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TABLE 11.9
Downstream Process
Recommended Online Monitoring for Critical Downstream Processing Steps
Process Stage Process Parameters Monitored Process Attributes
Harvest and clarication Flow rate
Pressure
Turbidity
Homogenization and IB
recovery
Purication pH
Concentration Dialtration volumes
Pressure
Number of cycles
Temperature
Flow rate
A280 (UV)
Conductivity
Binding conditions
Elution conditions
Concentration factor
Flow rate
Turbidity
Efciency
Filter membrane clogging
Clarity of the supernatant
Protein concentration (product)
Homogenization efciency
Protein concentration (product)
Amount of IBs
Protein concentration (product)
Purity and impurity prole (e.g., aggregates, residual
DNA, HCP, etc.)
Column performance/ resin lifecycle
Protein concentration (Product)
Purity prole
Impurity prole
Monitoring various parameters serves as feedback for control loops, optimizing processes. Data
from monitoring is stored in automation systems, allowing trend analysis and inclusion in batch
documentation (e.g., UV analysis, chromatogram from purication processes, pressure monitoring
in TFF systems (TMP)). pH, conductivity, temperature, and pressure are commonly monitored using
in- line or ofine probes or sensors (Table 11.9).
Timely monitoring of critical process parameters signicantly contributes to product quality,
understanding the process impact, and an efcient process. Sensor design depends on unit
operations and product/ protein type. For instance, in chromatography, in- line sensors should detect
proteins, nucleic acids, carbohydrates, salts, ow rates, and perform pressure safety checks. Off- line
instruments typically determine product quality resulting from processing conditions.
11.16.1 ultRaviolet MonitoRing
UV spectroscopy is crucial for downstream unit operations, measuring absorption from aromatic
acids (phenylalanine, tyrosine, tryptophan) around 280nm (sometimes up to 340nm) to scan different
proteins eluting from a column. UV monitoring aids chromatographic separation, with systems sometimes featuring an auto- zero function to normalize baseline UV absorption. In certain TFF applications,
UV cells monitor the protein breakthrough through the membrane. This is useful for high concentration protein. Alternatively, the UV sensors may also be installed in the retentate line to determine the
retentate recovery to minimize the nal product’s dilution and maximize the overall yield.
FTIR spectroscopy characterizes proteins based on amide bond vibrations in the polypeptide
(secondary structure), assessing structural changes.
11.16.2 Ph MonitoRing
pH measurements serve as in- process controls, buffer criteria, and a suitable reaction environment.
pH probes (glass or plastic) are calibrated before each operation but shouldn’t be exposed to extreme
conductivity values.
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11.16.3 conductivity MonitoRing
259
Conductivity measurements play a crucial role in monitoring chromatography and ltration
operations. They are utilized to oversee or regulate gradient operations, such as ion- exchange chromatography or hydrophobic interaction chromatography, where increasing salt concentration aids
elution. During dialtration operations, measuring conductivity helps track progress and determine
when the target process conductivity is achieved. Additionally, conductivity measurement offers
insights into lter equilibration for ltration operations and serves as an endpoint for Cleaning in
Place (CIP). Probes are strategically placed in the retentate and permeate lines to monitor buffer
exchange and measure conductivity during rinsing operations.
11.16.4 PRessuRe sensoRs
Pressure sensors are widely employed across bioprocessing operations, primarily serving as safety
measures for both equipment and operators. For instance, in TFF, pressure sensors monitor transmembrane pressure (TMP), detecting pressure differentials across cassettes and high system
pressure. If pressure spikes, it is critical to halt the feed pump. Similarly, in chromatography columns
and depth ltration systems, monitoring pressure drop over time serves as an indicative performance
parameter. In product wetted applications, in- line diaphragm sensors are typically used, while other
sensors or gauges cover non- product contact areas.
11.16.5 teMPeRatuRe
Temperature control is not universally required in downstream processing, but specic operations
occasionally demand controlled temperatures. These operations are conducted either in temperaturecontrolled manufacturing areas . For refolding operations that may require a controlled temperature,
tanks (jacketed vessels) are connected to a recirculation chiller to help keep the desired temperature.
