Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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Pr oduct puriﬁcation

pr ocesses

ngela Maria Moraes, Leda dos Reis Castilho,

and Sonia Maria Alves Bueno

12.1 Introduction

The development of methods and techniques for the separation and

puriﬁcation of biological macromolecules such as proteins has been a

prerequisite for advances in bioscience and biotechnology during the last

ﬁve decades.

The recovery and puriﬁcation of a bioproduct is carried out to isolate it

from its production system (cell culture, plant or animal tissues), and to

obtain the required purity and formulation. Before establishing a strategy

for recovery and puriﬁcation, it is essential to collect all available informa-

tion related to the protein and to the medium where it is found. Usually,

not only the theoretical information, but also preliminary experiments are

needed. It is also noted that the feasibility of a process on a laboratory

scale does not guarantee its feasibility on an industrial scale.

With the use of high performance materials and automated instruments,

protein separation is becoming a more controllable process. However,

some problems persist even with the use of sophisticated instruments.

Many difﬁculties are still found in determining the optimal extraction and

puriﬁcation conditions, as well as in selecting suitable methods for detect-

ing the protein and quantifying its biological activity.

A general scheme of the recovery and puriﬁcation process for a

bioproduct is shown in Figure 12.1. Despite the lack of a universal

strategy, some general directions (listed below) should be followed when

establishing the puriﬁcation protocol:

(i) choose separation methods based on different physical principles;

(ii) choose methods based on physical properties related to the greatest

differences between products and impurities;

(iii) ﬁrst remove contaminants that are most dissimilar to the product

and/or those that are most abundant;

(iv) assign the most demanding process to the end of the sequence.

12.2 Basic considerations

Before developing an isolation and puriﬁcation process for a protein, some

considerations should be made regarding the ﬁnal application of the

product, the most suitable source material and the available information

about the product, including existing data on manipulation and quantiﬁca-

tion (Harris and Angal, 1989).

From these preliminary data, the objectives and performance criteria of

the puriﬁcation sequence can be deﬁned and, thus, the fundamental basis

for designing the most suitable strategy is established.

12.2.1 Final application of product

Usually protein puriﬁcation represents the route for obtaining the product

in a pure form, suitable for different applications such as: activity and

structural studies, studies of the structure–function relationship of the

protein, and its clinical or industrial applications. The purpose of the

application determines the required purity of the protein, the acceptable

Solid–liquid separation

(cell extraction)

Cell disruption

Solid–liquid separation

(removal of debris)

Solid–liquid separation

(cell removal)

Bioreactor

Primary isolation

Intermediate purification

Final purification

Formulation

and packing

Intracellular

products

Extracellular

products

Figure 12.1

General scheme of a bioproduct recovery and puriﬁcation process. The dotted

arrows indicate integrated processes (see Section 12.4.6).

296 Animal Cell Technology

loss of activity, and also, the acceptable time and cost of the puriﬁcation

process.

Therefore, for activity-related studies, relatively small amounts of pro-

tein are needed and high purity is certainly not crucial, provided that the

activity-interfering species are removed. Cost in this case has relatively

low relevance, but speed is important to minimize activity loss.

Structural studies, on the other hand, require large amounts of highly

puriﬁed protein. Cost and time are of secondary importance, except in

studies regarding structure–function relationship, for which maintenance

of biological activity is important and thus, the puriﬁcation speed is

important.

Puriﬁcation cost and time are most relevant for industrial scale produc-

tion. The required amounts and purity levels are determined based on the

ﬁnal application of the protein. For example, for therapeutic applications

high purity is crucial, but the required amounts are relatively small.

The amount of puriﬁed protein obtained depends not only on the

amount of the raw material used, but also on the yield of the process. At

every stage of the puriﬁcation process, some product loss occurs. There-

fore, to maximize the yield, a minimum number of stages should be used.

However, decreasing the number of stages can affect the ﬁnal purity of the

protein. Some puriﬁcation methods give a higher yield than others and

thus, a systematic selection should be made that is aimed at maximizing

the yield at each stage, but also insuring the required ﬁnal purity is

obtained.

