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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5366_Библиотеки_им_академика_М_И_Перельмана.pdf
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Fig. 5.2 Example of a manufacturing process using the two-cell line approach
manufacturing process, including a potential position for the redox step, is presented in Fig.5.2. This approach solves the LC pairing problem by simply restricting LCs to one cell line and one process. While the reduction step likely reduces
the disulde bond formed between the HC-LC pair, the Fab does not dissociate,
due to the tight molecular interactions between the Fv, as well as the light chain
constant domain and CH1. Cell line generation for this type of multispecic is
much like a standard monoclonal antibody; vector congurations, CHO host cells,
and clone screening strategies used for the generation of the homodimer cell lines
are compatible with those used for standard mAbs. The only major difference
between generation of cell lines for mAbs and charged based multispecics is that
two cell lines are established. The need for multiple cell lines and at least separate
upstream manufacturing processes/Protein A chromatography steps can pose a
challenge for at scale manufacturing, which will be addressed later in this chapter.
It should also be noted that if observed, a half-antibody impurity after Protein A
purication is acceptable, as this species is competent for incorporation into a heterodimeric molecule during the redox step. Since the cell line generation approach
used for two cell line multispecics is highly similar to well-established approaches
for mAbs, the rest of this section will focus on cell line approaches for single cell
line multispecic molecules.
5 Process Development and Manufacturing Considerations for Multispecic…
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5.2.1.2 The Knob into Hole Approach
Based in part on a model proposed by Francis Crick for packing of amino acid side
chains, the concept of the knob into hole approach is to facilitate HC heterodimerization by mutation of an amino acid residue within the CH3 domain in one HC
from a large to a small amino acid (the “hole” chain) and then from a small amino
acid to a larger one (the “knob” chain) [16]. In the rst iteration of this strategy, the
hole chain harbored a tyrosine to threonine (Y407T) mutation, while the knob chain
bore a threonine to tyrosine (T366Y) mutation. Following co-expression from a
single cell line, the resulting Protein A puried pool was comprised of greater than
90% of the desired heterodimer [16]. Further protein engineering efforts obtained
via phage display screening yielded a change to the knob mutation, from tyrosine to
tryptophan (T366W), as well as two additional mutations in the hole heterodimer,
T366S and L368A, which improved molecule stability following cellular expression [17].
While this heterodimerization strategy is quite robust, it does not account for the
LC pairing problem. One simple way to alleviate this concern is through use of a
common LC for both antigen binding arms; however, not all targets may be easily
amenable to this approach. Another potential strategy to overcome LC pairing challenges is through domain crossovers [18]. In this approach, one Fab (fragment antigen binding) arm would retain its canonical LC/HC structure, while the other Fab
HC variable domain would be expressed as part of a “new” LC, either fused to CH1
(HC constant domain one) or CL (LC constant domain), and the LC variable domain
would be expressed as a “new” HC, and be fused to either CH1-Fc or CL-Fc (e.g.,
see Fig.5.1). This approach solves the LC pairing problem as the “new” LC would
not be competent for binding to the standard HC on the rst Fab arm and likewise
the same for the new HC’s ability to bind to the standard LC.However, depending
on the molecular format selected, new impurities can be generated [18].
Another strategy to overcome the LC pairing problem is through genetic engineering based upon either charge or hydrophobicity of the CH1-CL interface to
favor correct pairing [19]. In a previously described charged-based approach, a
negatively charged amino acid was substituted in the CH1 domain (T192E), while a
positively charged amino acid was introduced into the CL domain (N137K). These
mutated residues form a salt bridge, promoting specicity between this HC/LC pair
while helping to avoid improper pairing. A similar strategy can be used by swapping
out a pair of amino acids on each chain, one apolar pair of residues for a polar pair
(L143Q in CH1 and V133T in CL), and vice-versa (S188V on CH1 and
S176V on CL).
