Thida Chanyachukul

Introduction

The development of techniques and methods for the separation and

purification of product molecules have been an important prerequisite for many of the
advancements made in pharmaceutical manufacture. Improvements in materials and
utilization of modern instruments have made separation more predictable and
controllable. However, not all problems in purification system are solved by the
acquisition of sophisticated laboratory equipment and column packing, which give
high selectivity and efficiency. Difficulties still remain in finding optimum conditions
and choosing suitable methods. Our interest is focused on improvement of AIDS
drugs (such as protease inhibitors, HIV vaccine and IL-2), and then great care should
be taken in the selection of suitable techniques to obtain each efficient product.

I will overview first the different purification techniques and their principles of
operation. In the subsequent part some more detail in each purification work on all
three types of the drugs will be discussed. After that I will conclude with ideal and
suitable limits in purity degree.

Purification strategies

Today it is possible to base the purification of a pharmaceutical product on the

knowledge of its molecular properties, structural as well as function. Suggestions on
how to solve a purification problem can best be made if data on its structure and
function, including particular structural details, is available. Conversely, results from
the application of a particular purification method can often be interpreted in terms of
molecular properties, often in a detailed manner.

Production can be divided into “upstream” and “downstream” processing.

As defined here, upstream processing refers to the initial fermentation process, which
results in the initial generation of product. Downstream processing refers to the actual
purification of the product and generation of finished product format (i.e. filling into
its final product containers, freeze-drying if a dried product format is required)
followed by sealing of the final product containers. Subsequent labeling and
packaging steps represents the very final steps of finished product manufacture. 1The
quality and biosafety of a pharmaceutical is to a great extent, dependent on the
extraction procedures used to manufacture the purified product. On the one hand,
down stream processing has to ensure an effective and economic isolation of the
desired product from the culture broth or cellular material obtained during the cell
culture process. However, components that would contaminate the final product must
be reliably separated. Different types of components that should not be present in the
final product formulation have to be removed during these steps. These can be
classified into two groups as in table 1.

The removal of medium components and proteinaceous impurities is an

integral part of product isolation. Nevertheless, the removal of medium supplements,
such as antibiotics or cytotoxic substances must be guaranteed by the purification
strategy and appropriate tests have to be established to validate their efficiency.5

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Table1:

Categories of components that should not be present in the final

Pharmaceutical product
Components present due to process conditions :
- Host-cell-derived components : DNA and proteins
- Process-derived components : lipid, proteins, antifoam agents, antibiotics,
substances used for product isolation, cleansing
agents

Components present due to contamination :

- Adventitious agent : viruses, virus-like particles, bacteria, fungi,
mycoplasmas, transmissible spongiform encephalopathy agents

Because, for practical reasons, it is not possible to manufacture a 100% pure

product, acceptable concentration levels for the presence of impurities in the final
product formulation have been defined. For example, WHO defined the maximal
acceptable amount of DNA to be 100 pg per single dose of a biotechnological derived
protein drug.4

Working on reaction volumes of between 2-5 liters usually requires the use of
pilot-plant equipment. Many purification techniques are not practical when dealing
with large quantities of material. In general the most useful methods of purification
that can be applied to large quantities of material are recrystallization for solids,
and distillation or steam distillation for liquids. Chromatography should be avoided
if possible since it becomes a very expensive operation on large scale, but if it is
necessary, then medium pressure liquid chromatography (mplc) is the method of
choice.

Principle of purification strategies

Basic concept of Filtration6

The usual technique is to pass the solution, cold or hot, through a fluted filter
paper in a conical glass funnel. It is used to remove particulate impurities from liquids
or to collect insoluble or crystalline solids, which crystallize, from solution. When the
solid particles are too fine to be collected on a filter funnel because filtration is
extremely slow, separation by centrifugation should be used.

Basic concept of Gel filtration2

The separation may simply be regarded as due to the different amount of time
different solute stay within the liquid phase that is entrapped by the matrix. This time
is of course related to the fraction of the pores that is accessible to the solute. The
interpretation of this fraction in terms of pore dimension and gel structure, together

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with various expressions for solute size, results in slightly different equations for
relating the distribution coefficient to the size of the solute.