The purity of the target protein can be expressed in terms of the

percentage of total protein, although other types of contaminants can also

be important. For enzymatic studies, 80–90% of purity is usually suitable,

not taking into account the activity-interfering contaminants, which

should be removed. For structural studies, the required purity should be

equal to or higher than 95%. For therapeutic applications, any type of

contaminants should be removed and purity higher than 99.9% is often

required.

Additional puriﬁcation steps lead not only to additional yield losses, but

also to an increase in processing time and cost. For research purposes, the

process scales are usually small and the cost of an additional step may not

be important. On the other hand, at an industrial scale, the introduction of

an additional step may make a puriﬁcation process uneconomic. Removal

of residual contaminants is, in most of the cases, signiﬁcantly more

difﬁcult than the earlier puriﬁcation steps and involves several additional

steps, reducing the ﬁnal yield and increasing the processing cost and time.

In most cases, except for structural studies such as sequencing, the

puriﬁed protein should be as native and as active as possible. Attempts to

minimize denaturation and proteolysis should be made, avoiding typically

harsh conditions.

12.2.2 Selection of the protein source

As discussed in the initial chapters of this book, genetically modiﬁed

animal cells are commonly used to obtain proteins for therapeutic,

prophylactic, or diagnostic applications. The problem is that obtaining

Produc t purification processes 297

bioproducts from animal cell cultures is limited by the low protein

concentration observed in these systems, making the puriﬁcation process

difﬁcult and expensive. On the other hand, animal cells can secrete

recombinant proteins into the extracellular medium, allowing easier and

cheaper puriﬁcation, as compared with heterologous proteins found as

insoluble intracellular aggregates in bacteria.

To facilitate the puriﬁcation, the target biomolecule can be modiﬁed by,

for instance, coupling to it a peptide segment that can be recognized and

captured by an afﬁnity ligand attached to a chromatographic matrix. After

puriﬁcation, the inserted fragment can be removed by chemical or enzy-

matic cleavage (Scheich et al., 2003).

The culture medium containing the bioproduct should be processed as

soon as possible, to avoid the possibility of degradation, for example, by

oxidation or the action of proteases. When immediate processing of

extracellular products is not possible, it is recommended to remove the

cells and freeze the material, provided the bioproduct is stable to freezing.

12.2.3 Protein properties and manipulation

Knowledge of the physical and chemical properties as well as the cellular

location of the target protein helps in designing the separation process.

Whether the protein is intra- or extracellular, soluble or insoluble in the

cytoplasm, bound to the cell membrane or located within the organelles,

will affect the selection of the separation method and the type of buffering

system. Proteins bound to the cell membrane require detergents or organic

solvents for their solubilization.

Apart from the general problems of solution handling, protein solutions

should be handled to prevent denaturation, aggregation, and degradation.

Adequate cleaning and sanitization of all equipment is essential in the

manipulation of such biomolecules.

12.3 Cell disruption

Bioproducts are usually secreted from animal cells in culture, and can be

puriﬁed after cell removal by solid–liquid separation techniques (see

Chapter 11). However, the product can sometimes be found within the

cell and this requires its extraction from the cellular mass, which contains

numerous molecular species that can have high viscosity and proteolytic

activity, which increases the difﬁculty of sample handling.

The extraction of an intracellular protein usually involves a compromise

between recovery and purity. Optimization of the extraction conditions

should maximize the release of the target protein, while minimizing the

contaminants, which may be difﬁcult to remove. For this, it is important

to determine the conditions under which denaturation or degradation

occurs.

There are several available methods for disrupting cells or tissues. The

operational conditions can be optimized through the systematic variation

of parameters such as medium composition, time, temperature, stirring

rate, and size and shape of the blades. Selection of a suitable procedure

298 Animal Cell Technology

demands preliminary tests in which aliquots of the sample are taken at

different time intervals and analyzed for product concentration and

activity.

The recovery of intracellular proteins involves distinct cell disruption

procedures, depending on the cell characteristics. For the processing of

animal cells, which do not have a cellular wall, mild and moderate

techniques are commonly used. Mild techniques include cell lysis by

enzymatic digestion, chemical solubilization or autolysis and the use of

manual homogenizers and grinders, whereas the moderate techniques

involve blade homogenizers and abrasive grinding.