In terms of cell line generation, when comparing HC heterodimerization
approaches (charged-based vs. knob into hole), it may seem as though the knob into
hole approach would be favorable, as it only requires development of one cell line
per molecule, as opposed to two. This may be true in some circumstances, such as
in cases where multiple targets are being considered for a particular Fab, or multiple
constructs are being considered for a single target. However, the single cell linebased knob into hole approach may present more technical challenges that are
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difcult to overcome. These include the ability to screen for high levels of product
titer, as well as low levels of product-based impurities (e.g., homodimers and
mispaired LCs), and difculties in cellular genetic characterization. Cell line stability may also offer a challenge with the knob into hole approach, as a greater number
of peptide chains (which can also be more unique than LC/HC) are being expressed
in a single cell. Proper expression ratios must be maintained over generational age,
which may be more challenging for the cell, when compared to expressing homodimers in separate cell lines. The following sections offer considerations and strategies for overcoming challenges faced when generating single cell lines for the
expression of knob into hole multispecics.
T. Iskra etal.
5.2.2 Cell Line Generation Considerations forSingle Cell Line
(SCL) Multispecics
5.2.2.1 Stable CHO Host Cell Integration System—Random or Targeted?
Currently two prevailing stable CHO host cell expression strategies are used for the
production of biotherapeutics. One strategy, the random integration platform, results
in transgene sequences that are integrated within the CHO host cell genome at random numbers of copies and chromosomal locations. An alternative to this approach
is the targeted integration platform, wherein the transgene sequences are enzymatically inserted into pre-dened genomic loci [20]. Both systems have proven to be
efcient vehicles for the production of SCL multispecics [21, 22].
The indiscriminate nature of the random integration system, which is sometimes
viewed as a negative for the expression of standard monoclonal antibodies, can be
useful for the expression of SCL multispecics. The differing chromosomal locations of transgene vector integration can vary with respect to the amount of transcriptional activity they provide transgenes [23]; this variability can be used as an
advantage. Certain chains within the multispecic may exist at a higher molar ratio
within the molecule relative to other distinct chains (e.g., common LC multispecifics), which would necessitate higher expression of these genes. Further, some chains
that comprise the multispecic may not be transcribed and/or translated as efciently as others. By virtue of transgene insertion at arbitrary genomic locations,
random integration can provide a range of transcriptional efciency for transgenes
that can easily be harnessed through single cell cloning and screening for proper
product formation. In other words, it is possible to leverage the “random” nature of
the random integration system for the production of multispecics, but appropriate
analytical screening strategies are required; this will be discussed further below.
For targeted integration, typically the pre-dened loci for insertion of transgenes
are ones that provide high and stable transgene expression [24, 25]. Unless a battery
of insertion sites is identied, characterized, and developed, targeted integration
does not offer the benet of transcriptional differences that random integration has
the potential to provide. One potential benet of targeted integration is that
5 Process Development and Manufacturing Considerations for Multispecic…
113
determination of the likelihood of success/failure happens more quickly; analysis of
material generated from targeted integration transfection pools can generate meaningful results with respect to both product titer and product quality, whereas with
random integration a cloning step is more likely to be required. If targeted integration is found to be suitable for the expression of SCL multispecics, cell line stability is also less of a concern compared to random integration due to the predictable
stability of the integration site.
5.2.2.2 Expression Vector Considerations
The expression vector and transgene topologies serve as another potential source of
variability of transcriptional efciency; if appropriately leveraged, knowledge of
this variability can lead to higher total multispecic yields and lower product-related
impurities. Governing rules from expression of standard mAbs can serve as a foundation for the production of SCL multispecics. First, transcriptional analysis of
several standard mAb expressing cell lines has established that of the two chains,
the HC is limiting in its expression. In other words, there is a direct correlation
between HC transcript/peptide and mAb product titer [26]. In addition to this relationship, it has also been demonstrated that LC needs to be in excess to HC, as
mathematical modeling of the mAb synthesis pathway suggested that IgG assembly
time and LC-HC ratio are inversely correlated [27]. Cell lines which both naturally
and have been engineered to express mAbs typically express LC in excess [28, 29].