Ultrafiltration separates molecules

on the basis of size and shape9 Filtration devices used in industry 7

Even though gel filtration is an uncomplicated and straightforward technique,

there are some points worth consideration before starting the experimental work. The
actual sample may require a special pH, solvent, additives or pretreatment to yield a
true solution. The next step is to select the gel that will cope with the chosen solvent
and pH and that has a suitable separation range. Possible adsorption properties of the
gel must also be considered. The nature of the separation and the sample may put
demands on such parameters as resolution, separation time and sample load which in
turn are partly dependent on selected gel. The choice of column dimensions and the
packing efficiency of the column however, also affect these parameters. Obviously,
for different separation problems, such as desalting, preparative purifications or
analytical separations different requirements should be stressed. Economic factors and
the possibilities of scaling up may also are important.

Basic concept of chromatography2

The term chromatography refers to a group of separation techniques, which

are characterized by a distribution of the molecules to be separated between two
phases, one stationary and the other mobile. Molecules with a high tendency to stay in
the stationary phase will move through the system at a lower velocity than will those
which favor the mobile phase. The shape, rigidity and particle size distribution profile
of the gel matrix are important parameters, which govern the performance of the
stationary phase. Chromatographic techniques for analysis and purification of reaction
products are probably the most universally important of all the skills in which an
organic chemist requires expertise. Various chromatographic techniques which may
be used to separate proteins from each other. The basis upon which separation is
achieved is also listed in table 22

Table 2 :

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Chromatographic technique Basis of separation

Ion exchange
Gel filtration
Hydrophobic interaction chromatography
Affinity chromatography
- Immunoaffinity chromatography
- Dye affinity chromatography
Metal chelate chromatography
Hydroxyappetite chromatography
Differences in protein surface charge
at a given pH
Differences in size/shape of different
proteins
Differences in the size and extent of
hydrophobic patches on the surface
of proteins
Ability of a protein to bind in a bio-
specific manner to a Chosen
immobilized ligand
Immobilized antibodies may be used
as affinity absorbents for the
antigen that stimulated their
production
Purification of proteins which displays
ability to interact tightly with
selected dyes
Ability of certain proteins to complex
with zinc and copper
Mechanism not fully understood.
Involve ability of some proteins to
Bind to calcium and phosphorus
on the
surface of hydroxyappetite crystals

A variety of different chromatographic techniques are available which separate

molecules from each other on the basis of differences in various physiochemical
characteristics. In general, a combination of two to four different chromatographic
techniques is employed in a typical downstream processing procedure. Gel filtration
and Ion exchange chromatography are the among the most common. Affinity
chromatography is employed wherever possible as its high biospecificity facilitates
the achievement of a very high degree of purification.

There are often times when quite difficult separations need to be performed on
a fairly large scale. Even if the mixture will separate by flash chromatography, it may
be prohibitively expensive, especially if it is a step, which needs to be carried out
routinely. This is one occasion when mplc is very useful. The resolution of mplc is
somewhat better than flash chromatography, but another important feature is that the
columns are reused many times, thus avoiding the expense of throwing away large
quantities of silica.8

The mplc system is essentially a simplified and much cheaper version of an

hplc set-up. At the heart of the system is any type of pump, which will operate at 100
psi, with a controllable flow rate of up to 100 ml/min. The sample introduced into the
system via an injection valve, but this is a much simpler and less expensive valve than
that found in an hplc system. There is a choice of silica available for mplc, but for
most purposes ordinary “flash” (40-60 um) silica is used. A column packed with 15-

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20 um silica is more effective for difficult separations but there is a lot to be said for
sticking for only one, or perhaps two types of silica and becoming familiar with their
characteristics. In most cases getting a good separation is simply a matter of choosing
the appropriate size of column and the correct solvent. Other solid phases can also be
used on a mplc system alumina, ion exchange resin, Sephadex.8

mplc (medium pressure liquid

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chromatography)

It is very useful to have the mplc system linked to a fraction collector. An UV

or refractive index detector can also be incorporated into the system, or TLC can
simply analyze the fractions, as for flash chromatography. If the conditions are kept
constant the results from mplc are very consistent, so if you wish to repeat separation
of the same mixture, it is very easy to predict which fractions will contain each
component. And to define which fraction contains Ritonavir, you should have to
analyze them again by affinity chromatography or mass spectroscopy.

Basic concept of Ion exchange chromatography (IEC)2

Protein charge properties are used for fractionation purposes in several

techniques. Thus, electrophoresis depends on electrophoretic mobility, which in turn
is a function of charge density, while isoelectric focusing separates proteins according
to their isoelectric points, i.e. the pH of zero net charge. IEC not only depends on
charge density, but also on the distribution of charges on the protein surface, i.e.
charge anisotropy. Similarly separation in chromatofocusing reflects not only the pI of
a protein, but also on the shape of its titration curve in the vicinity of the pI. The
reason for the popularity of IEC is its versatility, its high resolving power, its high
capacity and its straightforward basic principle.