When the protein is present within a cellular organelle, these methods

can still be suitable. However, they may be preceded by isolation of the

organelles. Sometimes, the protein has low solubility in the extraction

medium, and produces a particulate system requiring speciﬁc techniques.

These proteins can be extracted through thermal, chemical, or enzymatic

treatments, and in some cases detergents are needed for solubilization. In

any case, it is essential that a suitable solvent is selected for protein

extraction.

In all puriﬁcation steps it is recommended that the pH of the protein

solution is controlled, to avoid denaturation or deactivation of the target

biomolecule. It is recommended that an operational pH condition in which

the target protein presents maximum stability is determined, although this

value may be different from the optimal for protein extraction.

Several factors affect the selection of the buffer solution, such as: the

optimum pH; the buffer anionic or cationic species (which can interfere in

the subsequent puriﬁcation steps); the pH variation with ionic strength or

temperature; the buffer reactivity with the proteins in solution; the bio-

logical activity (e.g. phosphates can inhibit or activate a protein in bio-

logical reactions); the interaction of the buffer with other components; the

buffer permeation in biological membranes; the toxicity; the light absorp-

tion at 280 nm; the cost (especially if used in large-scale processes); and the

protein solubility.

In many cases the target protein is bound to membranes or particles or

is aggregated as a consequence of its hydrophobic characteristics. In these

cases, detergents and chaotropic agents can be used to weaken these

interactions during cell disruption and extraction steps. The detergent

performance is highly dependent on pH and temperature.

Intracellular proteins usually expose thiol groups, which can oxidize

during the puriﬁcation process. Such groups may be protected by reducing

agents such as 1,4-dithiothreitol (DTT), 1,4-dithioerythritol (DTE), and

mercaptoethanol. Usually, reagent concentrations of 10–25 nM are sufﬁ-

cient to protect the thiol groups without reducing the internal disulﬁde

bonds.

The presence of metal ions may be harmful to a biologically active

protein owing to two main factors; the increase in oxidation of the thiol

groups by molecular oxygen, and the complexation with speciﬁc groups.

The chelating agent most commonly used to complex these metal ions is

EDTA (ethylenediaminetetracetic acid) at a concentration of 10–25 nM.

However, in some cases, application of metal ions such as calcium and

magnesium may be needed to stabilize certain proteins.

Produc t purification processes 299

The presence of proteases is a threat for protein stability. A simple

strategy for protection against proteolytic degradation is based on the fast

handling of the sample and use of low temperatures. One additional

precaution is the addition of protease inhibitors, especially during disrup-

tion and extraction steps. In some cases, there is a need for a combination

of inhibitors. Sometimes, pH adjustment is effective in inactivating pro-

teases without affecting the stability of the protein product.

Despite being more critical for intracellular products, protease action

may also occur in extracellular media. For example, in the expression of a

baculovirus vector, which is a lytic system, proteases are often expressed

and secreted during recombinant protein production, and the problem

may assume a critical level. A similar problem occurs in serum-free culture

systems. The absence of proteins such as albumin and macroglobulin

reduces the protection against proteolysis. In these cases, addition of an

antiproteolytic agent to the culture medium is recommended.

In order to avoid microbial growth in the protein solution during the

puriﬁcation process, buffer solutions should be routinely sterilized by

ﬁltration. Buffers such as phosphate, acetate, and carbonate at neutral pH

are highly susceptible to microbial growth. Buffers with pH values lower

than 3 or higher than 9 usually prevent bacterial growth, but can allow

fungal growth. To circumvent such problems, it is recommended to

proceed with the puriﬁcation steps as fast as possible, always using fresh

and ﬁltered buffers, storing solutions at 48C and, if possible, adding an

antimicrobial agent to the buffer solutions.

12.4 Protein puriﬁcation methods

After the extraction of the target biomolecule, puriﬁcation is the next step.

