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Best Practices for formulation and manufacturing of biotech drug products


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Maintaining product stability during the various drug product process unit operations is paramount to our ability to supply safe and efficacious biotech products to patients. New technologies are helping us ensure that we meet these challenges successfully and are able to embrace the Quality by Design paradigm. This article presents best practices to meet three of the significant technical challenges experienced in drug product manufacturing, namely, maintaining product stability during frozen storage, performing visual inspection of drug product vials, and controlling protein particulates.
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June 1, 2009 BIoPharm International 22(6) 32-48
Best Practices for Formulation and Manufacturing
of Biotech Drug Products
By Satish K. Singh,Nitin Rathore,Arnold McAuley,Anurag S. Rathore, PhD
How to maintain product stability and prevent particulates.
Maintaining product stability during the various drug product process unit operations
is paramount to our ability to supply safe and efficacious biotech products to patients.
New technologies are helping us ensure that we meet these challenges successfully
and are able to embrace the Quality by Design paradigm. This article presents best
practices to meet three of the significant technical challenges experienced in drug
product manufacturing, namely, maintaining product stability during frozen storage,
performing visual inspection of drug product vials, and controlling protein
Drug product manufacturing has its share of operational and technical
challenges. The large number of stock-keeping-units (SKUs) that we
typically manufacture for a single product, as well as the need for the
product to move outside the manufacturer's network and be delivered to
the patient, add to the operational complexity of drug product manufacturing. Further,
technical challenges associated with maintaining the purity, activity, and efficacy of the final
product during drug product processing must be overcome successfully.
This article is the 17th in the Elements of Biopharmaceutical Production series and presents
best practices to meet three of the significant technical challenges experienced in drug
product manufacturing, namely, maintaining product stability during frozen storage,
performing visual inspection of drug product vials, and controlling protein particulates.
Protein stability can be affected by a multitude of factors that interplay
during the manufacturing of biotech drug products. The need to
examine product stability over a broad range of process parameters has
been highlighted in the literature.
Such an examination can be achieved by characterization
studies at small scale using qualified scaled-down models or large-scale experiments
designed to examine worst-case scenarios related to changes in operating conditions.
development of a design space in the context of developing, scaling up, and transferring
freeze-dried products has been discussed in recent publications.
It has been pointed out
that when doing formulation and initial cycle development, the development scientist must be
aware of the type of equipment to which the product will be transferred in the next stage of
the product lifecycle.
Freezing and thawing large volumes of bulk protein solutions has become an important step
in biopharmaceutical manufacturing because the flexibility it affords makes it possible to
maximize productivity and align drug product logistics with market demands.
The stability of
therapeutic proteins during long-term storage has been highlighted as a key issue for product
safety and efficacy.
Storing drug substance for periods of time in the frozen state enables a
Technologies, Inc.)
Anurag S. Rathore
decoupling of drug substance manufacturing from drug product manufacturing. A successful
operation, therefore, requires an understanding of the fundamental aspects of freezing and
thawing proteins as well as the impact of the practical aspects of heat and mass transfer,
along with knowledge of the technology available.
Manufacturing sterile biotech products requires visual inspection of the final drug product
filled in sealed containers to ensure there is no contamination from foreign particulates.
Such inspection can be performed by humans or through an automated inspection machine
(AIM). Compared to manual inspection, automated visual inspection (AVI) offers more
consistency, higher speed, and improved quality of inspection. It also is cost efficient over a
longer period and for higher production volumes.
Problems arising from insoluble aggregate formation in biologics development along with
approaches to detect and characterize the aggregate species have been a focus for
regulators and the biotech industry lately. It has been suggested that our understanding of
aggregation pathways and how to inhibit aggregation remains relatively poor and that it is
challenging to characterize the whole size range of particulates for a given biologics
The United States Pharmacopeia and the harmonized versions of the European
and Japanese Pharmacopoeias set limits and cite enumeration methods for sub-visible,
foreign particulate matter in parenteral products.
A significant presence of particles,
whether they are product-related or foreign, may not only compromise the efficacy or the
drug product but also present a safety issue.
One important factor is potential
immunogenicity, which can be a result of poor quality of the protein product.
The quality
of a product can be affected by the presence of various degradation products such as
particulates and aggregates and also by chemical modifications of the protein molecule.
Protein particles typically are a result of the aggregation of structurally altered monomers and
or dimers, resulting in the insolubility of the species. Aggregates or multimers can be
categorized as either large or small in size.
