The overall trend in the biotherapeutics arena includes transitioning towards molecules that have higher value, potency, and thus smaller production volumes. Antibody Drug Conjugates (ADCs) and bispecific monoclonal antibodies (BsAbs) are two such product classes. Neither of them are new- they have been studied for many years. .
ADCs utilize antibodies as means to guide drugs to specific targets, where as a bispecific monoclonal antibody (BsAb) is composed of fragments of two different monoclonal antibodies that binds to two different types of antigens. The innovative research, and the advances in the field of bispecific antibodies/ADCs, and in turn bispecific antibodies that are themselves ADCs, holds great promise for the future development of therapeutics for a variety of diseases. 
Baeuerle and Raum present an extensive review of BsAbs in their article.  BsAbs are most commonly used for cancer immunotherapy where they are targeted to simultaneously bind to a cytotoxic cell as well as a target tumor cell to be destroyed. Targeting two antigens simultaneously can be a promising approach.  BsAbs can be expressed in mammalian and non-mammalian systems (example of the latter could be two Fab antibody fragments expressed using a microbial platform, and linking them through a chemical cross linker). The purification of these molecules can be complex and a platform approach is not always feasible for the downstream processing. These molecules can be unstable, diverse in their make-up, have aggregation tendencies, may or may not bind to Protein A, and could be of varied sizes, with varying impurity profiles. This paper will include a review of the challenges associated with the manufacturing of BsAbs in mammalian cell cultures, and the strategies that can be implemented to overcome those challenges.
Monoclonal antibodies closely resemble the naturally occurring immune response antibodies. As mentioned earlier, Bispesific antibodies (BsAb) are antibodies with dual specificity in their binding arms; they usually do not occur in nature and have to be created through the use of recombinant technology or somatically fusing two hybridomas (hybrid hybridoma/quadroma) or by chemical means. Quadroma technology is one the earlier methods used for production of bispecific antibodies. The antibodies secreted by quadroma including the bispecific antibody closely resemble conventional monoclonal antibodies, their molecular weight is approx 150kDa and they are relatively stable molecules. In addition to the dual-specific antigen binding fragment (Fab), these antibodies contain an Fcγ part, and thus can be considered trispecific. Triomab® (Trion Pharma) is one of the most advanced bispecific antibody format produced via improved quadroma approach: it uses the somatic fusion of a murine and a rat hybridoma cell line, expressing monoclonal antibodies with two IgG subclasses selected for their preferential pairing.
Figure 1: Examples of BsAbs
Another advanced format of bispecific antibody is BiTE® (Micromet/Amgen) – bispecific T-cell engagers. These antibodies are single polypeptide molecules of ~55kDa produced by recombinant linking the 4 variable domains of heavy and light chains required for 2 antigen-binding specificities, they are capable of efficiently redirecting T-cell cytotoxicity against various target cells without any requirement for pre- or co-stimulation of effector T cells.
A competing format of bispecific antibody called DART™ (“Dual-Affinity Re-Targeting” by MacroGenics) is based on the 2 polypeptide chains associated noncovalently in the diabody capable of targeting multiple different epitopes with a single recombinant molecule. The DART™ platform has been engineered to accommodate virtually any variable region sequence in a “plug-and-play” fashion with predictable expression, folding, and antigen recognition .
There are around 40 different formats of bispecific antibody in development by industry at the moment . These formats include tandem single-chain variable fragment (scFv), diabodies, tandem diabodies, two-in-one antibody, and dual variable domain antibodies (DVD-Ig) .
The primary challenges in bispecific antibody production include chemical manufacturing control issues, production yield, homogeneity and purity. While production of small amounts is typically straightforward, cost-effective manufacturing at large scale could require major efforts.
Biological activity of antibody-based therapeutic molecules is closely related to their chemical and structural stability. Degradation may occur at various stages of the antibody life cycle – from production/purification through formulation, storage and delivery. Two main categories of degradation are physical and chemical. Aggregation is the most common type of physical degradation and when it occurs the monomeric antibody units bind to each other forming dimers, trimers, tetramers or even higher molecular weight aggregates size of which could range from nanometers to microns. Aggregation could be induced by various stress factors antibody faces during its life cycle – temperature change, freeze/thaw, mechanical stress (agitation, pumping, filtration, filling), pH/conductivity change etc. Chemical degradation occurs via oxidation, deamidation, isomerization, cross-linking, clipping and fragmentation. Successful production of antibody based therapeutic requires careful assessment of the various degradation pathways possible for a molecule (both physical and chemical) and implementing control over those pathways .
Bispecific antibodies have an increased tendency to form high-molecular-weight aggregates compared to the parental immunoglobulins. The tendency to aggregate is often concentration dependent: in the example of a modular IgG-scFv bispecific antibody increase in aggregates up to 50% was found at concentration of 5 mg/ml. It was shown that the introduction of a VH-VL interchain disulfide bond to stabilize scFvs could help with preventing or reducing aggregation of the bispecific molecule to levels below 5% .
2.0 Harvest Clarification
The vast majority of the current therapeutic antibodies are still produced in mammalian cell lines in order to reduce the risk of immunogenicity due to non-human glycosylation patterns. Bispecific antibodies without any glycosylation could be successfully produced in bacteria.
