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11.3.2.1 Dilution
Downstream Process
Among refolding methods, dilution stands out as the most common due to its operational simplicity.
In this method, the solubilized IB is diluted into a suitable refolding buffer. This process reduces
protein and denaturant concentrations, creating an appropriate oxidative environment for disulde
bond formation. Maintaining low protein concentrations throughout the refolding process is crucial to minimize aggregation. Controlled dilution, such as pulse or drip dilution, for converting the
solubilized IBs into the refolding buffer over a dened time is a preferred method (slow dilution), is
preferred over rapid dilution into excessively large volumes, which can lead to increased aggregation or misfolding due to sudden environmental changes. Continuous mixing of the refolding solution and the denatured protein solution is vital to prevent aggregation. Choosing the optimal dilution
that suits the processing volume without compromising protein quality is essential.
11.3.2.2 Dialysis
Dialysis serves as another standard method to reduce or eliminate solubilizing agents by allowing
components to diffuse through buffer exchange. During dialysis, the solubilizing agent’s concentration gradually decreases, facilitating proper protein refolding. However, dialysis is a slow and
cumbersome process, especially when conducted on a large scale.
11.3.2.3 On- Column
Employing packed chromatography columns for refolding offers an appealing alternative to traditional methods. This approach encompasses various techniques:
• Denatured protein immobilization: Immobilizing denatured proteins on a matrix followed by
denaturant dilution aids in refolding through non- specic or afnity interactions, spatially
separating the protein. An example is afnity chromatography employing fusion tags for
on- column buffer exchange and renaturation, necessitating careful optimization of refolding
conditions based on protein- matrix interactions.
• SEC- based denaturant dilution capitalizes on partition coefcient differences between
proteins and denaturants, preventing aggregation by limiting diffusion of different protein
forms into the refolding mixture. The material eluted from the column is fractionated by size,
with denatured protein adsorbed, washed to remove denaturants, and eluted in the renaturing
solution via bind and elute method.
• Creating a refolding reactor involves immobilizing folding catalysts on the stationary phase
of a chromatographic column.
On- column refolding boasts advantages like suppressing unspecic intermolecular interactions,
achieving maximum protein concentration, and integrating purication with renaturation. However,
challenges like potential precipitation, column fouling, and cleaning difculty exist. Balancing
column chromatography operations with renaturation is crucial for successful refolding.
Denaturant removal facilitates the native protein conformation. Components in the refolding
buffer aid correct folding and native disulde bond (i.e., dilution, dialysis, or on- column) formation. The time required for complete refolding varies from a few hours to days depending on
the renaturation method (dilution, dialysis, or on- column). Optimizing refolding conditions signicantly impacts the overall yield and efciency of the process. During renaturation, two critical
processes occur: rst- order refolding and higher- order aggregation, competing for rst and higherorder reactions.
There is no one- size- ts- all refolding process; a method suitable for one protein may not work
for another due to various factors, including the refolding buffer, use of additives, and denaturant
removal rate. The optimal protein concentration reduces aggregation while considering refolding
conditions such as pH, temperature, refolding buffer volume, and processing time. Additives play a
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Downstream Process
TABLE 11.3
239
Advantages and Disadvantages of Common Protein Folding Methods
Refolding Techniques Advantages/ Disadvantages
Dialysis Time- consuming (can take several days)
Demands large buffer volumes
Dilution Most preferred
Slow dilution allows for control over protein aggregation; the dilution ratio is broad (ranging
from tenfold to a hundred- fold)
May demand large buffer volumes and handling capacity depending on the dilution ratio
On- column refolding Quick, effective, and straightforward
No volume limitations
Unlike dilution, working with high protein concentration is possible, eliminating cumbersome
equipment handling and even volume reduction steps
The method is highly dependent on the type of proteins
Size- exclusion
chromatography
Volume limitations, columns are designed to handle very small volumes
Aggregates formed on the column may be difcult to remove
Similar to on- column refolding, high protein concentrations can be used
crucial role in native disulde bond formation, proper folding, aggregation inhibition, and protein
stability. Achieving these goals often requires several additives to achieve these outcomes. pH and
temperature signicantly impact protein stability, disulde bond formation rate, and proper folding.
