Downstream Processing

INTRACELLULAR PRODUCTS

Cell Harvesting. The first purification step for intracellular products is cell harvesting.
Removal of the extracellular liquid is in agreement with the first general heuristic Remove the
most plentiful impurities first.
As seen in Figure, centrifugation and membrane filtration (both micro- and ultrafiltration) are the
only techniques used for large-scale cell harvesting.

Centrifugation has advantages for large and dense microorganims (diameter > 2 m and density >
1.03 g/cm3). For instance, centrifugation is very efficient for harvesting yeast. For smaller
microorganisms, various coagulation techniques can be used to increase the size of the settling
particles.

Membrane filtration has advantages for harvesting small and light cells. Another advantage of
membrane filtration is in product recovery. Cell loss during centrifugation is typically 1 to 5%.
However, with membrane filtration, essentially all cells are recovered unless there is cell disruption
(lysis) or ripped membranes.

Cell Disruption. This is usually the second step for intracellular products. Its purpose is to break
open the host cells and release the intracellular product. Disruption of bacteria and yeast is carried
out either by high pressure homogenizers or bead mills. For large capacities (several m3/h) only
high pressure homogenizers are practical. Osmotic shock is often used for release of periplasmic
products that accumulate between the cell membrane and the cell wall.

Prior to disruption the concentrate is often diluted (by 5-10%) with a lysis buffer to create
conditions that minimize product denaturation upon release from the cell. For hard-to-disrupt
microorganisms, multiple homogenizer passes at 500-1000 bar are required. Multiple passes are
also required if the product forms inclusion bodies. This allows the inclusion bodies to be released,
and also breaks the cell debris into very small particles, which facilitates the separation of inclusion
bodies from cell debris further downstream. Some product protein degradation occurs during cell
disruption due to high shear and oxidation.

Removal of Cell Debris. The cell debris that is generated by cell disruption is usually removed by
centrifugation or microfiltration. Other options include rotary vacuum filtration, press filtration,
depth filtration, extraction, and expanded bed adsorption (EBA) chromatography.

Soluble Product. When the product is soluble, it is recovered during cell debris removal either in
the light phase of a centrifuge or in the permeate stream of a filter. Centrifuges efficiently separate
only fairly large particles of cell debris (greater than 0.5 m Stokes diameter). Therefore, when a
centrifuge is used for cell debris removal, a polishing filtration step must follow to remove small
debris particles which might otherwise cause severe problems in processes downstream such as
chromatography. Various types of filters (e.g., depth, press, candle, rotary vacuum, membrane
microfilters, etc.) can be used for polishing. Alternatively, these filters can be used for cell debris
removal without a centrifugation step preceding them. It is very difficult to predict a priori which
filter performs best for a specific product. When microfilters are used for cell debris removal, some
degree of diafiltration is required to achieve an acceptable product recovery yield.

I nsoluble Product. When the product is insoluble and forms inclusion bodies, it must first be
separated from the cell debris particles, then dissolved and refolded (see the insulin example) .
Fortunately, inclusion bodies usually have a large diameter (0.3 1.0 m) and high density (1.3
1.5 g/cm3).
The inclusion bodies are recovered in the heavy phase of the centrifuge while most cell debris
particles remain in the light phase. The heavy phase is usually resuspended and recentrifuged 2-3
times to reach a high degree of inclusion-body purity. Resuspension in a solution of a detergent
and/or a low concentration of a chaotropic agent is often practiced to facilitate the removal of other
contaminants. The pH and the ionic strength of the solution are adjusted to reduce the
hydrophobicity of the cell debris particles and enhance their removal in the light phase. Final
product purity exceeding 70% is quite common.

Product Extraction / Adsorption. Product separation from cell debris can also be carried out by
extraction and/or adsorption. Organic solvents are commonly used as extractants for low molecular
weight products, such as various antibiotics. Aqueous two-phase systems have found applications
for recovery of proteins. The criteria for extractant selection are: the partition coefficient of the
product should be higher than the partition coefficient of the contaminants; the extractant should not
degrade the product; it should not be expensive; and it should be easy to recover or dispose of.