The inuence of temperature on pH and conductivity measurements is signicant. Calibration
and measurement should occur at the same temperature, or manual temperature compensation using
the meter’s control option becomes necessary. Some pH meters feature automatic temperature compensation functions. Given that buffers exhibit varying pH values at different temperatures, it is
crucial to use the buffer’s value at the calibration temperature. Most new meters include NIST calibration. Monitoring the temperature, pH, and conductivity of samples can be advantageous.
11.16.6 floW MeteRs
Flow meters are pivotal in controlling operational ow rates by providing real- time monitoring.
They are commonly integrated into chromatography and ltration systems. High- accuracy inline
and non- invasive ow sensors are frequently used as built- in components, capable of bidirectional
measurements within pipes. In tangential ow ltration (TFF) systems, mass ow or magnetic
inductive ow meters gauge ow on the retentate and permeate sides. Flow rates hold signicant
importance, especially during operational scale- ups, ensuring consistent system performance across
various scales. Magnetic owmeters, while less expensive than mass owmeters, require a minimum liquid conductivity for accurate ow rate measurement.
11.16.7 aiR sensoRs
Air sensors play a crucial role in chromatography systems by detecting the end of a sample or
buffer solution, thus preventing air from entering the system. This protective function safeguards
the column from potential damage caused by air and allows for automatic loading. Notably, the air
sensor operates as a non- invasive device.
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11.17 FLOW PATH
Downstream Process
In chromatography and ltration systems, the ow path stands as a vital component, which can
be constructed from either plastic or stainless steel. In bioprocessing operations, a disposable
ow path is preferred due to its ease of plug- in/ plug- out options. These pre- assembled and presterilized ow paths facilitate quick plug- and- play functionality. Moreover, disposable ow path
kits come with documentation on extractables and leachables to meet regulatory requirements
(e.g., Cytiva ow path kits, QuantaSep 300 SU system with disposable ow path Sepragen,
PROCONNOX by Repligen). Therefore, the ow path should accommodate low- volume
processes effectively.
11.18 PUMPS
Pumps hold signicant importance in both upstream and downstream operations, serving a wide
array of applications from pumping mediums and cultures to buffer solutions and delicate protein
solutions. The selection of an appropriate pump for a specic process involves careful consideration
of numerous factors, including ow rate requirements, liquid’s physical properties, shear sensitivity,
and pressure needs.
In a peristaltic pump, as uid enters the head, it gets trapped between two rollers. This movement
creates a void, leading the tubing to be occluded by the rollers. This alternation between uid
and void causes the ow to pulse rather than operate smoothly. Pulsations exist in all pumps but
having a pulse- free ow ensures better control of ow rates and more precise timed operations.
Methods to reduce pulsation include using a pump head with adjustable occlusion, employing a
multiple roller design, utilizing pulse- dampening mechanisms, or modifying the discharge tubing
layout.
Suction lines, often overlooked in pump systems, transport the source uid material through
piping or tubing. Priming the suction lines is necessary, and this feature may be part of the pump
itself – either self- priming or non- self- priming. Self- priming pumps can draw feed liquid even from
an empty pipe, while non- self- priming pumps require liquid in their lines before operation.
A shorter suction line can enhance operational efciency by reducing energy consumption and
extending the pump’s lifespan. Additionally, it helps minimize cavitation, a phenomenon that signicantly compromises the pump’s performance and durability. Cavitation arises from the formation of air pockets within the uid due to uctuating pressure, causing the liquid stream’s pressure
to drop below the vapor pressure of the pumped liquid. These minute bubbles collapse, leading to
damage to nearby components. Different pump types have specic suction line requirements for
optimal functioning. For instance, centrifugal pumps necessitate a direct and concise suction line.
The highest point on the suction line is the pump inlet, which must maintain a tight, air- leak- free
connection for proper pump operation. In magnetic drive pumps, keeping the suction line length to a
minimum and mounting it gradually to the pump prevents air pocket formation. The diameter of the
suction pipe should match or exceed the pump’s suction size. Diaphragm pumps, on the other hand,
require a non- collapsible, reinforced suction line.