Gradual pH reduction or temperature changes can aid correct folding and precipitate misfolded or
unnecessary proteins. Disulde bond formation is a critical step affecting renaturation rate, inuencing the overall process yield. Therefore, controlling reaction conditions is crucial to avoid rapid
environmental changes that hinder stable intermediate formation before aggregation, especially
when aiming for commercial- scale manufacturing.
To track refolding progress and assess the percentage of unfolded and correctly refolded
protein over time, representative samples at specic intervals undergo RP- HPLC analysis. This
information is particularly crucial in the early stages of process development when optimizing
conditions to maximize recovery. Table 11.3 lists a comparative description of various methods
of refolding.
11.3.2.4 Depth Filtration for the Clarification of Refolded Protein
After refolding, the protein is typically claried before chromatography purication. This clarication involves depth ltration using charged depth lters, normal- ow ltration, and sometimes
centrifugation to remove protein aggregates. Due to the diverse particle sizes in refolded proteins,
multiple separation steps may be necessary. Combining multigrade depth lters and membrane ltration as a single operation can often address the need for multiple separations. Centrifugation,
while still utilized, might not be practical, especially with large processing volumes. Minimizing
product loss and obtaining a high- quality ltrate are challenges in the ltration process to maximize
subsequent purication steps’ efciency and protect the column.
Cellulose depth lters are commonly used in the biopharmaceutical industry for this purpose.
Their thickness and depth trap suspended particles, aided by their positive charge attracting negatively charged particles in the feed stream. Graded structures, created by coupling multiple pore
size lters in series, maximize surface area and reduce fouling. The nal ltrate undergoes sterile
ltration before loading for chromatography. Optionally, adding a microporous membrane before
the sterile lter can prevent smaller particles from blocking the nal sterile lter, maintaining
throughput. Monitoring pressure and ltrate turbidity during depth ltration is essential.
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11.4 PURIFICATION
Downstream Process
After clarifying the refolded protein solution, the subsequent series of unit operations are dedicated
to purifying the protein, producing a highly pure product that meets specic purity requirements.
Certain proteins pose challenges in purication, necessitating the use of physicochemical- based
chromatography methods to optimize their yield. The selection of suitable chromatographic separation methods depends on variations in the characteristics of the target protein and other chemicals
present in the sample.
11.5 CAPTURING
The most appropriate chromatographic separation methods are chosen based on the properties of
the target protein and other compounds in the sample. The initial capture step is designed to bind
the product to the matrix (due to charge, specic interactions, or afnity) while preventing impurities from binding, facilitating their removal during elution. Elution of the product can be achieved
through a step gradient or linear elution method, resulting in a high product concentration but offering
a moderate degree of purication. For instance, to prevent interactions with proteases, emphasizing
high throughput and shorter processing times becomes crucial. Certain product variants may hinder
product recovery and contribute to further degradation that is challenging to remove afterward.
The chromatographic process and capture step strategy may vary depending on the target molecule. For non- antibodies, oligonucleotides, and polysaccharides, ion- exchange chromatography
(IEX) or hydrophobic interaction chromatography (HIC) are more commonly used. Conversely,
Afnity Chromatography, such as Protein- A or Protein G, is employed to capture antibodies.
In purifying refolded proteins, ion- exchange chromatography (IEX) and, to a lesser extent,
hydrophobic interaction chromatography (HIC) have proven effective as initial chromatographic
stages. These methods demonstrate increased refolded protein recovery by enhancing stability, reducing aggregation, and efciently addressing purication challenges associated with both process and
product- related contaminants. These methods, employing linear gradient elution for separation, are
utilized in purifying refolded proteins. Soluble proteins, expressed in the E. coli expression system,
are directly loaded onto the chromatography column to separate the target protein from other cellular
contaminants. This step occurs subsequent to recovering the proteins through cell disruption and
clarication, aiming to remove nucleic acids and lipopolysaccharides, as well as host cell proteins.
