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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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stability issues can still be encountered. One example is low pH instability that is
sometimes observed in the charge-based approach. The mutations incorporated in
the homodimer molecules to enable correct formation of the heterodimer can also
have the potential to adversely impact product stability. As described previously,
these mutations consist of charged amino acids that work to destabilize the homodimers when produced as full-length antibodies (parents) as the charges repel each
other [15]. In some cases, this destabilization is great enough that the full antibodies
can separate into half antibodies under normal conditions without the addition of
reductant. Additional molecular design can minimize or even prevent these issues
altogether, but in some cases, there may be no solution. In this circumstance, limiting exposure to the low pH condition required for elution from the Protein A capture
column, such as raising the elution buffer pH and/or neutralizing the product pool
quickly postelution may be necessary. Depending upon how sensitive the molecules are a Protein A capture step may not even be possible. Alternative capture
steps may be required; however, this will likely require additional development to
effectively design a process capable of delivering a product that meets acceptable
quality. This possibility further highlights the importance of performing molecular
assessment upfront on candidate molecules and, as needed, providing the necessary
time to potentially re-engineer a candidate molecule prior to moving forward with
development.
5.3.2.3 Reduction andOxidation (REDOX) Step
There are two strategies for producing multispecics using atwo cell line approach:
the homodimers can be produced as either half antibodies or full antibodies. In the
case of the half antibody approach, each half antibody contains a different binding
epitope. In order to ensure that these combine in the correct conguration to form
the desired multispecic, knob into hole technology is employed such that one half
antibody contains amino acid mutations to create the knob, while the other half
antibody contains amino acid mutations to create the corresponding hole in the Fc
region. These half antibodies are produced with the inter disulde bond cysteines
being cyclized or capped. For the full antibodies two completely intact antibodies
are produced such that each contains a different epitope. These two antibodies also
contain mutations in the hinge and CH3 region that enable reduction and ultimately
oxidation creating the desired multispecic. In both cases, a redox reaction is
needed, consisting of a reduction and oxidation of the disuldes for full antibodies
[15] or the removal of the caps or recyclization of the cysteines for half antibodies
[9]. Traditional reducing agents such as dithiothreitol (DTT), 2-mercaptoethanol
(BME), and tris(2-carboxyethyl)phosphine (TCEP) are very common reducing
agents for refolding or analytical analysis of disulde bonds [38]; however, these
are considered strong reducing agents and tend to reduce all of the disulde bonds
within an antibody, not just the inter-HC disulde bonds that must be reduced and
then reoxidized to form the desired multispecic. Mild reducing agents such as
reduced glutathione, 2-mercaptoethylamine, or cysteine have proven successful in
5 Process Development and Manufacturing Considerations for Multispecic…
121
these cases as they primarily impact the intra-HC disulde bonds or the caps or
cyclized cysteines without signicantly reducing the LC-HC or intrachain disulde
bonds [9, 15, 39]. The choice of reducing agent depends on several factors including
the ability to successfully reduce the molecule, availability, cost, toxicity, and the
ability to remove the reducing agent following the redox reaction.
There are several key parameters that should be considered when developing the
redox reaction step including buffering capacity/pH, temperature, reductant stoichiometry, molar ratio of proteins, mixing, and oxygen levels. Optimizing the reaction
timing or kinetics and the interplay of these parameters on timing/kinetics must be
understood when optimizing formation of the desired multispecic. Reduction and
oxidation occur under alkaline conditions and sufcient buffering capacity to control the pH above neutral is important. Higher pH conditions tend to drive the reaction faster [40]; however, elevated pH can destabilize the proteins and potentially
impact the charged species prole (e.g., asparagine deamidation).
Similar to pH optimization, the impact of temperature must also be well understood. Operating at elevated temperatures (30–37 °C) will drive faster reaction
kinetics than operating at room temperature but exposing the molecule to elevated
temperatures may also lead to instability. In most cases, reactions operating at room
temperature provides acceptable reaction kinetics and is more stable. This is more
amenable when considering manufacturing facilities as warmer temperatures
require jacketed or temperature controlled rooms or tanks [41].
In addition to the reductant employed, the ratio of reductant to protein is also a
critical parameter. There is a strong correlation between increased reductant and
more rapid reaction kinetics. This increase is seen until the reductant level is saturated [42]. There is also the potential to add too much reductant leading to degradation of the molecule due to reduction of additional disulde bonds. Beyond the ratio
of reductant to protein, the molar ratio of each half antibody or full antibody is also
a parameter that should be considered. A 1:1 molar ratio of either the full antibodies
or half antibodies to each other is typically employed during the reaction as this will
lead to full conversion to the multispecic, eliminating half antibodies or homodimers, thus simplifying subsequent purication steps [9, 43]. This ratio can be
adjusted in certain cases if there is a driver such as enabling a more efcient and
robust downstream process.