Regarding the most commonly used pumps in bioprocesses
• Peristaltic pumps are commonly used for single- use or disposable applications due to their
compatibility with exible tubing (e.g., silicone). However, these pumps have limitations,
such as susceptibility to tubing breakage due to constant movement and friction from the
pump head. Therefore, caution is advised against using them for highly viscous liquids or high
ow rates, which may risk product loss or contamination.
• Rotary Lobe Pumps: Suitable for high- viscosity liquids and large volumes, but susceptible to
damage from hard particles in the uid. Speed control via a variable frequency drive using a
ow meter is common.
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• Diaphragm Pumps: Predominantly used in downstream operations such as chromatography,
available in various pressure ratings suitable for high- pressure chromatography applications
requiring pressures more than 100 bar.
11.19 VALVES
Valves regulate product and buffer ow within the system, affecting pressure- ow characteristics.
Materials like stainless steel, polypropylene (PP), polyvinylidene diuoride (PVDF), and polyether
ether ketone (PEEK) are commonly used in valve construction. Various types of valves cater to
different biopharmaceutical manufacturing operations: pinch valves for soft, exible tubing in
single- use systems, diaphragm valves for low- pressure operations, and ball valves widely used in
HPLC systems.
11.20 IN- LINE FILTRATION (STERILE FILTRATION AND PARTICLE
FILTRATION)
Filtration is a vital operation in downstream processes, applied in stages such as harvest clarication, buffer and solution preparation, chromatography, and nal ltration for the nished product.
Operations like microltration, ultraltration, nanoltration, and direct ow ltration through
sterilizing- grade lters are commonly employed.
Standard ow ltration involves passing the feed directly through the membrane without crossow, known as direct ow or dead- end ltering. This method, often employed in chromatography
unit operations and sterile ltration like virus ltration, effectively protects the chromatographic
column by removing particles from the buffer and feeds, reducing bioburden. To optimize ltration parameters, it’s essential to assess the ltration area, porosity, and cartridge conguration.
Pressure sensors placed upstream and downstream of the lter unit monitor and control the ltration
operation by estimating pressure differentials, signaling when the lter requires replacement. For
particle and microbiological removal, membrane pore diameters range from 0.1 μm to 10.0 μm.
Pre- and post- use lter integrity testing is performed on all lters used in upstream and downstream
operations. Tests like the bubble point test, forward ow test, water intrusion test, and binary gas
test are conducted. These tests gauge a wet membrane’s ability to prevent gas from owing freely
is measured using the bubble point and forward ow tests. A dry hydrophobic membrane is utilized
as a barrier to the free ow of water, and a non- wetting uid is used in the water intrusion test
(HydroCorr test). A mixture of two gases with large permeability variances is used in the binary gas
test. The test entails determining the composition of the gas mixture upstream and downstream of a
wetted membrane. If the post- use integrity test fails, reltration is conducted.
When considering a bioproduction process, selecting and specifying equipment should primarily
align with operational needs. With the emergence of process intensication in bioprocessing, smallerscale equipment holds potential advantages, especially in meeting product quantity requirements for
toxicology and clinical studies. Additionally, smaller skids offer compatibility benets and facilitate
the implementation of single- use technologies.
Most downstream processing equipment is constructed from stainless steel, posing limitations
for operations requiring extreme conditions due to corrosion risks. Plastics like polypropylene,
EPDM, PTFE, and PEEK are commonly used in tubing, measuring cells, valves, and gaskets. pH
and UV sensors typically utilize glass components. Many elements, including stainless steel, are
considered extractable, necessitating collaboration between drug manufacturers and suppliers for
testing and documentation purposes. As the target product moves towards increased purity in the
nal drug substance or drug product preparation, the demand for sterile or aseptic conditions intensies, mandating hygienic design for process ow paths and product contact surfaces. Conventional
cleaning methods involve using caustic solutions of varying concentrations for tasks such as column
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Downstream Process
and lter membrane cleaning, equipment maintenance, and other consumables. If needed, steam
sterilization may also be applied. Single- use systems replace several components, signicantly reducing cleaning procedures and expediting turnover times.