Small aggregates can range from dimers to
multimers that can be detected by size exclusion chromatography and dynamic light
scattering, with a size range of 0.1 to 1 µm. Larger aggregates can be classified as sub-
visible which are 2 to 100 µm in size and detected by light obscuration methods such as the
HIAC Royco liquid particle counter and microscopy. Visible particles are detected by the
naked eye and can either be visible (>40 µm) or sub-visible and typically are detected by
visual methods or light obscuration instruments, respectively. The size range of the protein
particle can vary from <1 µm to >400 µm. In the sub-visible size range, injectable liquid
formulations must comply with the pharmacopeial limit of: not <6,000 particles for the 10 µm
range and not <600 particles for the 25 µm range.
Freezing biologics at large-scale is carried out in various ways, from improvised to custom-
designed systems. The simplest storage method involves filling the bulk solution into bottles
or carboys of appropriate size and storing in freezers. These containers are often made of
polyethylene or polypropylene, although steel (e.g., SS316L) can be used for small volumes.
Their advantage is simplicity. Disadvantages include a lack of active control and potential
variability between containers, as well as multiple container–closures to secure against
contamination. The procedure for preparation, loading, and placement in the freezer has to
be well defined to reduce this variability. Thawing is generally performed by placing
containers in a refrigerator or at room temperature. In the absence of an active thawing
mechanism, thaw times can be quite long (possibly days) depending on the size of the
container. During this period, significant concentration and temperature gradients can exist in
the container if it is not actively shaken or agitated. Practical handling considerations limit the
size to about 20-L carboys, although 50-L sizes are possible. The system is simple, however,
and if the protein formulation is stable under a wide range of freeze–thaw conditions and can
withstand cryoconcentration, the bottle or carboy system may be the preferred mode of
Commercially Available Solutions
Another solution that is available for freezing protein solutions at large scale uses stainless
steel vessels (cryovessels) from Sartorius-Stedim Biotech (Aubagne, France). These
cryovessels are available in multiple sizes (125-L, 200-L, and 300-L) and consist of a
jacketed stainless steel tank with an internal radial finned-heat exchanger. This effectively
divides the tank into six (or 10 for 125-L) longitudinal sections and has the effect of reducing
the heat-transfer distance and improved heat transfer across the entire volume. Dendritic ice
formation is promoted, thus avoiding the potentially damaging effects of cryoconcentration.
The vessels are cooled and heated by an external refrigeration system that circulates heat
transfer fluid through the jacket and fin system. The temperature profile of the heat transfer
fluid is programmable and results in reproducible temperature profiles in the vessel. The
vessel is kept stationary through the freezing process below 0 °C, but is gently agitated by
rocking during the thawing process. The lack of agitation during freezing prevents solutes
from moving and promotes the formation of dendritic ice. Agitation during thawing promotes
rapid mixing of the thawed material, thereby removing concentration hot spots and
maintaining uniform temperature in the solution with rapid thawing. The lowest working
temperature for the equipment is –60 °C.
A variation on the bulk freezing technology is the FreezeContainer from Zeta Holdings
(Styria, Austria). Jacketed vessels (currently limited to 300-L) are cooled or heated through
an internal circulation system (mounted in the lid). Heat exchange is accomplished by an
external refrigeration system by a circulating heat transfer fluid. The temperature profile is
programmable. The entire container is agitated during thawing.
A large-scale bag freezing system called Celsius from Sartorius Stedim Biotech uses upright
bags made of Stedim71 film (ethylene vinyl acetate product contact material) that are filled
with the solution to be frozen and held with slight compression between two plates that serve
as heat exchange surfaces. These plates are cooled or heated by circulating heat transfer
fluid from an external programmable refrigeration unit. The slight compression provides
improved contact and heat transfer resulting in a frozen bag in the shape of a pillow. The bag
is kept in frames so as not to stress the material during handling and transport. The sizes of
nominal bags are 16.6 L and 8.3 L, with fill volumes ranging between 4.2 L and 16 L, and 2.1
L and 8 L, respectively. Six bags can be simultaneously processed in the cryo unit.
Practical Considerations
The freeze and thaw behavior of proteins has been studied extensively, but primarily in small
or microscopic volumes and often in conjunction with lyophilization. The use of these small
volumes in literature studies makes the process aspects difficult to relate to the freezing and
storage of bulk proteins. A few studies have, however, elucidated fundamental aspects of the
impact of freezing on protein structure and interaction with ice and are reviewed by
Bhatnagar, et al.