Principle of ion exchange

chromatography2

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The basis for the IEC process is the competitive binding of ions of one kind, in
our case proteins, for ions of another kind, for example other proteins or salt ions of
the same charge, to an oppositely charged chromatographic medium, the ion
exchanger. The interaction between the proteins and the ion exchanger depends on
several factors: net charge and surface charge distribution of the protein; the ionic
strength and the nature of the particular ions in the solvent; pH, or strictly speaking in
the proton activity; and other additives to the solvent, such as organic solvents etc. It
is clear that the more highly charged a protein is, the more strongly it will bind to a
given, oppositely charged ion exchanger. Similarly, more highly charged ion
exchangers, i.e. those with a higher degree of substitution with charged groups, bind
proteins more effectively than weakly charged ones. Conditions, for example pH,
which alter the effective charge on either the protein or the ion exchanger will affect
their interaction and may be used to influence the ion exchange process. The term
strong and weak ion exchanger derive from the pKa’s of their charged groups and do
not say anything about the strength with which they bind proteins. At pH’s far from
the pKa, binding will be equally strong to either a weak or a strong ion exchanger.

IEC is eminently well suited to purification of either large quantity of proteins,

because of its high loading capacity or purification from large volumes of sample,
because of its ability to concentrate proteins from dilute solution. Although process
scale IEC is routinely carried out with column as large as 170 liters, even a standard
laboratory column with a volume of 500 ml is usually sufficient to concentrate and
purify 5 grams of protein from as much as 10 liters of a dilute sample. In practical
terms, large scale IEC differs mainly in the increased use of step, as opposed to
continuous gradient, elution procedures. Otherwise exactly the same general
procedures and principles are used as in a smaller scale. It is advisable to obtain this
increase in volume by choosing a column with a larger diameter to avoid excessively
long separation times. The multiple inlet design is suitable. The choice of buffer
systems for large scale work will naturally be influenced by cost considerations, but
the needs for adequate buffering capacity remain and should not be sacrificed for
small cost savings.

Basic concept of affinity chromatography2

Affinity chromatography owes its name to the exploitation of these various

biological affinities for adsorption to a solid phase. One of the members of the pair in
the interaction, the ligand, is immobilized on the solid phase, while the other, the
counterligand (most often a protein), is adsorbed from the extract that is passing the
chromatographic column as you can see in table 3. A typical separation by affinity
chromatography consists of four stages: adsorption, washing, elution and column
regeneration.

In many cases affinity chromatography is a very powerful method. This is

particularly true when the product of interest is a minor component of a complex
mixture. A property that needs special consideration is the association strength,
between ligand and counterligand. If it is too weak there will be no adsorption, if it is
too strong it will be difficult to elute the product adsorbed. It is always important to
find conditions, such as pH, salt concentration or inclusion of, e.g., detergent or other

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substances, that promote the dissociation of the complex without destroying the active
compound at the same time.

Table 3 :

Ligand Counterligand

Antibody antigen, virus, cell

Enzyme substrate analogue, inhibitor, co-factor
Lectin polysaccharide, glycoprotein, cell surface receptor, membrane
protein, cell
Nucleic acid nucleic acid-binding protein (enzyme or histone)
Hormone, vitamin receptor, carrier protein
Sugar lectin, enzyme or other sugar binding protein

Basic concept of distillation 6, 7

The distillation process involves boiling a

liquid, condensing the vapors, and directing
the resulting liquid into another vessel. The
liquid is heated to its boiling point, at which
point its vapor pressure equals the atmospheric
pressure. When the apparatus is at equilibrium
and the distillation is occuring.7 Almost
without exception, this method can be assumed
to be suitable for all organic liquids and most
of the low-melting organic solids. The
efficiency of a distillation apparatus used for
purification of liquids depends on the
difference in boiling points of the pure
material and its impurities.6

The conventional apparatus for simple distillation 8

Basic concept of Extraction7

Distillation is somewhat wasteful process. There is always material of

intermediate boiling point that represents a process loss. Extraction tends to be more
quantitative and requires a long time because of tendency for emulsification.
Laboratory studies to improve the speed of separation should be carried out. Solvents
used may have to be changed or temperature adjusted, or a preliminary purification
may have to be carried out by another method.