11.21 SUMMARY
The success of developing a robust downstream process and meeting product quality requirements
hinges on the efciency and recovery of each unit operation. Product losses can substantially impact
the economic feasibility of the product, emphasizing the need for a systematic approach in identifying the objectives and expectations of each unit operation. For instance, a three- stage purication plan does not necessarily require three chromatography phases in all manufacturing processes.
Decisions should be guided by purity standards and the intended usage of the protein. While a
platform process streamlines operations in a multi- product facility dealing with similar products,
adaptations to this approach warrant evaluation.
Downstream operations often have inherent limitations. Chromatography resins, for instance,
present challenges concerning their lifespan, lot- to- lot variability, cleaning, regeneration processes,
and viral clearance capacity in chromatographic procedures.
Optimization efforts targeting reduced processing times and process simplication through process intensication should be explored. Strategies like adjusting media volume based on impurity
levels can minimize resin and buffer consumption while enhancing economic viability. Prioritizing
process efciency, robustness, and scalability is crucial. Innovative approaches such as integrating
chromatographic steps into multimodal processes, continuous processing, and embracing single- use
systems that eliminate complexities associated with packaging, cleaning, and maintenance have
gained traction. Although their implementation remains limited, these technologies offer opportunities for enhanced productivity and exibility.
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12
Biopharmaceuticals
12.1 OVERVIEW
The development requirements for biopharmaceuticals differ signicantly from small chemical molecule drugs due to their larger molecular size and the variability of molecular structure. These properties impact the body’s immune systems in unique ways.
Firstly, as most protein drugs are administered via parenteral routes, the science of protein drug
formulation primarily focuses on injectable formulations. The choice of delivery route is limited
due to the instability of protein structures in many administration environments. Factors such as
acidity in the gastrointestinal tract, the large molecular size, high hydrophilicity hindering absorption across biological membranes, and high dose- response sensitivity restrict signicant variations
in bioavailability.
Secondly, proteins possess structural features like functional groups susceptible to oxidation, including methionine, cysteine, histidine, tryptophan, and tyrosine. These require common
approaches for stabilization. Additionally, conformational changes and aggregation, unique to large
molecules, necessitate specic formulation components.
Thirdly, proteins are sensitive to temperature, light, and agitation during storage, shipping, and
handling. The formulation challenges are compounded due to various factors not commonly considered
in small molecule drugs, potentially affecting the quality and efcacy of biopharmaceuticals.
The formulations of biopharmaceuticals vary signicantly based on the delivery route, predominantly parenteral, but advancements are diversifying to noninvasive routes. The choice
of administration route considers practicality and probability factors, requiring a detailed
understanding of biopharmaceutical interactions with the administration route’s environment.
Computational tools over the past two decades aid in rapidly creating decision matrices for optimizing formulations.
Different biological barriers confront each route of administration due to anatomical and physiological characteristics. To surmount these barriers, various formulation approaches have been
developed, incorporating advancements in information technology, biotechnology, nanotechnology,
and sophisticated medical devices, including electric or magnetic forces and sonic waves, to maximize noninvasive drug delivery effectiveness.
A formulation aims to deliver a biopharmaceutical active to the administration site, crossing biological barriers into the bloodstream and ultimately reaching the site of action. Given the high likelihood of product degradation during shelf- life, proprietary technology, including numerous patents,
has been developed to enable a dosage form to deliver the drug effectively to the site of action.
An appendix in this chapter details the physicochemical properties of proteins and peptides, crucial for designing formulations.
DOI: 10.1201/9781003392026-12
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Formulation of Biopharmaceuticals
Due to the high cost of biopharmaceutical development, scientists engage in evaluating intellectual property related to biopharmaceutical manufacturing and delivery systems. Chapter 8 provides
details on intellectual property management, essential reading for scientists involved in the formulation of biopharmaceuticals.
Managing biopharmaceutical stability involves either chemically modifying the molecule or
selecting proper excipients. These combinations ensure stability without compromising product
safety or efcacy. Structure modications, such as creating protein scaffolds and PEGylation of
molecules, among other technologies, are elaborated in Chapter 1.