Scenarios in which the HC is forced to higher expression than LC result in HC
aggregation, as well as a decrease in cell viability [30]. The simplest way to account
for these rules in developing expression vectors for mAbs is to construct a vector
positioning the LC upstream of the HC, using identical promoters and polyadenylation signals that confer very high expression. In this topology, the LC would
theoretically not be subjected to transcriptional interference in the same way that the
HC would (from the LC), thus providing a boost in expression relative to HC such
that the LC would not be limiting. Other options for optimizing LC-HC expression
ratio include varying promoter/terminator strength, and varying gene copy number
within the vector.
If opting for a single vector approach, the position of each chain within the vector
must be carefully considered. Chain position within the vector has the potential to
impact transcriptional activity within the cell, which can have downstream effects
on overall product yield, product quality, and therefore even efcacy and safety. To
compensate for any transcriptional interference effects on chains positioned further
downstream within the vector, the use of stronger promoters/terminators, or inclusion of multiple copies of the same gene should be considered such that desired
molar balance in the nal product can be achieved. The use of different promoters/
terminators may be preferable in this instance, as they can aid in ease of vector construction, sequencing, and genetic characterization of production cell lines.
Multivector strategies could also be employed. In this scenario, several vectors
could be constructed having either some or all transgenes, in different orientations.
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Selection of cells that have taken up the expression vector is usually accomplished
through the vector harboring a gene expressing either (a) a protein that confers
resistance to an antibiotic such as geneticin or hygromycin or (b) a protein that
complements an auxotrophic phenotype, such as glutamine synthetase. In the random integration host system, cells are amenable to integrating several copies of the
vectors harboring these genes as a means of surviving the selection. To further
enhance clonal diversity, cell transfections could be performed using different ratios
of vectors. A single selection scheme could be employed, such that all vectors have
the same selectable marker. This approach would require intensive screening for
clones producing the desired product quality, and cell line stability could also
become an issue. To limit these issues, multiple selection schemes could be used
(e.g., one vector harboring a geneticin resistance gene, another harboring a hygromycin resistance gene); however, this approach may limit clonal diversity, as cell
selection would be more stringent.
For a targeted integration approach to the production of SCL multispecics, variations in vector topology serve as the only sure way to inuence expression differences between genes coding for the different molecule chains. Due to the genetic
loci of insertion being controlled across the entire transfection pool cell population,
if a vector topology is chosen that results in poor expression/molecule product quality, it will be very difcult/impossible to screen for a high-expressing clone.
Therefore, an upfront evaluation of multiple vector congurations is paramount for
the expression of SCL multispecics in a targeted integration system [21].
Fortunately, this can be facilitated by the targeted integration platform, as these
types of screens can be performed prior to single cell cloning, on material generated
from the cellular transfection pools. Depending upon the exact mechanism/genetics
of the targeted integration host cell system, pool screening results should not be
expected to exactly mirror results from the clones; however, general trends should
remain consistent; pools with higher expression and better product quality should
yield better clones. Once cellular pools harboring a certain vector topology yield
adequate results, clones can be generated, which can then be subjected to further
screening. Thus, in the absence of a clear understanding of the relative expression
of the individual polypeptide chains, and a similar understanding of vector positional effects, early vector screening is essential.
5.2.2.3 Cell Line Screening Strategy Considerations
When compared to cell line screening for the expression of standard monoclonal
antibodies, clone screening for SCL multispecics typically requires (a) screening
a larger number of clones, and (b) different clone screening analytical tools (for
illustrative example, see Fig.5.3). The natural existence of monoclonal antibodies
coupled with decades of experience and learnings can facilitate development of
standard monoclonal antibodies without much upfront analytical screening. In the
absence of these benets, SCL multispecics need tools that can provide both
meaningful information and rapid results such that clone culling can happen as
Monoclonal ant ibody
Single Cell Line (SCL) Mult ispecif ic
5 Process Development and Manufacturing Considerations for Multispecic…
115
Screening Criteria Illustrave Result
Screening step:
Screening Criteria Illustrave Result
Fig. 5.3 Comparison of example cell line screening strategies and potential results for both a traditional monoclonal antibody and a single cell line (SCL) multispecic. Since SCL Multispecic
cell line screening strategies need to consider product quality results more than traditional monoclonal antibody cell line screening, these typically employ more screens with lower success rates,
resulting in the need to screen signicantly more clones
efciently as possible. As the cell line generation process progresses and the number of clones that are being analyzed decreases, different, more intensive molecular
screening can take place. Because of this, an upfront assessment of the possible
impurities that may be encountered, development of a robust screening strategy, and
inventory of other cell screening reagents (e.g., homodimer impurities and target
antigens) must be completed to ensure a successful cell line generation campaign.