Afnity chromatography, primarily used for monoclonal antibody (mAb) purication, is also being
explored for the large- scale purication of various recombinant proteins (e.g., recombinant insulin,
plasminogen, GCSF, IFN- alpha, Follicle Stimulating Hormone). The key requirements for this capture step are a high degree of recovery, product stability, and the ability to process high capacity.
11.6 INTERMEDIATE PURIFICATION
Following the capture step, high- resolution methods such as hydrophobic- interaction chromatography (both anion and cation exchange), size- exclusion chromatography, and reversed- phase chromatography are employed to remove most impurities like host- cell proteins, nucleic acids, and
endotoxins. It’s uncommon to use an anion exchange (AEX) as a capture stage followed by cation
exchange (CEX) chromatography or vice versa. These choices heavily rely on the target proteins
and the impurity prole.
Lower ow rates, gradient elution, and matrices with smaller particles are used to enhance resolution. Typically, the purity of the nal product after these stages reaches nearly 99%.
Throughout the chromatography process, there’s an inevitable loss of some product at each
purication step. Hence, there’s a constant drive to minimize the number of purication steps in
biomanufacturing processes. Reducing steps not only enhances processing speed but also improves
the yield of unstable proteins. After the capture step, an evaluation of the product quality, purity,
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Downstream Process
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impurity prole, and objectives of intermediate and nal purication steps determines the necessity
of an intermediate purication step. Some processes might skip an intermediate purication step,
favoring a polishing step to economize biomanufacturing operations.
11.6.1 Polishing
The polishing phase in purication aims to eliminate traces of aggregates (both low and high
molecular weight proteins), degradation products, or other product variants. Previous purication
processes have signicantly reduced product- related contaminants. This phase prepares the puried product for formulation or storage as an intermediate product. Size- exclusion chromatography
and reversed- phase chromatography serve as two polishing procedures for achieving the ultimate
product purity.
Occasionally, desalting and buffer exchange are necessary to modify the puried protein composition. Size exclusion chromatography effectively accomplishes this by allowing the removal of
low and high molecular weight contaminants while enabling the transfer of the protein into the
desired buffer. This facilitates nal adjustments to the conditions of the puried protein for storage.
Purication steps may or may not occur sequentially, and sometimes various intermediate processing steps, such as product concentration by tangential ow ltration (TFF), salt precipitation,
or desalting, might be integrated between chromatography steps. Desalting is commonly performed
when there’s a modication in buffer components, either before ion exchange to remove salt and
enable the protein sample to bind to the column, after purication to eliminate low molecular weight
contaminants, or after purication for the nal pure protein.
A generalized downstream process scheme for soluble proteins is depicted in Figure 11.6.
Figure 11.7 illustrates both a three- step and a two- step purication scheme for a protein expressed
as inclusion bodies (IBs).
Following the purication process, the concentrated puried protein primarily undergoes TFF to
produce the drug substance (either lyophilized or in a sterile solution) before storage for subsequent
processing into a drug product.
11.7 MAMMALIAN SYSTEM PURIFICATION
In mammalian cell- based protein production, the product is typically secreted into the culture
medium. Consequently, the initial step in product recovery involves separating the cells from
the culture medium through depth ltration, centrifugation, or alternative separation methods
(Chapter 5). Because the protein is secreted into the media, unlike in E. coli, the separation process
is less extensive.
The claried harvest undergoes purication and concentration steps to attain the desired
product, meeting specied purity and safety targets. Understanding the protein’s properties and
its contaminants is crucial in devising the purication strategy. Additionally, proteins produced in
a mammalian system may carry a risk of viral contamination. Thus, downstream operations must
include steps targeting viral inactivation and removal.
Figure 11.8 outlines a general process for mAb purication. mAbs share a common feature
through the Fc region, enabling the development of a platform process. Structurally, mAbs exhibit a
symmetrical conguration consisting of identical heavy and light chains linked via disulde bridges.