Once the molecules are reduced, the oxidation can occur naturally over time by
ensuring sufcient oxygen is present. Oxidation can be accelerated through the
addition of either the oxidized form of the reducing agent and/or metals that are
known to promote oxidation. Ensuring sufcient levels of oxygen may consist of
either sparging with oxygen or providing an air overlay while continuously mixing.
If there is insufcient oxygen, then other means such as chemical oxidation or buffer exchange to remove the reductant can be considered.
Another consideration is the potential for undesired reduction of the molecule as
has been observed during harvest and clarication operations using depth ltration
[44]. Although not a part of the desired process, it has been observed that in harvest
and clarication using a depth lter there is the potential of reduction of disulde
bonds. Depending on the format chosen, this might have signicant impact on the
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molecule. This may result in undesirable impurities such as fragments and should
be carefully considered and or evaluated for each molecule and appropriate corrections made to prevent reduction during harvest and clarication.
5.4 Manufacturing ofMultispecics
The production of multispecic antibodies is complex; although extensive development work is done to ensure that processes are robust, efcient, consistent, and
scalable there are several important considerations when manufacturing these molecules. The need for additional process operations, stability data to support hold
times and manufacture scheduling considerations are just a few. An evaluation of
both multispecic approaches, KiH and electrostatic, may initially favor the single
cell line KiH approach, as this eliminates the need for a second series of upstream
process operations, but other aspects of each process need to be considered in determining which process to use.
As such, one aspect to consider is protein stability and the potential need for
additional hold times. In the case of a two-cell line multispecic process, the operation of the production bioreactors may require additional hold time if the bioreactors are operated sequentially. This challenge may be circumvented by operating
homodimer production bioreactors in parallel. Considering the risk mentioned
above, if operating two bioreactors and one fails and hold times are not sufcient to
cover the time needed to generate a back-up reactor, this would have signicant
impact on both manufacturing time and cost. Another consideration with the twocell line approach is that one homodimer might be used for other multispecic molecules. Thus, it is extremely important to carefully consider homodimer stability in
the early design of the manufacturing process for these types of multispecics.
Another consideration for manufacturing of multispecics is cost. If total process yield is equal, the single cell line approach would likely be favored in terms of
cost, as two bioreactors run either in parallel or sequentially will have higher overall
costs due to an increased need for more raw materials, equipment, and personnel.
Extending this, the cost of failure for a two-cell line approach would also be higher.
In our experience, however, total process yield between the single cell line and dual
cell line approach is not equal, with the dual cell line approach typically having
signicantly higher yield. This demonstrates the need for a careful risk-benet analysis when considering cost of the multispecic heterodimerization strategy. Other
variables that may need consideration here are anticipated product demand and drug
substance stability.
Considering the complexities associated with the production of multispecics,
scheduling and equipment utilization should not be ignored. Production using two
cell lines either means that the manufacturing suite is occupied longer if the bioreactors are run sequentially or more bioreactors are occupied if the homodimers or half
antibodies are produced in parallel.
5 Process Development and Manufacturing Considerations for Multispecic…
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In addition to these considerations, when manufacturing multispecic proteins
thoughtful handling of product contact waste streams is necessary for multispecic
molecules with high potency designations. Multispecic oncology therapeutics traditionally have very high potency and therefore can reach potentially toxic concentrations within the bioreactor. As a result, special considerations are needed in
treating product contact waste streams and properly protecting personnel during the
handling of such potent biologics.
As the realm of biotherapeutics expands from the standard monoclonal antibody
to multispecic modalities, complexities and challenges are introduced to nearly
every step of development. Early development choices, such as molecular format
and heterodimerization strategy, can have a profound impact on subsequent development steps. To make the optimal choice, one needs to consider all program
aspects, including but not limited to cell line generation, process development, manufacturing, as well as product safety proles, and material demands.
5.5 Case Study: Challenges inRemoval ofBispecic Product
Related Impurities
A case study is presented to highlight some of the challenges that multispecics (in
this case a bispecic) may encounter compared to traditional mAbs. This case study
highlights development strategies and process adjustments that were made to enable
manufacture of a product with acceptable quality. The case study focuses on using
a single cell line to produce the bispecic, utilizing the knob into hole technology.