An unavoidable feature of freezing is cryoconcentration as
water converts to ice and excludes the solutes (and
protein), ultimately creating a viscous glassy matrix (Figure
1). This can affect the embedded protein in a number of
ways. If the buffer salts are prone to crystallization because
of saturation, significant pH shifts can occur. Among the
common buffers used for biologics, the sodium phosphate
buffer mixture is particularly susceptible, and the pH can
change from seven to near four on precipitation of the
dibasic salt; the actual value is dependent on strength and
Even if the salts do not precipitate, buffer pH is sensitive to temperature, and
therefore, pH shifts will occur during freezing and in the frozen state. Other excipients in the
formulation can also cryoconcentrate. Although there is a complex dependence on factors
such as the rate of cooling and composition, phase and state diagrams provide some insight
into the cryoconcentrated system. If sodium chloride (NaCl) is present, a eutectic is formed
at –21.2 °C which has a concentration of 23.3% w/w, i.e., an approximately 25-fold increase
from 0.9% w/w normal saline. For most carbohydrates (including disaccharides), the
concentration of solute in a maximal freeze-concentrated glass is around 80% w/w.
Reactions that could lead to incompatibilities in the matrix are slowed down because of the
low temperature, but the cryoconcentration of solutes can counteract this effect. Reactions
such as oxidation can be enhanced, especially because the solubility of oxygen increases as
temperature drops, while ice formation also excludes gases. Other potential incompatibilities
among the solutes, including the protein, can be exacerbated. Proteins also interact with the
ice surface with a consequent perturbation of their native structure. Proteins can partially
denature at the ice interface through weakening of hydrophobic bonds as well as adsorption
on the ice surface.
This phenomenon is largely reversible after thawing, although some
fraction of the protein may become irreversibly damaged. More importantly, depending on
the storage temperature (in relation to the glass transition temperature of the
cryoconcentrated mass), this loss of protein structure can result in aggregate formation
because the partially unfolded molecules interact with other species around them. Storage
above the glass transition temperature (Tg') of the matrix will allow greater mobility for this to
occur. Similarly, other solutes (e.g., NaCl, glycine, mannitol, sorbitol) can phase separate,
crystallize, or undergo phase transitions over time if frozen into nonequilibrium states during
the freezing process, leading to protein destabilization.
Maximally freeze-concentrated
carbohydrate solutions relevant to biologics formulation tend to have a Tg' below –30 °C.
Less than maximally freeze-concentrated systems have even lower Tg' levels. Proteins
themselves have Tg' levels in the range of –10 to –15 °C, but freezing without
cryoprotectants is generally not viable.
Practical storage areas always have a degree of
temperature variability within which they are controlled. Temperature fluctuations, especially
in the vicinity of and above the Tg', can be especially detrimental because the rates of the
processes described above will increase significantly more than would be expected based on
the nominal storage temperature.
The large-scale storage systems discussed here attempt to control
the rate of heat removal and thereby obtain a reproducible process.
In the case of bottles or carboys, the exact pretreatment and
placement of the containers and load in the freezer must be
defined. Similarly, the thawing conditions and placement must be
established. The active systems from Sartorius-Stedim can be
programmed to provide reproducible temperature profiles in the
bulk. The actual profile is determined by the load, but a range of
loads (fill volumes) can be defined, qualified, and validated. During
operation, after a small degree of supercooling, ice formation is
Figure 1
Figure 2
generally nucleated throughout the bulk, although the growth is faster at the edges than in
the center. Depending on the rate and nature of ice growth in relation to diffusion rate,
solutes get trapped between growing ice crystals. Thus, concentration gradients are
generated and "frozen-in" in such systems. An example of concentration gradients observed
in bottles is shown in Figure 2(a). Such gradients can persevere if thawing is carried out
without mixing, as shown in Figure 2(b). Our in-house observations show that proteins and
other excipients cryoconcentrate to the same extent. Cryoconcentration occurs in the
cryovessels and bags.