In addition, product separation from debris and simultaneous concentration can be achieved by
adsorptive techniques. Various types of adsorbents (e.g., ion exchange, reverse phase, affinity, etc.)
can be used. This type of purification requires the disrupted cells and product to be mixed in a
stirred tank with an adsorbent. A washing step, where most of the cell debris particles and
contaminants are washed out, follows product adsorption. More recently, expanded bed adsorption
(EBA) chromatography has shown promise for separating proteins from cel debris particles. The
feed is pumped upwards through an expanded bed.Target proteins are bound to the adsorbent while
cell debris and other contaminants pass through.
A washing step removes all weakly retained material. An elution step follows that releases and
further purifies the product.


EXTRACELLULAR PRODUCTS

Biomass Removal. In agreement with the second generic heuristic, remove the easiest-to remove
impurities first, biomass removal is usually the first step of downstream processing of extracellular
products. This step can be accomplished by using one (or more) of the following unit operations:
rotary vacuum filtration, disk-stack or decanter centrifugation, press filtration, microfiltration,
ultrafiltration, flotation, etc. Since each unit operation has advantages and disadvantages for
different products and microorganisms, the selection of the best unit operation(s) for a given system
can be difficult.

Rotary vacuum filtration, especially with precoat, is the classical widely used method for removal
of mycelial organisms. Rotary vacuum filters can operate continuously for long periods of time. In
addition, the filtrate flux in these units is usually higher than 200 L/m2-h and may reach 1,000 L/
m2-h. The most important disadvantage of this type of unit is the problem with the disposal of the
mixture of filter-aid and biomass.

Filter-aid is added in equal or higher amounts than biomass. Stringent environmental laws have
made it costly to dispose of such solid materials. Therefore, if the disposal cost of filter aid is
relatively high where a new plant is going to be built, alternative unit operations should be
considered for biomass separation. However, if the disposal cost of filter aid is relatively low, a
rotary vacuum filter is a good choice. The citric acid process, which is described later in this
chapter, offers an example where rotary vacuum filtration is used for biomass removal.

Centrifugation. Disk-stack and decanter centrifuges are frequently used at large. Of the two, disk-
stack centrifuges operate at higher rotational speeds and remove smaller and lighter
microorganisms. However, with the use of coagulating agents, the decanter centrifuge performance
improves, and choosing between the two types becomes more difficult. It appears that the only
criterion being used to choose disk-stack as opposed to decanter is the ability to remove small, light
microorganisms. Centrifugation does not require filter aid, which is a significant advantage
compared to rotary vacuum filtration. In general, the centrifuge paste contains 40-60% v/v
extracellular liquid. In order to recover the product dissolved in that liquid, the paste is usually
washed and re-centrifuged.

Membrane filtration. With membrane filters (micro- and ultrafilters) the extracellular product
passes through the membrane while biomass and other particulate components remain in the
concentrate. Concentration is usually followed by diafiltration to increase the product recovery
yield. Membrane filters are used for biomass removal mainly in recovery of low molecular weight
products, such as antibiotics from mycelia. For high molecular weight products, applications are
limited to cases where the amount of solids is rather small as in cell culture.


Intermediate Recovery Stages

The primary recovery stages just described are followed by the intermediate stages, where the
product is concentrated and further purified. If the product is soluble, product concentration is
usually the first step. If the product is denatured and insoluble, first it is dissolved and refolded
and then it is concentrated and purified.

Product Concentration. After primary separation, the product is usually in a dilute solution.
Volume reduction by concentration is in agreement with heuristics 1 and 2. Common concentration
options include ultrafiltration, reverse osmosis, evaporation, adsorption, precipitation, extraction,
and distillation.

Ultrafiltration. is used extensively for protein solution concentration. The molecular weight cut-off
of the membrane is selected to retain the product while allowing undesirable impurities (mainly low
molecular weight solutes) to pass through the membrane. The low operating temperature and the
purification achieved along with concentration are some of the advantages of ultrafiltration over
evaporation. The typical operating trans-membrane pressure is 2-5 bar and the average flux is 20-50
L/m2-h.

Reverse Osmosis filters employ membranes with smaller pore sizes and are used for concentrating
medium to low molecular weight products (e.g., antibiotics, certain amino acids,
etc.).

Evaporation. Thin-film rotating evaporators can operate at relatively low temperatures (40-50 C)
under vacuum. These units compete in the market with ultrafiltration and reverse osmosis for
concentrating both low and high molecular weight compounds. One disadvantage of evaporation
compared to ultrafiltration is the lack of any purification during concentration.

