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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.
ABSTRACT
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.
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.
1
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.
BACKGROUND
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.
1
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.
2
The
development of a design space in the context of developing, scaling up, and transferring
freeze-dried products has been discussed in recent publications.
3
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.
4
The stability of
therapeutic proteins during long-term storage has been highlighted as a key issue for product
safety and efficacy.
1
Storing drug substance for periods of time in the frozen state enables a
(Althea
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.
5
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
formulation.
6
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.
7–9
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.
10
One important factor is potential
immunogenicity, which can be a result of poor quality of the protein product.
11,12
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.
13
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.
ENSURING PROTEIN STABILITY DURING FROZEN STORAGE
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
operation.
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.
14
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.
15
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
rate.
15
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.
16
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.
17
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.
18
Maximally freeze-concentrated
carbohydrate solutions relevant to biologics formulation tend to have a Tg' below –30 °C.
19
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.
19–20
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.
21,22
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
region.
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.
23
For
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
evaluated.
AUTOMATED VISUAL INSPECTION
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
products.
24–26
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.
27–28
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
consistent.
PARTICULATES IN BIOPHARMACEUTICAL FORMULATION
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.
29
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
particles.
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.
10
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.
30
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
bubble.
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
particulation.
31
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
development.
ACKNOWLEDGEMENTS
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,
asrathore@biotechcmz.com [http://mailto:asrathore@biotechcmz.com]
Rathore is also a member of BioPharm International's editorial advisory board.
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(Althea Technologies, Inc.)
Anurag S.
Rathore
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