It is possible to select the entire sequence of puriﬁcation steps for isolating

a particular protein based on its properties, and on the fact that every

protein presents a unique combination of characteristics. A strategy should

be established to minimize the number of stages, leading to the maximum

protein yield, minimum cost, and the required purity for the product’s

ﬁnal application.

Table 12.1 shows the most commonly used puriﬁcation techniques and

related protein properties to provide separation. Every technique should

be evaluated with regard to different parameters such as:

(i) the capacity, i.e. the sample amount that can be handled;

(ii) the resolution, i.e. how efﬁciently proteins are separated from each

other;

(iii) the yield;

(iv) the cost.

A balance should be made between process parameters and performance

expectation of each puriﬁcation stage. In the early stages, capacity and cost

are important parameters, whereas in the ﬁnal stages, high resolution is

more relevant.

The sequence of stages in a puriﬁcation process usually starts with low

resolution steps such as precipitation and liquid–liquid extraction, which

300 Animal Cell Technology

are low cost and allow the processing of large amounts of material and

high total protein concentrations. In the intermediate steps, a combination

of chromatographic techniques is usually employed, followed by a ﬁnal

puriﬁcation step focusing on the removal of product-related impurities

(e.g. aggregates or isoforms) and/or those existing at very low concentra-

tions. In this ﬁnal stage, afﬁnity chromatography and molecular exclusion

are very usual processes.

The currently most relevant separation and puriﬁcation techniques for

processing biomolecules from animal cell cultures are discussed in the

sections that follow. They are classiﬁed according to the protein character-

istic on which the separation is based, such as solubility, molar mass,

electrical charge, adsorption properties, and biological afﬁnity for ligands.

12.4.1 Separation processes based on solubility

Protein precipitation

Precipitation is used as a separation step during the early stages of a

puriﬁcation process, usually followed by chromatographic separations,

and also as a concentration method prior to an analysis or to a subsequent

puriﬁcation step.

The solubility of a protein in an aqueous solution relies on the distribu-

tion of the hydrophilic and hydrophobic groups on its surface. Protein

precipitates are formed by the aggregation of protein molecules caused by

changes in pH, ionic strength, or solvent dielectric properties, as well as by

the addition of a miscible organic solvent, other inert solvents, and

Table 12.1 Characteristics of typical puriﬁcation methods: properties on which separation is

based and their performance parameters

Principle of

separation

Technique Capacity Yield Resolution Cost

Solubility Liquid–liquid extraction High High Low Low

Fractional precipitation High High Low Low

Size Microﬁltration, ultraﬁltration, dialysis High Medium Low Low

Molecular exclusion chromatography Medium-low High Medium-low Medium

Electrical SDS-PAGE Very low High High Medium

charge Electrofocusing Very low High High Medium

Ion exchange chromatography Medium Medium Medium Medium

Speciﬁc

interaction

with ligands

Afﬁnity chromatography Medium-low Low Very high High

Surface Reverse phase chromatography Medium Medium High High

hydro-

phobicity

Hydrophobic interaction

chromatography

Medium Medium Medium Medium

Adapted from Wheelwright, 1991.

SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Produc t purification processes 301

polymers. Precipitation is based on the fact that proteins are electrolytes

of high molar mass. The method is suitable for fractionation of protein

mixtures, since each protein is composed of different amino acids that

deﬁne its behavior. The precipitates can be recovered by ﬁltration or

centrifugation, and the remaining traces of both the precipitating agent

and the redissolved precipitate can be removed by dialysis, diaﬁltration, or

desalinization in a molecular exclusion column. Detailed reviews on the

topic are given by Scopes (1994) and Kumar et al. (2003).

The most widely used precipitation agents for protein puriﬁcation are

ammonium sulfate and polyethylene glycol (PEG), which are discussed

below.

Salting-in and salting-out

Neutral salts have a pronounced effect on the solubility of proteins,

especially if they are globular. At low concentration, salts increase the

solubility of many proteins in a phenomenon known as salting-in. This

solubilization is a function of the solvent ionic strength, which depends on

the concentration and on the electrical charge of the cations and anions

that constitute the salt. These effects are caused by changes in the ioniza-

tion of dissociating groups of the protein.