A three- phase purication strategy, akin to purifying the refolded protein, can be employed for
the claried harvest (CHO process).
• Capture: Isolate and concentrate the product.
• Intermediate Purication: Remove most bulk impurities (e.g., host cell proteins, nucleic acids,
DNA, endotoxin, and viruses).
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Downstream Process
FIGURE 11.6 Scheme for purication of a soluble protein expressed in E. coli (three- step purication
strategy)
• Polishing: Achieve high purity by eliminating trace impurities and other product- related
substances/ impurities.
Chromatography methods are chosen and optimized to t into the purication strategy, aiming to
achieve the desired outcomes. The number of steps employed varies depending on protein properties, target purity criteria, yield, impurity identity, and intended use.
Beginning with Protein- A chromatography followed by a series of ion- exchange chromatography steps, most operations are executed in a bind and elute mode, except for anion exchange chromatography, which operates in a ow- through mode for the desired protein.
11.8 CAPTURE– PROTEIN- A CHROMATOGRAPHY
The capture step (e.g., IEX, Afnity Chromatography) designed to isolate and concentrate the
product should also effectively remove critical contaminants. Typically, this step substantially
increases product concentration and purity, particularly if a highly selective afnity medium is
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FIGURE 11.7 Multistep purication schemes for expressed proteins
utilized. Other capture methods, such as crystallization and precipitation, are viable but entail additional processing steps (e.g., solid- liquid separation) and are not as successful on an industrial scale.
The primary capture for mAbs from the claried harvest begins with afnity chromatography,
frequently utilizing Protein- A chromatography. The consistent Fc- portion among mAbs, which is
the targeted binding region for Protein- A, has made it the predominant choice in downstream purication for capture.
Protein- A chromatography involves antibodies binding to an immobilized protein- A ligand in a
specic and reversible manner. Protein- A, a 56- kDa surface protein found in Staphylococcus aureus,
possesses ve binding domains that can bind to the Fc region of immunoglobulin G (IgG). Both
native and recombinant forms of Protein- A are utilized as ligands in Protein- A resins. The ligand,
typically coupled to a matrix such as cross- linked agarose (e.g., Sepharose®), exhibits high capacity,
capable of binding at least two antibody molecules per single coupled Protein- A molecule.
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Downstream Process
FIGURE 11.8 Overview of a mAb purication process
FIGURE 11.9 Protein- A, and it is binding to an immunoglobulin
Source: E A S– Own work, Creative Commons Attribution
Recombinant Protein- A, engineered for enhanced binding capacity, displays robustness and high
capacity due to modications to the ligand, including single domain multimers/ single point coupling
and multi- point attachment. These alterations offer additional benets, such as chemical and
thermal stability, enabling Protein- A to withstand a wide pH range of 2– 11 and resist denaturing
and chaotropic agents (e.g., urea, Gu- HCl). Figure 11.9 illustrates the structure of Protein- A and its
protein- binding mechanism.
Three major varieties of Protein- A resins are available commercially, based on matrix composition.
• Agarose Based: e.g., Protein- A- Sepharose® Fast Flow, MabSelect (Cytiva).
• Glass or silica- based: e.g., ProSep vA and ProSep vA Ultra (Millipore).
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• Organic Polymer- based: e.g., polystyrene- divinylbenzene POROS A™ and MabCapture™
(Applied Biosystems).
These resins are accessible for both laboratory- scale and commercial- scale processes, ranging
from less than 1 cm to 2 m column diameter. They exhibit resistance to high concentrations of GuHCl, urea, reducing agents, and low pH.
Protein- A also demonstrates an afnity for specic variants of the Fab region, which proves
benecial for purifying Fab fragments. While Protein- A remains the most widely utilized purication system for human and humanized mAbs, certain antibodies may not strongly bind or bind at all
to the Protein- A ligand. This selectivity, however, can be advantageous in specic cases.
