNA polymerase. See the internet
printout from the site of Carolina Biological Company.









It’s a qualitative or quantitative test of whole or fragmented DNA in a
sample, making use of the enzyme DNA polymerase. See the internet
printout from the site of Carolina Biological Company.


Polymerase Chain Reaction (PCR)

Restriction Mapping DNA Sequencing

56

Training Effectiveness Worksheet;

Name: ________________________

1)

What type of natural biochemical does a gene encode? __________


2)

What holds both DNA double helices together and protein alpha helices
together? _____________ _______s


3a&b) Give at least 2 differences between a Biologic & a Pharmaceutical:

a)_______________________________________________________________,

b)_______________________________________________________________


4a&b) Name 1 advantage and 1 disadvantage of using E. coli fermentation


vs mammalian cell culture to make recombinant

-
楮瑥牦敲潮?

愩‱ 慤v慮t慧攠楳 i彟彟彟彟彟_彟彟彟彟彟_彟彟彟彟彟彟_彟彟彟彟彟

戩‱⁤楳慤v慮瑡g攠楳 i彟彟彟_彟_彟彟彟彟彟彟_彟彟彟彟彟_彟彟彟彟彟


㔩†⁗ 慴⁣ v慬敮琠扯湤 捡c
浩m
景牭 扥瑷敥e

-
楮瑥牦敲潮潮潭敲o


瑯 灲潤畣攠潬楧潭敲猿o 彟彟彟彟_彟_彟彟彟彟_








57

Training Effectiveness Worksheet

6)
Name either one specific form of electrophoresis
or


one HPLC method that helps detect aggregates/oligomers of IFN?
_________________________________


7)

What is the opposite of gradient elution in column chromatography?

________________ elution


8)
Give at least one advantage of a PEGylated therapeutic protein?

_______________________________________________________________


9)
In pH 8.2 Tris Buffer, what
overall

charge (+ or
-
) does Intron A have,
given that its isoelectric pH is ~6.2 ?
_______________


10)
For a therapeutic protein, is there ever one best stability
-
indicating
assay? _____









58

Bibliography

Process Validation in Manufacturing of Biopharmaceuticals
, ed. Rathore, A. S., Sofer, G.; 2005

Industrial Microbiology and Biotechnology

2
nd

ed., ed. Demain, A. L., Davies, J. E.; 1999

“[FDA] Guidance for Industry: Q5E Comparability of Biotechnological/Biological Products Subject to
Changes in Their Manufacturing Process,” June 2005

“[EMEA] Guideline on Comparability of Medicinal Products Containing Biotechnology
-
Derived Proteins as
Active Substance: Quality Issues,” December 2003

ICH Q5C, “Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological
Products,” November 1995

“Bacteriophage Therapy” Biotech Journal, March 2003

[EU Directive] Note for Guidance, “Production and Quality Control of Medicinal Products derived by
Recombinant DNA Technology,” July 1995

[EU Directive] Note for Guidance, “Production and Quality Control of Cytokine Products Derived by
Biotechnological Processes,” February 1990

ICH Q5C = [EU Directive] Note for Guidance, “Quality of Biotechnological Products: Stability Testing of
Biotechnological/Biological Products,” July 1996

ICH Q5B, “Quality of Biotechnological Products: Analysis of the Expression Construct in Cells Used for
the Production of rDNA Derived Protein Products,” November 1995

[FDA, CBER] Guidance for Industry, “For the Submission of CMC Information for a Therapeutic
Recombinant DNA
-
Derived Product or a Monoclonal Antibody Product for In Vivo Use,” August 1996

ICH Q6B, “Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological
Products,” March 1999

“Evaluation of a New Reagent for Covalent Attachment of Polyethylene Glycol to Proteins,” Biotechnology
and Applied Biochemistry 15, (1992), Zalipsky, S., Seltzer, R., Menon
-
Rudolph, S.






59

Bibliography

“PEGylation: An Innovative Approach for Protein Delivery,” Drug Delivery Technology, Vol 5, June 2005,
Patel, M, et al.

“The BioPharm International Guide to Fermentation and Cell Culture” 2
nd

Edition, March 2003

“U.S. Pharmacist, “Interferons: An Overview,” Al
-
Hasso, Shuhia; volume 26:06

[FDA] “Draft of Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals
(1993)”

[EU] GMPs, Annex 2: Manufacture of Biological Medicinal Products for Human Use”

[FDA] “Biotechnology Inspection Guide Reference Materials and Training Aids,” November 1991

ICH Q5D, “Derivation and Characterization of Cell Substrates Used for Production of
Biotechnological/Biological Products,” July 1997

[PDA] “Technical Report No. 42, Process Validation of Protein Manufacturing” October 2005

[PDA] “Technical Report No. 14, “Industry Perspective on the Validation of Column
-
Based Separation
Processes for the Purification of Proteins,” June 1992

[Brinny] Final Response to FDA 483s from Inspection of S
-
P Brinny 15
-
25 Aug
-
2005

The Biopharm Guide to Bioanalytical Methods, December 2001

Supplement to BioPharm International, Guide to Bioanalytical Advances, September 2005

Product Quality Research Institute, “Development of Scientifically Justifiable Thresholds for Leachables
and Extractables,” April 2002

1996 sBLA for New Isoform Conversion Process of Intron A

Isoform diagrams from Brinny’s Bart Cronin

Brinny’s final 2005 FDA 483 responses provided by Pat Nagle