However, as the ionic strength is gradually increased, protein solubility

begins to decrease. At high ionic strengths, the protein may be precipitated

from the solution. This effect is known as salting-out. This phenomenon

occurs when a salt is added to a system and the ions are solvated by water

molecules displaced from the protein. Therefore, at high salt concentration

the hydrophobic protein groups are exposed, enabling the formation of

hydrophobic interactions between the protein molecules, thus causing

aggregation. Larger proteins or those with more available hydrophobic

regions will aggregate faster and this can be the basis of fractionation. The

effectiveness of the salt is largely dependent on the anion, multivalent

anions being the most effective. The order of selectivity may be described

by the Hofmeister series (Ersson et al., 1998):

Anions: PO

3–

. SO

2–

. CH

COO

–

. Cl

–

. Br

–

. NO

–

. ClO

–

. I

–

. SCN

–

Cations: NH

. K

. Na

. C(NH

)

Ammonium sulfate is frequently used for protein precipitation, due to

its high solubility in water, which allows the solvent to reach very high

ionic strengths. Usually, protein precipitates produced through salting-out

do not denature and the protein activity is easily recovered. Addition of

neutral salts can also protect proteins against proteolysis and microbial

contamination.

Precipitation with polymers

Precipitation of proteins by the addition of anionic or cationic polymers

occurs through the neutralization of charges and formation of high molar

mass aggregates. Polymers commonly used for this purpose are carb-

oxymethylcellulose and chitosan. Neutral polymers, such as PEG and

methylcellulose can also be employed. In such cases, the mechanism is

302 Animal Cell Technology

similar to precipitation with organic solvents, that is, by change in the

dielectric constant of the medium, although at lower concentrations

(around 20%). Higher concentrations result in viscous solutions, making

the recovery of the precipitate difﬁcult. Polymers used in protein precipi-

tation are expected to:

(i) present reversible solubility response to medium changes in pH and

temperature;

(ii) form compact precipitates;

(iii) have low cost; and

(iv) be inert and nontoxic.

The polymer must have a high molar mass. PEG at a range of 6–20 kDa

is commonly used (Ersson et al., 1998). Precipitation with PEG was used,

for instance, by Deml et al. (1999) as one of the puriﬁcation steps for the

isolation of hepatitis B virus surface antigen (HBsAg) from the insect cell

culture supernatant of genetically modiﬁed DS-2 (Drosophila melanoga-

ster Schneider-2) cells.

Liquid–liquid extraction

Extraction is based on the transfer of a solute from one phase to another,

according to its partition properties between two immiscible phases. In

biotechnological processes, the most widely used extraction is liquid–

liquid involving the partitioning of biomolecules, organelles or cells be-

tween the phases (Harrison et al., 2003).

In a bioprocess, two types of liquid–liquid systems are used. When

molecules are stable in organic solvents, as occurs with low molar mass

molecules (such as antibiotics), the extracting liquid can be an organic

solvent immiscible with the aqueous phase originally containing the target

biomolecule. However, in the case of proteins with a tendency to denature

or degrade in the presence of organic solvents, aqueous two-phase systems

are used (Franco et al., 2005). Such systems are usually formed by a

polymer phase and a salt solution, or by two polymers, both soluble in

water but immiscible with each other. The most commonly used systems

are formed by PEG in one phase and a salt or other polymer (such as

dextran) in the other. Under ideal conditions, the protein of interest is

recovered in the upper phase (usually PEG) and the contaminants and

particles (cells, fragments of cells, and organelles) in the lower phase

(Ersson et al., 1998).

The partitioning coefﬁcient K (equation 1) describes the distribution of

a solute, in equilibrium, between two liquid phases. This coefﬁcient is a

function of several factors, such as the molecular size, pH, temperature,

concentration, and type of components in both phases. In Equation (1), y

and x are the solute concentrations in the extract and in the original

solution, respectively.

K ¼

(1)

Ideally, K should be maximized with a minimum volume of the extract-

ing phase. Partition coefﬁcients close to 1 implicate in large solvent

Product purification processes 303