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Basic concept of crystallization or recrystallization

The most commonly used procedure for the purification of a solid material by
crystallization from a solution involves the following steps: 6
(a) The impure material is dissolved in a suitable solvent, by shaking or vigorous
stirring, at or near the boiling point, to form a near-saturated solution.
(b) The hot solution is filtered to remove any insoluble particles.
(c) It is then allowed to cool so that the dissolved substance crystallizes out. The rate
of cooling has to be controlled to obtain the crystal size desired. Fast cooling will
cause the generation of many nuclei, producing a large number of small crystals
rather than the growth in size of those already formed. Also, fast cooling will
cause inclusion of by-products in the crystals.7
(d) The crystals are separated from the mother liquor, either by centrifuging or by
filtering, under suction, etc. Usually, centrifuging is much to be preferred because
of the much greater ease and efficiency of separating crystals and mother liquor,
and also because of the saving of time and effort, particularly when very small
crystals are formed or when there is entrainment of solvent.7
(e) They are washed free of mother liquor with a little fresh cold solvent, then dried.

Medium to large scale crystallization

(> 1 g) a set-up along the line 8

Crystallization for purification should be carried out only as a last step. The
crystallization process gives a yield of about 90% at best. A 10% yield loss is quite
high. Of course, the material in the mother liquor can be purified with additional
processing which are much more quantitative such as extraction or chromatography. 7

Plant-scale purification of vaccine and IL-2 protein

During the cultivation process, the cells either secrete the desired protein into
the culture medium or the product accumulates in the cells. In either case, the first
step of the purification procedure is the separation of cells and cell debris. This is
achieved by centrifugation or microfiltration techniques. The efficacy of this step is
influenced by the viability of the cells and by the medium composition. The product-
containing fraction (either by culture broth or a crude cell extract) is then concentrated
by ultrafiltration or diafiltration, precipitation, high-affinity absorption or extraction
steps, which reduce the volume and prepare the material for the chromatographic
steps used for the final product purification.3 This diagram overviews a generalized

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 Thida Chanyachukul

downstream processing procedure employed to produce a finished-protein product

biopharmaceutical (vaccine or IL-2) 3

Fermentation

If protein is expressed if protein is expressed

Intracellularly extracellularly

Recovery of producer cells Removal of cells from media

(centrifugation or filtration) (centrifugation or filtration)

cellular disruption (homogenization)

removal of cellular debris

(centrifugation or filtration)

initial purification / concentration concentration of product-

containing(ultrafiltration / ion exchange or extracellular media
(ultrafiltration or
precipitation) precipitation)

chromatographic purification; usually 2-4 chromatographic step

Adjustment of potency and addition of excipients

sterile filtration and aseptic filling

freeze-drying

powder preparation liquid preparation

sealing of final product container, labeling and packing

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 Thida Chanyachukul

Plant-scale purification of Ritonavir

silica gel
+
chromatography
2-aminoaldehyde diols (white solid) bromoacetate (white solid)

precipitation
/ filtration

silica gel

chromatography filtration
diamine (white solid) compound X (white solid) epoxide (white solid)

distillation

silica gel

chromatography
resin compound compound XXXIIIa Ritonavir

Hydrophobic distillation
Interaction chromatography

Affinity chromatography

filtration

RITONAVIR
(final product)
CRYSTALLIZATION

LABELLING AND PACKAGING

The purification steps presented in the above plant scale synthesis are designed
in a modulate fashion, but the full processes are listed in the section of designing plant
(Kanathip’s report). In the full processes, informations from the patent 10 number
5846987 and simulation of superpro program are documented. In general, however,
industry tends to avoid hydrophobic and affinity chromatography even they are

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 Thida Chanyachukul

conceptually the best method to specify and separate Ritonavir from other organic
molecules which has the same molecular property. (Affinity chromatography will use
protease enzyme to coat in the column, then pass the solution through, if it contains
Ritonavir after eluting you will get it and use mass spectroscopy to define again, MW
is approximately 720). Because both are very expensive and complicated, it is not
practical in making protease enzyme in the plant scale level.

To elute out the product from chromatography, we adjust pH, change ionic
strength (isocratic or gradient), use another more potent hydrophobic interaction or
other potent substrate analogue.

Crystallization may be repeated until the substance has a constant melting

point or absorption spectrum, and a substance may be redistillated in a fractionating
column until it distils repeatedly within a narrow, specified, temperature range.