Because a degree of cryoconcentration is unavoidable and the
protein is most vulnerable when the solute concentration is high but the mass has not been
completely immobilized, it is best to freeze as rapidly as possible. Doing so minimizes the
time the protein spends in the partially frozen high concentration but still mobile transition
Once processed, these bulk containers must be stored for a period of time. The storage
temperature is determined by the nature of the formulation as well as practical and logistical
considerations. Bottles and carboys can be placed in deep freezers at any desired
temperature that can be tolerated by the material of construction. The glass transition
temperature of high density polyethylene (HDPE) is –145 °C for the amorphous portion
(brittle temperature quoted as –100 to –70 °C), making it suitable for most applications.
polypropylene (PP), the glass transition temperature ranges from –15 to –10 °C requiring
that such containers be handled carefully in cold storage. Similar care is required for
containers based on ethylene vinyl acetate (EVA), which has a transition around –15 °C
although the brittleness temperature for film-grade EVA is stated to be as low as –75 °C or
below. Large stainless steel cryovessels have an operational temperature limit around –60
°C, although custom-built warehouses are required if storage below –20 °C is needed.
The process of thawing, while simple in principle, must be controlled properly to ensure that
the wall temperatures at the heat transfer surfaces do not exceed allowable limits for the
product. To ensure that the thawed material does not overheat while a remainder is still in
the frozen state, the mass should be agitated during processing, thereby ensuring efficient
heat transfer as well as preventing hot spots. Finally, a comparable system to perform long-
term stability studies is required to support regulatory filings. In the case of a bottle or carboy
storage, smaller units are used. Simple dimensional considerations imply that these cannot
be completely representative. For the cryovessels and bag freezing systems, small-scale
models are available to do process development. Their utility as stability models must be
Various light transmission or camera-based commercial systems are currently available in
the market and can be used to perform automated visual inspection (AVI) of sterile drug
The automated inspection machine (AIM) used in this study uses a light
transmission–based static division system to detect particles of foreign contaminants in vials
filled with liquid product. The vial containing the product is spun at a specified speed followed
by the application of brakes to stop the rotating vials. As the vial stops, the particles continue
to stay in motion while being suspended in the liquid, thereby causing interference in the
incident light that is detected by the sensor. The performance of an automated visual
inspection system should be qualified and characterized before its usage for inspecting drug
products filled in sealed containers such as vials and syringes. A Knapp study can be
conducted to establish the human capability for visual inspection and to set the performance
acceptance criteria for the AIM.
In the case study presented here, process parameters
that affect the performance of the AVI system, such as machine parameters, product
formulation, and fill configuration were evaluated. A standard vial defect set was prepared by
seeding filled clean vials with a single glass bead of size 70 µm, 100 µm, and 400 µm. Each
seeded vial contained only one glass bead of a specific size. Two vial sets comprising 24
vials each (six clean, and six of each particle size) were run through the AVI system 32 times
and the detection results (accept/reject) for each vial were recorded. At the end, percent
detection rate (% DR) of the machine for each vial was evaluated as the ratio of the number
of times the vial was rejected by the machine and the number of times the vial was
inspected. Studies using product mimic solutions were designed to evaluate the effect of
each process parameter on the defect detection rate by the machine.
Role of Machine Parameters
Key machine parameters affecting the process performance
include spin speed (how fast the vial is spun, measured in
rpm), brake setting (how quickly inspection is performed after
applying the brakes), sensitivity (signal to noise ratio),
inspection view height (based on liquid meniscus height), and
background light intensity. Sensitivity and background light
settings were optimized and held constant throughout this
study. Inspection view height was altered based on meniscus
height. Various formulations of different viscosities were then
tested by changing the brake and spin speed settings. Figure 3
shows that increasing the spin speed improves the detection
rate, the impact being more significant for high viscosity products. Detection of the glass
particle by the machine requires the particle to move and stay suspended in the detection
window. Higher spin speeds transfer more momentum from vial to liquid and then from liquid
to the glass particle, thereby resulting in larger particle movement over longer durations.
Inspecting the vials quickly after applying the brakes (a higher brake setting) also seemed to
help the detection rates.
Role of Product Formulation
In addition to machine parameters, the product formulation
also can have a significant effect on the ability of the machine
to detect particles. Similar to the observations in Figure 3, we
observe deterioration in the detection rate as product viscosity
increases. For low viscosity solutions, a spin speed of 1,600
rpm is sufficient to achieve detection rates of >80%. However,
for high concentration products with viscosities >4 cP, process
performance deteriorates significantly at low spin speeds.