12.2 PROTEIN STRUCTURE
12.2.1 basis
A comprehensive understanding of protein structure and the associated risks in formulating these
products is necessary to comprehend the complexities of formulating biopharmaceutical products.
Proteins consist of amino acid chains with reactive groups forming multidimensional structures,
dening their chemical and physical properties are based on this reactivity.
Protein instability arises from both chemical and physical reactions. Chemical instability involves
the formation or dissolution of covalent bonds within a polypeptide or protein structure, leading to
oxidation, deamidation, reduction, and hydrolysis. Physical or conformational instabilities encompass dissociation, denaturation, accumulation, and precipitation. When a chemical event, like oxidation, triggers a physical reaction like aggregation, the protein degradation pathways synergize.
Unlike small molecule drugs, physical changes in biopharmaceuticals, apart from the PK prole,
can signicantly impact safety.
Table 12.1 lists several formulation variables triggering uncertainty, grounded in the chemistry
of chemical and biopharmaceutical molecules. However, biopharmaceuticals have a distinct effect
on protection and efcacy.
Understanding the intricate degradation mechanisms causing chemical and physical instability in
biopharmaceutical formulations has historically posed challenges. However, advancements in analytical chemistry have signicantly facilitated this comprehension. The FDA- approved Appendix
Physicochemical Properties of Proteins and Peptides, as of March 2020, serves as a comprehensive
compilation of critical product properties. This database enables formulators to adopt a focused scientic approach in developing formulations by comparing and selecting stable formulations based
on similar molecular properties.
Compared to small molecule drugs, biopharmaceuticals are less tolerant of minor variations in
solution chemistry. Their stability in terms of composition and conformation is conned within
a narrow pH and osmolarity range. To maintain solubility throughout the product’s shelf life,
TABLE 12.1
Impact of Formulation and Environmental
Factors on the Degradation of Proteins
Factor Impact
Buffer species Deamidation
Light Photo decomposition
Metal ions Hydrolysis, oxidation
Other excipients Maillard reaction
Oxygen Oxidation
pH Hydrolysis, deamidation
Temperature Most routes
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TABLE 12.2
265
Protein Biopharmaceuticals Stability Issues
Problems Potential Causes Possible Solutions
Cleavages Protease impurity, other unknown
mechanisms
Covalent aggregation Disulde scrambling, other unknown
mechanisms
Cyclic imide pH around 5 pH optimization
Deamidation pH < 5.0 or pH >6.0 pH optimization
Non- covalent aggregation Solubility, structural changes, heat, shear,
surface, denaturants, impurities
Oxidation Active oxygen species, free radicals,
metals, light, impurity
Surface denaturation,
adsorption
Low protein concentration, specic
afnity, protein hydrophobicity
pH, product purity, inhibitors
pH, inhibit non- covalent aggregation
pH, ionic additives, amino acids, surfactants,
protein concentration, raw material purity
Excipient purity, a free- radical scavenger, active
oxygen scavengers, methionine
Surfactants, protein concentration, pH
supportive formulation components are often necessary for multiple molecules. Even lyophilized
protein products are susceptible to substantial degradation, unlike small- molecule drugs, which tend
to be highly stable in most lyophilized formulations.
Table 12.2 succinctly outlines prevalent stability issues encountered in protein formulation development along with potential solutions. However, given the unique nature of each molecule, this list
should serve as a guideline rather than an exhaustive directive.
12.2.2 Physical degRadation
Protein degradation stems from various factors including hydrophobic surfaces, boiling, lyophilization, reconstitution, interaction with organic solvents, shaking, and other physical and chemical
inuences. Physical stressors can lead to denaturation, adsorption, accumulation, or deposition on
container walls.
12.2.2.1 Structural Changes
Biological macromolecules like antibodies possess a three- dimensional tertiary structure crucial
for their folded state, involving complex intramolecular and intermolecular interactions with functional amino acid groups and external environments. Non- covalent interactions (electrostatic, van
der Waals, hydrogen bonding, hydrophobic) play a pivotal role in maintaining the native structure’s
stability. Disruption of this interaction balance by external sources leads to structural changes,
rendering large molecules unstable. For instance, in an aqueous solution, more soluble amino acid
residues interact with solvent molecules while non- polar residues are shielded, creating a hydrophobic core.