Figure 1 shows result of the simulation on Superpro designer for base case of
308 kg/batch. Noted that the reaction 1, 2, 4, 5 and 7 contribute to less than 10% but
the reaction 3, 6, 8 and 9 contribute to more than 90% of total equipment cost for
purification in base case as seen in Figure 1.

Base case : 308 kg/batch

Total equipment cost : 16,562 M$
Purification equipment cost : 7,505 M$

Fig1 : Purification cost based on each reaction

rxn9 rxn1 rxn2

rxn8
7% 1% 1%
rxn7 8%
3% rxn3
42%
rxn5 rxn4
rxn6 2%
1%
35%

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 Thida Chanyachukul

Sensitivity analysis on the plant throughput (kg/batch) (Figure 2) shows that

reaction 3 and 6 dramatically increase in the equipment cost for purification when the
throughput increases. Whereas the reaction 8 and 9 are not too much sensitive to the
throughput.

Fig. 2 : Sensitivity analysis

total purification equipment cost ($US)

18000000
16000000

14000000
12000000 77 kg/batch
154 kg/batch
10000000
308 kg/batch
8000000
618 kg/batch
6000000 928 kg/batch
4000000

2000000
0
rxn3 rxn6 rxn8 rxn9
reaction

When we compare the equipment cost for purification to the total capital cost
you can obviously see from Figure 3 that when the throughput increases, the
equipment cost for purification and total fixed cost also increase. But the rate of
increase of both costs is not the same, as the total fixed cost is increasing sharper than
purification cost suggesting that the higher throughput, the less equipment cost for
purification you have to invest compared to the total equipment cost.

Fig 3 : Cost analysis based on different yields

70000000

60000000

50000000

40000000

30000000

20000000

10000000

0
77 kg/batch 154 kg/batch 308 kg/batch 618kg/batch 928 kg/batch

purification total fixed cost

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 Thida Chanyachukul

Conclusion

Purity is a matter of degree. All commercial available chemical substances are

in some measure impure during manufacture. The important question, then, is not
whether a substance is pure but whether a particular sample is sufficient pure for some
intended purpose. By suitable manipulations it is often possible to reduce levels of
impurities to acceptable limits, but absolute purity is an ideal which can never be
shown to be attained. When setting out to purify a chemical, it is desirable that the
starting material should be of the grade commercially available. In general, at least
two different methods should be used in order to ensure maximum purification.

Things that should be looked into more detail are the time necessary to carry
out each purification step and the variation of product yield. For example, the cost of
operating the plant will go up if each step takes a long time. Reaction 1, 2, 4, 5 and 7
contribute to less than 10% whereas reaction 3, 6, 8 and 9 contribute to more than
90% of total equipment cost for purification. Sensitivity analysis states that reaction 3
and 6 dramatically increase in total equipment cost for purification when throughput
increases. But the reaction 8 and 9 are not too much sensitive when throughput
increase. In case of different throughput , it can be suggested that the more you
produce, the less you invest for purification operation.

The decision to market the product in liquid or powder form must be

determined experimentally, as there is no way to predict the outcome for any
particular material. Some may remain stable for month- or even year- in solution,
particularly if stabilizing excipients are added and the solution is refrigerated. Other,
particularly when purified, may retain biochemical activity for only a matter of hours
or days when in aqueous solution.

References

1. Berthold, W. and Walter, J. Protein purification: aspects of processes for

pharmaceutical products. Biologicals 1994;22:135-150.

2. Janson JC. And Ryden L. Protein purification; principles, high-resolution

methods, and application: VCH publishers, Inc. 1989.

3. Hesse F. and Wagner R. Developments and improvements in the manufacturing of

human therapeutics with mammalian cell cultures. Trends in Biotechnology.
2000 ;18(4):173-180

4. WHO study group. Acceptability of cell substrates for production of biologicals.

WHO technical report series; 1987: 747, 1-29.

5. Walter, J and Allgaier, H. Validation of downstream processes. In: Mammalian

Cell Biotechnology in Protein Product.1997; 453-48.

6. Perrin DD., Armarego WLF. and Perrin DR. Purification of laboratory Chemicals.
2nd ed. : Pergamon Press. 1980.

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7. White HL. Introduction to industrial chemistry : John Wiley & Sons, Inc. 1986.

8. Leonard J., Lygo B. and Procter G. Advanced practical organic chemistry. 2 nd ed.
Blackie academic & Professional. 1996.

9. Walsh G. Biopharmaceuticals : Biochemistry and Biotechnology. John Wiley &

Sons, Ltd. 1998.

10. Kampf DJ. Patent number 5846987. 8 Dec. 1998.

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