Creation of cell lines intended to exclusively express the homodimers should be
strongly considered, as this reagent is not only a critical component of cell line
screening, but may be useful for other facets of development, such as assay development for characterization and release testing. For these, cell line screening and generation will likely not need to be as extensive, and dependent upon the expression
system used, transfection pools are often sufcient for their production.
One of the rst screens that should be employed while clone screening for IgGlike multispecics is the ability to make material capable of binding to Protein A
(Fig.5.3). This screen should be implemented shortly after single cell cloning, perhaps even as quickly as upon outgrowth of the single cell cloning vessel. While this
assay will give a general idea as to whether a given clone is expressing IgG-like
molecules, it does not discriminate between clones expressing high amounts of HC
homodimers, or mispaired LCs. Therefore, results from this screen should be treated
as preliminary. If feasible, taking forward all clones that can produce Protein A
bound material should be considered, allowing for the highest amount of clonal
diversity. If utilizing the domain crossover approach [18], the framework of the
VH-CH1 peptide should be considered. Binding of HC to Protein A can be accomplished through interactions other than the HC Fc domain, in particular for HCs
with the VH3 framework domain [31, 32]. If VH3 is used for the VH-CH1, the
resulting peptide will be independently capable of binding to certain Protein A
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resins, resulting in articially high titer values. In this case, preliminary titer determinations that do not rely solely on Protein A binding may be used.
Either simultaneous to, or shortly after the initial Protein A screen, the ability of
clones to express molecules that can bind to both/all target antigens should be considered (Fig. 5.3). For this assay, we typically employ ELISA (enzyme-linked
immunosorbent assay) screening, which offers a robust combination of information
and speed to complement a clone screen strategy, as hundreds to even thousands of
clones can be efciently subjected to this screening. In theory, two ELISA screens,
one for each antigen, could be conducted, although it would be preferable to design
a format which can provide information on the ability to simultaneously engage
both antigens in the same assay, as this has the potential to eliminate clones producing only mixtures of incorrect homodimers. Much like a Protein A titer assay, the
ELISA assay can be used to determine protein quantities. For those clones that can
bind to both antigens, ratios between material bound to one antigen versus another
or versus total Protein A bound material can be established and used as screening
criteria. It should be noted, however, that these ratios represent a snapshot in time of
an early screening stage. If the cell line experiences genetic drift, these values may
not remain static throughout cell line development. Results from these screens
should be treated semi-quantitatively, and ratio ranges to determine progression to
the next round of screening should be kept large.
Once clones producing material capable of simultaneously binding to both target
antigens have been identied, product quality must be examined (Fig.5.3). One
such screening assay to be considered for this purpose is analytical size exclusion
chromatography (SEC). This method is useful for obtaining high-resolution separations of desired protein molecules from other product related species, such as
HMMS and degraded forms. For detection of smaller fragments, methods based on
gel electrophoresis should be considered. These methods can either utilize standard
polyacrylamide gel electrophoresis or capillary electrophoresis, the latter of which
may lend itself better to high-throughput screening. The electrophoretic methods
are suitable for identifying clones producing material with signicant size differences, for example, missing one or more of the desired polypeptide chains, but may
not be suitable for identifying clones producing product-related impurities that are
similar in size to the desired product (e.g., homodimers). If better resolution is
required than what can be provided by electrophoretic methods, mass spectrometry
may be needed, although screeningmaterial from large numbers of clones by this
method can prove cumbersome.