An alternative to Protein- A is Protein G, an immunoglobulin binding protein found in group G
of streptococcal bacteria. It differs from Protein- A in its binding specicities. Depending on the IgG
being puried, either of these alternatives can be effectively employed in the capture step. Some key
features of these two ligands include:
Protein- A: It displays broad species reactivity, binding well to IgG from various species including
humans, rabbits, mice, and cows.
Protein G: Exhibits stronger binding to IgG from mouse rats. The binding site to albumin, present
in the native protein G, has been eliminated in the recombinant form.
Protein- A might serve as a better option for segregating certain IgG subtypes and other classes
of antibodies, as well as for eliminating other antibodies, such as cross- species IgG contamination
from horse or fetal calf serum. When combined with agarose beads, some binding afnities alter; for
instance, rat IgG does not bind to Protein- A but binds to Protein- A- Sepharose (Table 11.4).
TABLE 11.4
Antibody Subtypes and Recommended Purification Media
Species Antibody Subtype Protein- A Binding Protein- G Binding
Human IgG
IgG1
IgG2
IgG3
IgG4
IgA1
IgA2
Fab
scFv
Mouse IgG
IgG1
IgG2a
IgG2b
IgG3
Rat IgG
IgG1
IgG2a
IgG2b
IgG2c
Source: Adapted from Abcam
Strong
Strong
Strong
Weak
Strong
Weak
Weak
Weak
Weak
Strong
Weak
Strong
Strong
Strong
Weak
Weak
None
None
Strong
Strong
Strong
Strong
Strong
Strong
None
None
Weak
None
Strong
Medium
Strong
Strong
Strong
Medium
Medium
Strong
Weak
Strong
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Downstream Process
Chromatography commonly employs afnity gravity ow or a low/ medium- pressure system.
The binding capacity is determined by both binding strength and ow rate. Consideration should
be given to ligand leakage, with Protein- A point attachment to Sepharose showing minimal leakage
under various elution settings. These ligand contaminants are eliminated during the polishing using
SEC. Afnity chromatography, as an initial capture step, offers several performance attributes that
streamline the process development for new molecules, such as robustness to solution conditions for
loading and washing.
In large- scale mAb manufacturing, Protein- A- Sepharose (Cytiva) stands as the most frequently
used capture step. Its binding ability surpasses that of protein G Sepharose. Additionally, Cytiva’s
high- performance medium yields high resolution along with sharp and concentrated peak elution.
Table 11.5 presents an example of commercially available Protein- A chromatography medium.
Evaluation of each resin for protein A leachability is essential.
Furthermore, the formation of protein aggregates should be considered as a selection factor.
Protein A resins exhibit a dynamic binding capacity of 15– 100 g mAb/ L resin, which varies
depending on the mAb type, adsorbent, and ow velocity (Table 11.5).
The suggested typical binding and elution conditions for this media are outlined in Table 11.6.
To optimize the performance of Protein- A chromatography, it is crucial to ne- tune certain process variables. This includes optimizing binding conditions, such as buffer composition and pH, to
ensure efcient binding. It also involves considering washing conditions, including the ideal composition of the column wash solution, as excessive washing steps can potentially weaken ligand
binding and decrease yield. Equally important are the elution conditions, as any variations in these
parameters can impact Protein- A’s afnity, purity, and impurity levels (Table 11.6).