Higher spin speeds of 2,200 and 2,800 are needed to achieve
the same detection rate. Other product properties such as
density (relative to defect particle density) and surface tension
also may affect the performance of an AVI system. Figure 4 compares the detection rate
(average of 100 µm and 400 µm) of the machine for two mimic solutions of equal viscosity
(2.3 cP) but different surface tensions. The formulation without polysorbate (PS-20) had a
higher surface tension and was more challenging for the AIM than the one with polysorbate
for the detection of 400-µm particles. The results highlight the importance of using a
representative mimic solution that mimics not only a product's viscosity but other properties
as well.
Role of Fill Configuration
Figure 3
Figure 4
The fill configuration of the product SKU also affects AVI
performance. The automated inspection of syringes can be
more challenging than vials because the smaller radius of
syringe barrels reduces the momentum imparted to particles
for a given spin speed. The fill volume of the liquid in the
container also can have a bearing on the ability of the
machine to detect particles. Figure 5 compares the machine
performance for low and high fill volumes for two different vial
sizes. For both 5 cc and 10 cc, the detection rate (plotted as
the average of 100 µm and 400 µm) is lower for very low fill
volumes. When the fill volume is reduced, the inspection
window available to the sensor becomes smaller, thereby reducing the detection rates.
Intermediate fill volumes did not impede performance as much as the low fill volumes.
The automated visual inspection of biopharmaceuticals is a key process step in the fill–finish
process and offers several benefits over manual inspection, including higher speed, better
detection, and improved process consistency. The machine should, however, be qualified
and characterized before its usage for product lot inspection. Machine parameters, product
properties, and fill configuration are all important factors that determine the performance of
the AVI system. In addition to actual product, appropriate mimic solutions can be used to
characterize the effect of these parameters and design an AVI process that is robust and
Particles may be generated as a result of large-scale manufacture or because of an inherent
property of the protein molecule. The large-scale manufacturing of protein drug products
involves processing steps such as purification, formulation, freeze–thaw, filling, shipping, and
storage. Stresses that are introduced during these steps can cause instabilities that can lead
to aggregation and particulation.
In this case study, we discuss some important factors that
may be used to control particulation for a monoclonal
antibody product. The data presented are from the
formulation development of a monoclonal IgG2 antibody. A
typical example of visual particulation in a
biopharmaceutical liquid formulation is shown in Figure 6.
The particles in these formulations can be counted by
instruments such as the HIAC Royco liquid particle counter and characterized by purifying
the particles and subjecting them to protein analysis. During formulation development,
screening studies are performed to study the effect of factors such as pH, buffering agents,
excipients (sugars, surfactants), and protein concentration to minimize the presence of
The Effect of Polysorbate
Figure 5
Figure 6
Figure 7 shows the effect of including a low amount
(0.004% by weight) of Tween 80 (polysorbate 80) on the
particle counts in a particular IgG liquid formulation.
Samples were stored at 4 °C for three months and then
analyzed for particulates. These IgG preparations were
either derived from a hybridoma cell line or a Chinese
hamster ovary (CHO) cell line. In the absence of Tween,
the hybridoma-derived material had higher particle counts
compared to the CHO-derived material. This difference
may be a result of the inherent nature of the protein
molecule or differences between the two processes. A size
range of 2, 5, 7.5, 10, 20, and 25 µm is shown at 10 and 20 mg/mL. As evident from the
graph, the 2 µm counts are orders of magnitude higher than the other size range and should
be an important consideration in the particle analysis.
The Effect of pH
Figure 8 shows the effect of varying pH on particulate
counts for 10-, 20-, and 25-µm particle size. Samples were
formulated in a poly buffer system with a common excipient
to maintain osmolality. Samples were then stressed over 24
h using a tumbling apparatus. The tumbling action was
used to represent agitation stress that may be experienced
during the transportation of drug product. This particular
antibody is more stable in the acidic pH range from 5 to 6.
At neutral and basic pH, however, the particle counts are
significantly increased. The exact reason for this particle
increase as a function of pH is not known, but it may be related to changes in the surface
charge distribution of the molecule as the pH is increased from 5.0 to 7.5, causing the protein
to become less soluble. It was also noted that formulations containing higher particulate
counts showed increased dimer levels by size exclusion chromatography (data not shown).
Detecting Particles with Flow Imaging Technology
Recently, flow imaging technology has emerged as an orthogonal technique to measure
subvisible particles, in addition to light obscuration–based techniques.