For some molecules, the size of homodimer impurities may be too close in size
to the desired heterodimer to be able to differentiate for screening purposes. In these
cases, other methods of separation must be explored. This can include separation
based on attributes such as charge, hydrophilicity, and hydrophobicity. These types
of screens will require sufcient amounts of homodimer impurities to serve as controls, which can be generated via their own cell line generation efforts, as mentioned above.
Once clones have been identied that can produce high amounts of the properly
assembled molecule, they should continue to be assessed using more traditional
5 Process Development and Manufacturing Considerations for Multispecic…
clone screening strategies. This includes phenotypic assays such as performance in
a small-scale bioreactor process, using cells that have been passaged to an appropriate generational age. Upon harvest, it is prudent to conrm molecular structure with
one of the previously established assays to determine the impact (if any) that the
generational age and bioreactor environment have on molecular assembly. It has
been our experience thus far that analytical results from material harvested from a
small-scale vessel (e.g., plate well or shake ask) have been comparable to material
from a more representative process, such as a fed-batch bioreactor.
Just as a standard mAb, stability of the expression construct to the limit of invitro
cell age (LIVCA) needs to be conrmed. In addition to loss of product yield, loss of
transgene(s) expression can also have signicant impact on product quality for multispecics. If screening clones transfected with different vector congurations, it is
good practice to maintain clone vector diversity at least through the cell line stability assessment. Certain vector congurations may be able to yield high/optimal
expression and product quality, but unable to maintain this phenotype over cellular
generational age. Traditional methods of ensuring transgene stability (quantitative
PCR, Southern/northern blotting, etc.) may have difculty in discerning between
the genes coding for the different chains of a multispecic molecule. For this reason, variant codons that can provide enough heterogeneity between similar chains
should be considered and incorporated where possible.
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5.3 Process Development ofMultispecics
5.3.1 Upstream Process Development
The production of multispecic antibodies is similar to traditional antibodies in
terms of upstream development and optimization. Disulde bonds between LC and
HC as well as between the two HCs comprising an antibody molecule play an
essential role in maintaining structure. As a result, more attention may be required
to ensure parameters that might affect the oxidative or reductive capacity of the cell
culture are carefully determined and well controlled.
The production bioreactor can be operated in a wide variety of ways including
batch, fed batch, perfusion (either more traditional steady-state, or with uctuating
viable cell densities [dynamic]), or even hybrid modes that combine perfusion and
fed batch [33]. The choice of the bioreactor operational mode as well as the operating conditions is optimized for each unique molecule. Commonly manipulated and
important parameters include seeding density, pH, temperature, and the composition of media and feeds as well as the volume and timing of addition of feeds, and
cell culture duration. The adjustment of these and other parameters enables not only
the ability to produce suitable quantities of multispecic antibodies but also ensue
the product is of high quality and potential issues such as covalent dissociation,
clips, and aggregation are minimized or eliminated (Chap. 4 in this volume by
Gregory W.Hiller).
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5.3.2 Downstream Process Development Considerations
The main objective when developing a downstream process for any protein is to
develop a robust, high-yielding, and scalable process that can reproducibly deliver
high-quality product. Downstream processes normally start with the removal of
cells and cellular debris followed by purication of the product of interest through
a series of chromatography and ltration steps. Once fully puried, the product is
concentrated and exchanged into a stable buffer solution for long term storage.
Downstream development for multispecic antibodies shares much with typical
monoclonal antibodies, but there are a few unique challenges that these molecules
provide that require additional attention. Using mammalian systems, the product
along with impurities is secreted from the cells into the culture medium. Similar to
mAbs, downstream processes for multispecics are designed to remove host cell
DNA, host cell proteins (HCPs), adventitious viruses, endogenous noninfective retroviral particles, aggregated product, endotoxin, process additives, and residual
media components.