Advantages include the ability to concentrate to a higher final solids concentration and the ability
to handle large throughputs.

Precipitation is often used for concentration and purification. Blood protein fractionation and citric
acid production constitute typical applications. Addition of salts, solvents, and polymers and
changes in pH, ionic strength, and temperature are commonly used to selectively precipitate
compounds of interest. Precipitation often follows an extraction carried out by a polymer/salt (e.g.,
PEG and potassium phosphate) aqueous two-phase system. When the product is recovered in the
polymerrich phase, precipitation is accomplished by addition of more polymer. It is important for
economic reasons to recover and recycle the precipitating materials. Precipitation is also used to
remove contaminants, i.e. nucleic acids, by adding MnSO4 and streptomycin sulfate
.
Distillation is used for concentrating and purifying organic solvents, such as ethanol, acetic acid,
etc.

Product Renaturation. Eukaryotic proteins produced by prokaryotic microorganisms often form
insoluble inclusion bodies (IBs) in the host cell. Inclusion bodies can be dissolved rapidly using
solutions of strong chaotropes, such as 6 M guanidine hydrochloride or urea, in the presence of a
reducing agent, such as 0.5 M 2-mercaptoethanol or 50 mM dithiothrietol . The dissolved protein is
then allowed to refold to its native conformation by removing the chaotropic agents through
diafiltration or chromatography and diluting the solution down to total protein concentration of 20-
50 mg/L. Dilution is necessary for minimizing intermolecular interactions, which occur during
product refolding and can lead to product inactivation. Addition of small amounts of reduced
glutathione (2-5 mM) and oxidized glutathione (1-2 mM) and incubation at 35-40 C for 5-10 hours
completes the refolding process. Thus, when choosing an upstream process that forms IBs
consideration must be given to the large volumes, and hence large waste streams, that are produced.


Final Purification Stages

The final purification steps are dependent on the required final product purity.
Pharmaceutical products require high purity while industrial products require lower purity. For
products of relatively low purity, such as detergent enzymes, the final purification step is
dehydration or more generally a solvent removal step. For high purity products, the final
purification stages usually involve a combination of chromatographic and filtration steps. If the
final product is required in solid form, then, a dehydration or solvent removal step follows.

Chromatography is typically done later in a process in agreement with the third generic heuristic,
make the most difficult and expensive separations last. With the previous separation steps, a large
fraction of contaminants is removed, thereby reducing the volume of material that needs to be
treated further. In fact, a 50-100 fold volumetric reduction is quite common for high value
biological products, resulting in a protein content of 1-5% w/v in the feed stream to
chromatographic units.
Recent advances in expanded bed adsorption (EBA) chromatography promise to position
chromatographic steps in the primary recovery stages. As mentioned earlier in this chapter (see
Product Extraction / Adsorption), EBA chromatography units can be used to capture, concentrate,
and purify product directly from fermentation broth that contains whole cells, cell debris and other
particulate components. Consequently, EBA chromatography has the potential to eliminate some of
the typical primary recovery steps, such as biomass and cell debris removal, product concentration,
etc.
A sequence of chromatographic steps is usually required to achieve the desired final product purity,
and the fourth and fifth generic heuristics are good guides for selecting and sequencing such steps.
For instance, according to the fifth heuristic, an ion exchange step should not be followed by
another step of the same type. Instead, it should be followed by a reverse phase, affinity or any
other chromatography step that takes advantage of a different separation driving force.

Membrane filtration steps are commonly employed between chromatographic steps to exchange
buffers and concentrate the dilute product solutions.

The insulin and monoclonal antibody examples that are presented later in this chapter provide
additional information on selection and operation of chromatographic separation units.

Dehydration or Solvent Removal is achieved with dryers. Spray, fluid bed, and tray dryers are
used when products can withstand temperatures of 50-100 C. Freeze dryers are used for products
that degrade at high temperatures. Freeze dryers require high capital expenditures and should be
avoided if possible.


Bioseparations Science and Engineering
ROGER G. HARRISON, University of Oklahoma, PAUL W. TODD, Chief Scientist, Space
Hardware Optimization Technology, Inc., SCOTT R. RUDGE, Vice President of Operations
for FeRx, Inc., and DEMETRI PETRIDES, President, Intelligen, Inc.