The molecule presented in this case study is a diabody fused to an Fc domain
(diabody-Fc) [45]. The diabody portion of the molecule is comprised of two peptide
chains, each chain consisting of an scFv (single chain fragment variable) region
with VL (variable light)/VH (variable heavy) domains having afnity for different
targets. In the nal molecule, each Fv is comprised of a VL from one chain and a
VH from the other chain (Fig. 5.4). Compact packaging of the properly paired
VL-VH is facilitated by short linkers in the scFv peptides; these structural constraints favor the formation of the diabody without domain exchange [46]. To favor
Fig. 5.4 Molecular architecture of the molecule described in the case study
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T. Iskra etal.
high yields of the heterodimer, knob into hole technology was engineered into the
Fc domains [47]. The choice of the knob into hole molecular design was in part
facilitated by the relatively low number of theoretical product related impurities that
would be predominantly present (two—the K/K homodimer and the H/H homodimer) in the expression of the two chain diabody-Fc format.
While molecular design inhibits biological activity of the K/K and H/H homodimers, a downstream process capable of generating drug substance with low homodimer impurity is desired. This is especially important in this case study as drug
substance stability was impacted by homodimer levels of approximately 4% and
HMMS impurity levels of approximately 3%. This impact was eliminated when
these two impurities were reduced to ≤1% for each impurity. As outlined in the case
study below to robustly deliver stable drug substance, strategic decisions had to be
made during both the design of the cell line and throughout biomanufacturing process development. Consideration and understanding of the biophysical characteristics of the homodimer impurities helped to drive several project decisions which
resulted in a robust process capable of delivering drug substance with acceptable
quality.
5.5.1 Vector Orientation andCell Line
The host cell line selected for the expression of this diabody-Fc molecule utilizes
targeted integration, which harbors one landing pad previously engineered into the
host cell at a dened chromosomal location, competent for receipt of the vector
payload [20, 24]. Given the use of this host, cell line generation efforts were likely
to yield clonal cell lines harboring one integration of the expression vector.
To express the molecule, the vector contained a single copy of both the gene coding for the hole and knob transcripts, under the control of identical transcriptional
promoters and terminators. With chromosomal integration and transcriptional promoting signals being equal, the conguration of the cell line vector can be manipulated to differentially impact expression levels of hole and knob transcripts.
Since recombinase-mediated cassette exchange in this host cell is directional,
vector conguration will dictate which chain is upstream of the other in the host cell
genome. In our experience, when comparing similar genes/transcripts at a xed
chromosomal location, the upstream chain is slightly more transcriptionally active
than the downstream gene. For example, in Fig.5.5, a vector conguration using
orientation 1 (the knob transcript preceding the hole transcript) will bias for higher
expression levels of knob protein while orientation 2 (the hole transcript preceding
the knob transcript) will bias higher expression levels of hole protein.
Knowledge of heterodimerized knob into hole (KiH) Fc domains and corresponding homodimerized H/H and K/K Fc domains has been gained through X-ray
crystal structures [36]. As previously discussed, homodimerized H/H Fc domains
are more open than KiH and K/K Fc domains. The differentially exposed surface
5 Process Development and Manufacturing Considerations for Multispecic…
125
Host Cell Landing Pad
TK
PAC
Vector Cassee
Exchange
KNOB HOLE
(RMCE)
Trace Moderate
Moderate Trace
HOLE
KNOB
Fig. 5.5 For targeted integration systems, vector orientation impacts the types of product-related
impurities observed. Host cell landing pad diagram adopted from Zhang etal. (2015). RMCE—
recombinase mediated cassette exchange; HH—hole-hole; KK—knob-knob; TK—thymidine
kinase gene; PAC—puromycin N-acetyltransferase gene
areas of H/H homodimers provide unique handles for removal through various protein purication techniques.
In this case study, orientation 2 was selected for the vector design to bias higher
expression levels of the hole protein. In doing so, the H/H homodimer impurity, an
impurity easier to separate from the heterodimer product than K/K homodimer, had
levels of approximately 20% H/H in the Protein A pools (Fig.5.5) while the K/K
homodimer was undetected.
5.5.2 Downstream Process Design: Removal ofImpurities
5.5.2.1 H/H Removal
By intentionally designing the single cell line to preferentially produce the hole HC,
H/H homodimer was observed in the Protein A captured pool. H/H levels of over
20% were observed after Protein A capture. Although it is easier to remove the H/H
homodimer, due to the signicant presence of this impurity, signicant downstream
removal capacity is required.
Through high-throughput resin screening and additional development experiments, two differentiating biophysical properties between H/H homodimer and the
heterodimer product were identied and leveraged to design a robust downstream
process:
1. H/H bound more tightly to anion exchange (AEX) resin than heterodimer
(Table5.1).
2. H/H had reduced low pH stability than heterodimer with H/H converting to
HMMS during low pH exposure (Fig.5.6).