Multispecic antibodies often contain additional impurities not found in typical
mAb processes, such as homodimers, fragments, or product with unwanted subunit
pairings/mis-parings [8, 34, 35]. As detailed earlier, chain mispairing can result in
several different impurities that make removal challenging (Fig.5.1). The separation of the product of interest from these impurities can be approached through
traditional purication strategies. These include optimization of existing or traditional process steps through changes such as collection criteria, pH, buffer composition, or inclusion of multiple buffer/wash steps. In some cases, optimization of
existing/traditional unit operations may not be sufcient and additional or less commonly employed steps would be required. For example, additional chromatography
modes may be required that exploit different separation mechanisms based on
charge, size exclusion, metal afnity, or hydrophobicity. The order of these steps
can also have an impact, so assessing the order of the various process steps should
be considered. For example, removal of a specic impurity in an early stage of the
process might lead to an overall downstream process that is simpler and more robust.
The following sections describe some of the purication challenges as well as
potential product stability issues that may be encountered when working with multispecic antibodies. In addition, a case study is presented to further highlight the
complexity of multispecic antibodies as compared to more traditional mAbs.
5.3.2.1 Unique Impurity Challenges
Unique impurities are common when producing multispecic antibodies. The type
and amount of these impurities can depend upon both the molecular architecture as
well as host cell line used (some example impurities are shown in Fig.5.1). Common
impurities consist of knob/knob (K/K) and hole/hole (H/H) homodimers for the
knob into hole (KiH) approach or full antibody or half antibody homodimers for the
5 Process Development and Manufacturing Considerations for Multispecic…
119
dual cell line approach. Other impurities that can be found consist of mispaired HC
and LC combinations as well as fragments (HC/LC variants) [8, 9]. The type of
fragments found can consist of heterodimer molecules where one or more LCs is
absent. Another common impurity can occur when LCs are swapped resulting in
undesirable molecular structures.
Product-related impurities add to the difculty in achieving multispecic drug
substance that meets an acceptable purity specication. Homodimers, for instance,
are often very similar to the desired product in terms of size, shape, charge prole,
and hydrophobicity. Thus, multiple purication approaches using different modes
to achieve drug substance of acceptable quality may be required. Although homodimers are typically similar to the desired product, some are different enough that they
can be more easily separated from the heterodimer. As an example, examining the
crystal structure of K/K and H/H homodimers compared to the desired heterodimer
showed that the K/K homodimer much more closely resembled the heterodimer
than the H/H homodimer [36]. The H/H homodimer has a slightly more open structure than either the K/K or the multispecic molecule which exposes different
patches of amino acids and consequently provides a means to separate this impurity
from the heterodimer [36]. Due to the similar structural nature of the K/K homodimer to the heterodimer, it can be more difcult to remove than the H/H homodimer.
To avoid more complex downstream processes, it is important to use a cell line and
cell culture process that maximizes the output of desired heterodimer while minimizing product-related impurities. As previously discussed, by optimizing the
expression vector and cell line, different transgene transcript ratios can be achieved
to limit formation of unwanted species.
Depending upon the format chosen, product-related impurities such as multispecic antibodies with LCs swapped or the loss of a LC(s) can also challenge the
downstream process. These impurities, sometimes referred to as fragments, not only
pose challenges for the downstream process but can sometimes form HMMS thus
presenting an additional purication challenge [8, 9, 37]. Depending on the level
and nature of fragment(s) during the production of a multispecic, there might be
one or more additional downstream process steps needed to reduce levels to acceptable limits. mAb-like impurities that contain swapped lights chains are the most
problematic as these molecules are very similar to the intended multispecic antibody. Removing these mispaired molecules downstream is a signicant challenge
and relies heavily on exploiting as many minor differences as possible, often leading to more complicated purication processes [8]. As mentioned earlier, an appropriate engineering of the molecule is the best way to prevent mispairing of
the HC-LC.
5.3.2.2 Stability Concerns
Through a combination of molecular design and genetic engineering, many of the
initial issues encountered in the development of multispecic antibodies can be
resolved. However, depending upon the molecular architecture used, product