The amino acid sequence determines protein folding into its biologically active form. However,
proteins can also unfold, transitioning to an intermediate or denatured state from their native structure. Such variants tend to assemble into more stable complexes, such as aggregates, due to higher
free energy. Aggregates consist of multiple monomers held together by covalent or non- covalent
bonds. Dilution, for example, can dissociate native monomer cluster aggregates due to monomer
association. Precipitation or irreversible aggregates can occur as a result of the nucleation of different
monomers. Aggregation is caused by a variety of stressors, the most common of which are temperature, mechanical agitation, and freeze/ thaw stress during manufacturing. Aggregation can be caused
by a pH change, and high temperatures can cause conformational destabilization or partial/ complete
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Formulation of Biopharmaceuticals
unfolding. IgG4 forms more soluble aggregates than IgG1 at lower pH and higher temperatures, for
example, due to lower conformational stability caused by lower unfolding temperature and changes
in tertiary structures.
Because protein tertiary structures are vulnerable to environmental physical stress, structural changes
in mAbs can occur at any stage during the manufacturing process, starting from protein expression to
processing and storage. Structural transformation is attributed to non- physiological conditions during
manufacturing processes contribute to structural variants in the nal product. Stressors such as buffer
selection, fabrication techniques, and container type may exacerbate these issues.
12.2.2.2 Aggregation
Aggregated proteins pose signicant concerns in biopharmaceuticals, diminishing bioactivity and
increasing immunogenicity. Macromolecular protein complexes can trigger an immune response,
recognizing the protein as foreign and prompting an antigenic reaction.
Buffer selection signicantly impacts product stability. For example, acetate buffer causes precipitation in IgG3 formulations, unlike when arginine and histidine are used. Adjusting buffer salt
concentrations can mitigate phase changes. The lyophilizate’s pH is crucial in preventing recombinant vaccine antigen aggregation, often necessitating additional stabilizers such as trehalose.
Non- native proteins should be avoided in nished mAb- based biopharmaceutical products. When
protein solutions are drawn from vials, aggregation might occur, leading to inconsistent dosing.
For instance, aggregating IFN– promotes the development of neutralizing anti- drug Abs (NAb),
hindering the IFN receptor from binding and thus reducing clinical efcacy. Induced NAbs can disrupt the normal function of endogenous proteins, especially hormones and cytokines. NAb interference with endogenous erythropoietin led to Eprex®- related severe anemia and pure- red cell aplasia
(PRCA) in patients receiving recombinant human erythropoietin. The NAb formation was due to
aggregation, often facilitated by prelled syringes containing ingredients like polysorbate 80 and silicone oil. Therefore, any progress in the formulation or manufacturing process of biopharmaceuticals
requires further research following the ICH Q5E guidelines (https:// datab ase.ich.org/ sites/ defa ult/
les/ Q5E%20Gu idel ine.pdf). The creator of Eprex did not conduct essential studies and believed
that minor formulation changes would not impact erythropoietin’s protection or efcacy.
Aggregation is a prevalent issue in protein production and storage. Exposure to liquid– air, liquid–
solid, or liquid– liquid interfaces enhances a protein’s aggregation tendency. Mechanical agitation,
including shaking, stirring, pipetting, or pumping through tubes, and freezing and thawing also contribute to aggregation. Solvent conditions such as temperature, protein concentration, pH, and ionic
force can affect the nature and amount of aggregates formed.
Several mechanisms lead to protein aggregate formation, including domain swapping (ds), strand
association (sa), edge- edge- association (ee), and beta- strand stacking. Aggregators include protein
multimers such as dimers, trimers, tetramers, and large polymers. These aggregates can be noncovalent or covalent (disulde- linked). Non- covalent aggregates are completely soluble in a clear
solution, partially soluble in a turbid solution, and mostly insoluble as sediment at the container’s
bottom. Interactions between exposed hydrophobic groups in proteins trigger non- specic proteinto- protein interactions, leading to aggregation. While covalent aggregation is irreversible, weakly
associated non- covalent aggregates might be reversible but progress to multimers in most cases.