In this setup,
samples are made to flow through a microfluidic cell, and digital images of suspended
particles are captured. The images are then analyzed by the software to count particles and
estimate their size. In addition to being an orthogonal technique to light obscuration, flow
imaging also has the advantage of making it possible to view the particle in question. The
image and the aspect ratio (ratio of longer to shorter dimension) helps differentiate if the
particle is an aggregate or silicon-oil droplet, some other foreign particle, or even an air
Figure 7
Figure 8
Figure 9 shows particulate analysis using microflow imaging (MFI) of a
different IgG2 monoclonal antibody. Particulation behavior of this
molecule stored in a glass prefilled syringe was compared to a glass vial,
in a formulation that lacked polysorbate. Data are plotted as total particle
counts for a variety of particle ranges (from 2 to >125 µm). Under these
conditions, the prefilled syringe produced significantly more particles than
the glass vial, across the different size ranges (up to >50 µm). This was
most likely caused by the phenomenon of silicon-oil–induced
Characterizing and controlling particulates through a rational formulation
screening process is an important part of protein drug development. Careful analysis of
particle generation through downstream processing, storage, and transportation should be
an important consideration in drug development. Furthermore, particle detection and
quantification using advanced techniques has become an integral part of biopharmaceutical
Nitin Rathore would like to thank Cylia Chen and Oscar Gonzalez of Amgen, Inc. for their
support in conducting these studies and Wenchang Ji, Erwin Freund, and Ed Walls for
review and useful feedback. Arnold McCauley would like to thank Sekhar Kanapuram, Hyo
Jin Lee, Alexis Leuras, Lyanne Wong, and Rahul Rajan, all from Amgen Inc., for providing
data, editing the manuscript, and helpful discussions.
Satish K. Singh, PhD, is a research fellow at Pfizer Inc., global biologics, Chesterfield, MO,
Nitin Rathore, PhD, is senior scientist in process development and Arnold McAuley, PhD,
is scientist in formulation and analytical research, both at Amgen, Inc., Thousand Oaks, CA.
Anurag S. Rathore, PhD, is a consultant, Biotech CMC Issues and a faculty member in the
department of chemical ?engineering at the Indian Institute of Technology, Delhi, India, []
Rathore is also a member of BioPharm International's editorial advisory board.
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(Althea Technologies, Inc.)
Anurag S.
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Figure 7
... Usually, the batches are split into smaller volumes to fit inside bottles, carboys, or bags for storing, transporting, freezing, and thawing (2). However, this may require multiple disposable containers, and consequently, multiple containerclosures are involved, leading to some risk of contamination (3,4). Although the disposable bags offer some advantages, the potential break of a bag or tubing assemblies, leading to contamination and loss of product, is a serious concern, compared with bottles/carboys (2). ...
... In particular, damage of bags during freezing and handling at low temperature is regularly reported (5,6), e.g., during transportation. The bottle/carboy systems are simple and the preferred mode of operation of many companies, especially when biopharmaceutical formulations are robust and stable under a wide range of freeze-thaw conditions (3,4). Notwithstanding, freezing, handling, and transportation of bottle/carboys at low temperature also present risks. ...
Bottles and carboys are used for frozen storage and transport of biopharmaceutical formulations under a wide range of conditions. The quality of freezing and thawing in these systems has been questioned due to the formation of heterogeneous ice structures and deformation of containers. This work shows that during freezing of bulk protein solutions, the liquid at the air–liquid interface freezes first, forming an ice crust and enclosing the liquid phase. As the enclosed liquid freezes, internal pressure rises, pushing the liquid phase through the porous ice crust towards the air interface, leading to interfacial stress and protein aggregation. The aggregation of bovine serum albumin was more intense in the foam-like ice mound that was formed at the top, where bubbles were entrapped. This was characterized experimentally with the assistance of magnetic resonance imaging (MRI). An isothermal cover is proposed to prevent the early freezing of the liquid at the air interface, attenuating substantially interfacial stress to proteins and releasing hydrostatic pressure, preserving the shape and integrity of the containers.
... 18 Also, manufacturers of biologicals encounter challenges specific for protein manufacturing and formulation. [19][20][21] The aim of this study was to assess the challenges experienced by companies developing ATMPs in Europe. Experiences were collected via a survey distributed among identified ATMP developers active in Europe. ...