126
Table 5.1 Impurity data from AEX resin screen
Conditions Load impurity levels: 20% HMMS, 23% H/H
pH Anion
level
Lower Low 6 16 78
Medium 5 7 88
Higher 3 5 92
% HMMS—Eluate
pool
%H/H—Eluate
pool
T. Iskra etal.
% heterodimer—Eluate
pool
Fig. 5.6 Impact of low pH on product of interest (POI), hole/hole homodimer (H/H), and high
molecular mass species (HMMS) levels for knob into hole (KiH) demonstrating conversion of H/H
homodimer to HMMS under low pH conditions
5.5.2.2 HMMS Removal
HMMS is a very common product-related impurity observed during mAb manufacture. Several different chromatography modalities (such as ion exchange, hydrophobic, multi-modal) under optimized conditions have proven capable of separating
HMMS from the monomer mAbs. Similarly, these modalities can also be applied to
bispecic antibodies.
In this case study, HMMS levels of up to 20% were observed following Protein
A capture, thus requiring signicant downstream removal capacity for this productrelated impurity.
Both anion exchange (AEX) and cation exchange (CEX) chromatography provided robust HMMS clearance (Tables 5.1 and 5.2). CEX was observed to have high
HMMS capacity, but unlike AEX, had very limited H/H capacity. Identifying more
than one step capable of removing HMMS was necessary given the high level of
HMMS observed in the Protein A capture pool.
5 Process Development and Manufacturing Considerations for Multispecic…
Table 5.2 CEX impurity results from resin screen
Conditions Load impurity levels: 5% HMMS, 5% H/H
pH Anion level % HMMS—pool %H/H—pool %Heterodimer—pool
Lower Low NMT 0.2 4 96
Lower Medium NMT 0.2 4 96
Higher Low 1 5 94
Higher Medium 1 5 94
NMTnot more than
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5.5.2.3 Balancing H/H andHMMS Removal
Due to the high level of H/H homodimer and HMMS present signicant removal
capacity for these impurities was needed to build a robust downstream process capable of delivering >99% product purity. To achieve this, several strategies were
employed during the design of the downstream process based on previously
described experimental observations.
The AEX resin demonstrated limited capacity for HMMS removal, but reasonable capacity to remove H/H homodimer. Although the reduction was signicant,
the AEX was still limited and could only decrease H/H levels to approximately 5%.
The CEX resin demonstrated capacity to remove all the HMMS but could not
remove any of the H/H homodimer. The combination of these two process steps
(AEX and CEX) demonstrated that the process was capable to clearing all the
HMMS, but additional capacity to remove the H/H homodimer was required to
achieve the desired drug substance H/H homodimer purity targets. To increase the
capacity of the process to remove H/H homodimer, a strategic placement of a low
pH step within the downstream process was used to drive a conversion of H/H
homodimer to HMMS.In doing so, H/H homodimer, a difcult to remove impurity,
would be converted to HMMS, which is an easier to remove impurity from the process stream.
The placement of the low pH step was inuenced by the anion concentration
ranges that were in use for the AEX and CEX steps. Robust operating conditions for
the AEX resin were achieved at high pH, low anion concentrations (Table5.1), and
robust operating conditions for the CEX resin were achieved at low pH and low-tomedium anion concentrations (Table5.2). Placement of the low pH step following
Protein A capture, a traditional location for mAb processes, would have had a negative impact on AEX step performance. The introduction of anions into the AEX load
through the low pH titration of the Protein A pool would shift the AEX into a
medium anion operating space, impacting impurity removal over this step.
Placement of the low pH titration after the AEX step introduced additional anions
into the CEX load but well within the anion concentration range and robust operating space for impurity removal over the CEX.
By rearranging the placement of the low pH hold from a traditional mAb process, the capacity for unique bispecic process impurity removal increased while
negating the need for in-process pool dilution or an intermediate dialtration step.
128
Fig. 5.7 Downstream process overview for knob into hole molecule case study
Table 5.3 In-process impurity levels and yields for KiH case study
Step Yield (%) HCP (ppm) DNA (ppb) rProA (ppm) H/H (%) HMMS (%)
Harvest 90 230,000 2,000,000 N/A N/A N/A
ProA pool 90 500 10 10 20 20
AEX pool 65 BLOQ BLOQ BLOQ 3 4
Low pH pool 98 BLOQ 7
CEX pool 85 BLOQ
VRF 98
UF/DF 98
BLOQ below limit of quantitation, VRF virus retentive ltration, UF/DF ultraltration/dialtration, N/A not applicable
T. Iskra etal.
The overall downstream process is illustrated in Fig.5.7 with typical in-process
impurity levels highlighted in Table5.3.
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