Strongly associated non- covalent aggregates are not reversible through dilution and can precipitate.
Aggregates are categorized based on their size range: (1) submicron particles (<1 µm), also known
as soluble particles; (2) sub- visible particles (1– 100 µm), and (3) visible particles (>100 µm size).
Aggregation models exist in various forms and sizes. In the “Native to Unfolded to Aggregate”
model, hydrophobic interactions lead denatured or unfolded molecules to aggregate. These
interactions occur due to the release of submerged hydrophobic regions, inducing aggregate formation. As temperature increases, unfolding intensies, and since reactions typically follow rst- order
kinetics, the reaction rate rises in this model.
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The “Native to Intermediate to Unfolded” model involves the intermediate stage producing the
aggregate. These misfolded intermediates are thermodynamically stable and are part of the native
state ensemble. Therefore, protein aggregation isn’t necessarily an abnormal state and can occur
even under conditions favoring the native state.
Protein aggregation comprises two stages: nucleation expanding to a critical mass after the nucleation process. Although turbidity measurements track aggregation extent, they aren’t always reliable
predictors. When native and folded proteins interact, irreversible non- native structures with elevated
non- native intermolecular- sheet formations may develop. Solution conditions such as pH, salt
species, concentration, co- solutes, excipients, and surfactants signicantly inuence the aggregate’s
onset, rate, and nal morphology. The precise existence of an aggregate is determined by the relative
intrinsic thermodynamic stability of the native state.
Biopharmaceuticals can aggregate at various process stages, including hold points, shipping, and
long- term storage due to necessary physical and chemical manipulations in production, downstream
processing, formulation, and lling. Agitation of protein solutions (shaking, stirring, or shearing) at
air- liquid interfaces aids protein molecules in joining and unfolding, exposing hydrophobic regions,
fostering aggregation.
Agitation- induced aggregation is observed in proteins like recombinant factor XIII, human growth
hormone, hemoglobin, erythropoietin, and insulin. Preventing signicant protein activity loss or visible particulate matter formation requires minimizing foaming caused by agitation in manufacturing
and product use. Antimicrobial preservatives used in multidose formulations, such as benzyl alcohol,
accelerate protein aggregation by producing partially unfolded protein conformations. Increasing
preservatives’ amounts can increase hydrophobicity, decreasing aqueous solubility. Phenol and mcresol signicantly destabilize proteins, with m- cresol causing protein precipitation and phenol promoting both soluble and insoluble aggregates.
Freezing and thawing can occur several times during growth, which may reduce the shelf- life of
the product and have a signicant effect on protein aggregation. When water- ice crystals form at
the container’s rim, the effect is known as “salting out” (where heat transfer is greatest). Protein and
excipients become increasingly concentrated in the eventually freezing center of a container.
Precipitation and accumulation occur during freezing owing to high salt and protein concentrations,
which will not be completely reversed upon thawing. Thyroid- stimulating hormone, for example,
retains its potency for up to 90 days when stored at −80°C, 4°C, or 24°C but loses more than 40%
of its potency when frozen at −20°C due to subunit dissociation.
Repeated freezing and thawing cycles have a cumulative effect on subvisible and visible particulate generation. Buffer component crystallization during freezing can induce pH changes, with
potassium phosphate buffers exhibiting smaller pH transitions than sodium phosphate buffers.
Oxidation can accelerate pH- dependent reactions, leading to precipitate appearance within minutes.
The causes of aggregation related to the manufacturing process are listed in Table 12.3.
TABLE 12.3
Process- Related Causes of Protein Aggregation
Process Factor
Administration Diluents, component materials and surfaces, leachable
Fermentation/ Expression Inclusion bodies
Fill/ Finish Surface interaction, shear, contamination (e.g., silicone oil)
Filtration Surface interaction, shear
Freeze/ Thaw Cryo- concentration, pH changes, Ice- solution interfaces
Lyophilization Cryo- concentration, pH changes, ice- solution interfaces, dehydration
Purication Shear, pH, ionic strength
Shipping Agitation, temperature cycling
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