... 18 Also, manufacturers of biologicals encounter challenges specific for protein manufacturing and formulation. [19][20][21] The aim of this study was to assess the challenges experienced by companies developing ATMPs in Europe. Experiences were collected via a survey distributed among identified ATMP developers active in Europe. ...
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Advanced therapy medicinal products (ATMPs) hold promise as treatments for previously untreatable and high-burden diseases. Expectations are high and active company pipelines are observed, yet only 10 market authorizations were approved in Europe. Our aim was to identify challenges experienced in European ATMP clinical development by companies. A survey-based cohort study was conducted among commercial ATMP developers. Respondents shared challenges experienced during various development phases, as well as developer and product characteristics. Descriptions of challenges were grouped in domains (clinical, financial, human resource management, regulatory, scientific, technical, other) and further categorized using thematic content analysis. A descriptive analysis was performed. We invited 271 commercial ATMP developers, of which 68 responded providing 243 challenges. Of products in development, 72% were in early clinical development and 40% were gene therapies. Most developers were small- or medium-sized enterprises (65%). The most often mentioned challenges were related to country-specific requirements (16%), manufacturing (15%), and clinical trial design (8%). The European ATMP field is still in its early stages, and developers experience challenges on many levels. Challenges are multifactorial and a mix of ATMP-specific and generic development aspects, such as new and orphan indications, novel technologies, and inexperience, adding complexity to development efforts.
... Here in part 2, we examine some technologies available for large-scale freezing and storage and provide guidance on rational development of formulations and processes for this unit operation. An abridged version of this review was published elsewhere (2). ...
This chapter will describe the current state of the art and expectations of performing inspections of drug product with a focus on those defects that are visible to the eye. Visible attributes cover cosmetic and functional defects using both manual and automated techniques that use appearance as the key characteristic. The section will cover inspection attributes, compare and contrast manual and machine-based inspection with regulatory expectations, limitations, technologies, and provide several examples through case studies.
Commercializing therapeutic proteins involves a series of processes aimed at maintaining safe and efficient protein drug solutions before final patient administration. Common operations include important steps such as preformulation, drug product formulation, sterile filtration, freezing, thawing, and freeze-drying intended to stabilize the protein drug before fill-and-finish, and during storage and transportation. Freeze-thaw operations used in the biotechnology industry still are generating debates regarding safety problems because methods to freeze and thaw samples can affect the purity, activity, safety, and efficacy of the final product. This article presents a Quality by Design (QbD) approach to define a safe freeze-thaw space where a protein's quality is not affected by the freezing or thawing method used.
This chapter provides an outline to some of the key events that have led to the current level of interest in peptide and protein drugs and defines the terms "peptide" and "protein." A consensus has not been properly reached with respect to the use of these latter two terms, with the 51 amino acid, mature human insulin being a good example of the ambiguity, since it is generally described as a peptide but also as a protein by some. As a guide, peptides can be considered to be up to 50 amino acids in length, with proteins being larger than this. This boundary corresponds approximately to the upper limit of routine peptide synthesis in the solid phase. The rise of molecular biology as a tool by which to generate biopharmaceutical drugs-those that include proteins, DNA, conjugates, viruses, etc., initially far outpaced the development of delivery technologies. These biomacromolecules almost inevitably do not survive the stomach and intestinal environment due to pH and the presence of proteases. Nor do they readily transit the epithelial barrier due to the presence of cell-cell tight junctions, the semipermeable cell membrane, and, for intestinal epithelia, efflux proteins such as P-glycoprotein and the cell glycocalyx. Furthermore, biomacromolecules are very much larger than organic drugs, have short plasma half-lives, are involved in active transport processes, are susceptible to chemical and physical degradation, and are very potent. The need to overcome these challenges to their delivery has resulted in huge volumes of research, drawn from many disciplines including pharmaceutical materials, chemical engineering, biophysics, analytical methodology, cell and molecular biology, and in vivo studies.
IntroductionFormulation and Stability of Protein SolutionsFormulation of VaccinesReferences
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An important responsibility of a formulator for a biologic that is not often recognized is the definition of storage conditions (container/ closure and temperature) of the bulk drug substance prior to its conversion into drug product. This article examines the options for storage of biologic drug substance in a liquid state in either disposable polymer bags or in stainless steel (SS316L) tanks. Data from developmental studies at small-scale has shown that all of these options may be appropriate depending on the length of storage required, as well as the stability determining characteristic(s) of the biologic. In the example presented, aggregation and oxidation are the main determinants. Apoint to note is that the development studies usually performed at small scale represent a worst-case scenario in terms of the container (surface area to liquid volume) ratio compared to full-scale systems. Performance in full-scale systems will generally be better than that observed in these model systems.