Protein- A chromatography is a widely favored method for capturing recombinant mAbs due
to its resilience. Depending on the intended function of the target protein, such as for diagnostic
TABLE 11.5
Purification Options for mAbs and Fc Fusion Proteins Using Commercial Protein- A
Chromatography Media
Protein- A Chromatography
Media Features
MabSelect PrismA Optimal productivity, cleaning, sanitization, bioburden control, alkaline stability, continuous
and batch processes, long resin life, easy cleaning, suitable for small- scale purication
and screening, prepacked columns suitable for large- scale commercial manufacturing
MabSelect SuRe LX
MabSelect SuRe
MabSelect SuRe pcc Continuous processes, long resin life, easy cleaning, prepacked large scale columns
MabSelect Low- to- medium titers, limited number of cycles, suitable for small- scale purication and
MabSelect Xtra High titer, higher ligand density, limited number of cycles, suitable for small- scale
TOYOPEARL AF- rProtein- A
HC- 650M
ProSep Ultra Plus Large scale, cost- effective, suitable for high titer, increased capacity, and productivity,
Poros MabCaptureA Highest dynamic binding capacity, process exibility (shorter bed height, faster ow rate)
Higher ligand density, good performance, long resin life, easy cleaning, prepacked
columns suitable for large- scale commercial manufacturing
suitable for commercial manufacturing, suitable for small- scale purication and
screening
screening, prepacked columns suitable for large- scale commercial manufacturing
purication and screening, prepacked columns suitable for large- scale commercial
manufacturing
IgG binding ability of >65 g IgG/ L resin
process exibility, prepacked, ready- to- use, and disposable columns, high throughput,
not suitable for cleaning with sodium hydroxide
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TABLE 11.6
247
Binding and Elution Conditions Commonly Used With Protein- A Sepharose®
Chromatography Purify IgG From Different Species
Species Subclass Protein- A Binding pH Protein- A Elution pH
Human IgG
IgG
IgG
IgG
Mouse IgG
IgG
IgG
IgG
Rat IgG
IgG
IgG
IgG
1
2
3
4
1
2a
2b
3
1
2a
2b
3
6.0– 7.0
6.0– 7.0
8.0– 9.0
7.0– 8.0
8.0– 9.0
7.0– 8.0
~7.0
~7.0
≥9.0
≥9.0
≥9.0
8.0 to 9.0
3.5– 4.5
3.5– 4.5
≤7.0
3.0– 6.0
4.5– 6.0
3.5– 5.5
3.0– 4.0
3.5– 5.5
7.0– 8.0
≤8.0
≤8.0
3.0– 4.0 (using 3 M potassium isothiocyanate)
Source: Adapted from Afnity Chromatography Vol 1: Antibodies, Cytiva LifeSciences (Formerly GE Healthcare)
purposes, purication using only Protein- A chromatography may sufce. During this stage, process-
related contaminants such as host DNA, HCP, and viruses are signicantly removed.
Despite the widespread use of Protein- A chromatography, it does have certain limitations. The
high cost of the resin, the procedures required for its repeated use that could potentially lead to
cross- contamination, and the impact of caustic cleaning solutions like 0.1 N NaOH on the resin’s
performance are notable disadvantages. Another concern is the non- specic binding of impurities,
such as host cell proteins, DNA, and other cell culture- derived impurities. Consequently, research
is underway to explore alternatives, such as ion exchange and other methods, to overcome these
limitations associated with Protein- A chromatography. Expanded- bed chromatography is also being
considered as a benecial method for binding proteins from crude cell culture media.
11.9 INTERMEDIATE PURIFICATION AND FINAL POLISHING
The intermediate purication step aims to separate the target protein from contaminants like
other proteins, viruses, endotoxins, and DNA. The specics of this step, including resolution and
specications, heavily rely on the properties of the Protein- A eluate sample and the nal product
specications that need to be met. If the protein purity achieved from the capture step is high, the
intermediate step might be unnecessary, and the process can proceed to one or more polishing steps
to further enhance the nal product’s purity. High resolution in separating the sample’s protein
components is expected during the nal polishing. Therefore, the binding capacity, resolution, and
selectivity for the intermediate purication step are critical, especially if the capture step product
had lower purity, as a signicant number of impurities may still be present. Eluting the target protein
might involve using a continuous gradient or a multi- step elution method.
11.10 POLISHING
The binding of impurities, such as host cell proteins, DNA, and other cell culture- derived impurities,
in chromatography is non- specic. For instance, if the target protein has a neutral to basic pI, the
ow- through mode would be suitable to eliminate contaminants. In contrast, the chosen conditions
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