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A key concept in the Quality by Design paradigm is design space - a multidimensional space that encompasses combinations of product design and processing variables that provide assurance of suitable product performance. This article discusses design space in the context of developing, scaling up, and transferring freeze-dried products to a manufacturing setting. Smooth technology transfer starts with a robust formulation and an appropriate container and closure system. The design space is developed as an envelope in a graph of sublimation rate, shelf temperature, and chamber pressure. One boundary of the design space is established by failure of the formulation under aggressive cycle conditions. Other boundaries of the design space are determined by equipment performance, including refrigeration capacity, condenser capability, heating capacity, or limitations of the dynamics of water vapor flow within the system. Definition of this design space assures a thorough understanding of both the product and the process, and it minimizes the probability of unpleasant surprises in the technology transfer process.
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Aggregation continues to be one of the major concerns in development of biotherapeutics. However, understanding of aggregation pathways and inhibition of aggregation remain relatively poor. Protein particulates form in a wide range of size and shape, and it is extremely challenging to comprehensively characterize the whole size range of particulates in a given biologics formulation. This article highlights the major issues due to insoluble aggregate formation in biologics development. In particular, the detection gap for the subvisible size range is discussed.
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Subambient thermal analysis with differential scanning calorimetry and thermomechanical analysis has been used successfully to optimize the lyophilization of proteins based on the thermal behavior of solution components. In deionized water, frozen protein solutions exhibit two glass transition temperatures (Tgs): the first ca. − 80 °C (Tg″), and a second ca. − 10 °C (Tg′); the latter appears to be related to the collapse phenomenon. Proteins lyophilized in buffers with low Tg values such as Tris (− 81 °C) or sodium acetate (− 80 °C) are collapsed during primary drying at − 20 °C. Substitution of salts which crystallize readily during freezing and have eutectic melting temperatures (Tes) above − 20 °C, e.g., sodium carbonate (Te = 3 °C), prevents the protein solutions from being collapsed during the same process. The crystallization of these salts during freezing, which may lead to undesirable changes in pH, can be prevented by use of excipients such as sugars. To remove the amorphous fraction in partially crystallizable eutectic salts which exhibit very low glass transition temperature values, an adequate annealing at temperatures between devitrification and eutectic melting is necessary. Annealing is not effective for wholly amorphous glass-forming salts. However, the final Tg′ of protein solutions which contain such salts can be raised by adding excipients or by increasing the protein concentration.
During the past decade, the importance of amorphous water-soluble substances has been increasingly recognised within the food and pharmaceutical industries. In response, Amorphous Food and Pharmaceutical Systems brings together current leading experts to contribute to this unique cross-disciplinary account of the subject. Coverage includes: water-compatible amorphous solids (physical, chemical behaviour), low water content systems (water as plasticizer); applications in food and pharmaceutical sciences and industries (processing and stability) along with state-of-the-art technology in food and pharmaceutical systems. This timely publication will be welcomed by academic and industrial researchers and professionals in the pharmaceuticals, food, materials and polymer sciences.
Large-scale freezing and thawing is commonly used in biopharmaceutical manufacturing but is not well understood. Freeze-thaw variations can exist within or between batches, and nonuniform processes raise serious validation concerns.
Two novel approaches to cryopreservation have spurred the development of large-scale systems for biopharmaceutical products: freezing and thawing using internal heat transfer surfaces and freezing and granulation using volumetric heat transfer. Both have been applied to large volumes of biological solutions.
A set of equations have been developed that allow the T′g, C′g intersection point of the equilibrium liquidus curve with the kinetically controlled glass transition curve on the state diagram to be calculated for many aqueous frozen solutions of mono-, oligo- and polysaccharide products. These equations allow the T′g and C′g intersection point on the state diagram to be calculated from just knowledge of the glass transition temperature and the corresponding specific heat change for the pure solute and pure water. C′g and T′g values currently have to be determined experimentally, which can be both time consuming and tedious. The availability of these equations should now allow C′g and T′g values to be calculated for many more solutes. Having more ready access to this information should be of particular benefit to food scientists, because this position on the state diagram is known to be particularly technologically significant in terms of understanding and predicting the processing and storage properties of frozen foods.