For bispecific antibodies expressed in mammalian cells (most commonly with CHO and PERC6® cell lines, HEK293, BHK and NS0 being also used), cell culture conditions mimic those used for monoclonal antibody processes. As such, harvest clarification methods are also similar. For process volumes ≤ 2,000L, companies often employ normal flow filtration for primary and secondary clarification. Depth filtration is the most common normal flow filter used at this step. For batch sizes greater than 2,000L, it is more economical to utilize centrifugation for primary clarification and normal flow filtration (using depth filtration) for secondary clarification. The use of depth filters can also reduce impurity (i.e. HCP and DNA) levels, which can alleviate some of the strain on the downstream purification steps. As titers and cell densities increase, the use of flocculation polymers or acid precipitation is becoming more common at harvest. The addition of flocculants (typically cationic polymers) or acid to the bioreactor prior to harvest can lead to lower impurity levels and can increase secondary clarification filter capacities (post centrifugation). For some bispecific antibodies, due to their potency, titers can be lower than standard Mabs, which can influence critical parameters (i.e. cell densities, viability, particle size distribution) that can affect the harvest clarification process.
As in the case of monoclonal antibodies the next unit operation after clarification in the downstream process of a bisepecific antibody molecule is the capture step. In the case of full-length bispecific IgG molecules or IgG-like BsAbs (i.e. those containing the Fc-region of an antibody) this initial chromatography step can be done using a Protein A media. BsAbs produced by quadromas assemble randomly and produce some bispecific molecules as well as the parent monospecific IgG types. These molecules can be initially purified by Protein A but other chromatographic methods are needed to separate the target bispecific molecule from the product related impurities.
Full length BsAbs can also be generated by expressing individual antibodies separately. For example, the two antibodies can be mixed together under optimized chemical conditions to produce the BsAbs , use knobs-into-holes technology  or produce half antibodies separately to name a few approaches. In these cases the use of Protein A for capture is the same as for traditional monoclonal antibodies. Each antibody or half-antibody can be purified in this single step to a level that is enough to perform the in-vitro generation of the BsAb molecule.
The capture step of bispecific antibody molecules that do not contain the Fc region of an antibody has been achieved using a different chromatography types. Some BsAbs of this type are engineered to contain a histidine tag which allows the use of immobilized metal affinity chromatography (IMAC) for the initial chromatography step .
Other small bispecific antibody molecules containing the variable region of the kappa light chain can be captured using Protein L affinity chromatography . Although the binding capacities for IMAC and Protein L are generally lower than for Protein A it is important to note that the molar capacities are not as low because the molecular weight of these molecules could be 2-3x smaller than a monoclonal antibody.
When affinity chromatography methods are not an option for capture the use of ion exchange (IEX) or hydrophobic interaction chromatography (HIC) has also been reported . The use of IEX or HIC for capture can be challenging to optimize for bispecific antibodies as it is for their monoclonal counterparts. Although acceptable purities can be achieved using IEX or HIC for capture the process development efforts required to achieve the same levels as with affinity chromatography modes are generally higher.
Additional purification and polishing chromatography steps are required after the initical capture of BsAbs. In cases where a biologically derived ligand was used as capture (Protein A or Protein L) these process-related impurities have to be removed in these steps. Product-related impurities like aggregates and fragments need to be reduced to acceptable levels and in the case of full-length BsAbs the parent monoclonal antibodies or unwanted BsAbs variants need to be separated as well. In cases where the charge difference between the BsAb antibody and the other full-length variants is relatively large cation exchange chromatography operated in bind and elute mode can be sufficient to achieve the target purification .
Product-related impurities such as aggregates are generally present at a higher concentration than in a monoclonal antibody process particularly for bispecifics that are not full-length antibodies. These impurities can have very similar physicochemical characteristics to the product such as hydrophobicity and net surface charge. To remove this impurities it is required to employ one to three IEX or HIC steps but given the similarities between the molecules the resolution for these chromatography steps is generally lower than in a MAb process. Lower yields may need to be sacrificed to achieve the target purity and optimization of these steps can be more difficult than in a MAb process. Alternative methods of operation these chromatography types including multicolumn countercurrent solvent gradient chromatography have shown improvements in yield without sacrificing purity . However, the use of multicolumn processing strategies has not been implemented at large scale in the biomanufacturing industry for a commercial product.
4.0 Sterile Filtration – Post Harvest Clarification and Process Intermediates
Upstream sterile filtration in bispecific antibody processing is similar to that of monoclonal antibody processing. Media components influence the sterile filtration of bioreactor supplements and process intermediates (post primary and secondary clarification). Symmetric PVDF membranes are better suited for the sterile filtration of PEG and hydrolysate-containing media types. Asymmetric PES membranes offer higher fluxes and capacities than symmetric membranes and are recommended for downstream purification process intermediates. Care and attention should be paid to sterile filtration at each step, with respect to product recovery, capacity and operating flux.
5.0 Virus Filtration
Some bispecific antibody molecules are similar in size to virus filter membrane pore sizes (20nm), which can lead to significant process challenges. For these types of molecules, asymmetric PES parvovirus filter should be evaluated first, with and without prefiltration. If product recovery is an issue, regulatory agencies have accepted the use of non-parvovirus filters (i.e. Planova® 35N). For smaller bispecific antibody molecules, asymmetric PES parvovirus filters are recommended. The use of a membrane-based prefilters should be used to normalize the feed (with respect to aggregate and impurity levels), increase capacity and reduce operating costs for this process step. Special care should be taken when outlining the virus validation step as this will dictate achievable process loadings.
6.0 Ultrafiltration and Diafiltration
In the view of an increasing trend towards the development of dosage forms for alternative routes of administration, in particular the subcutaneous (sc) route, the final ultrafiltration and diafiltration (formulation) step in bispecific antibody processes can present unique challenges due to the high viscosity of the highly concentrated product. Protein-protein interaction at high concentration is a major factor that may influence opalescence and viscosity. New product offerings from filtration companies designed to address formulation of high concentration Mabs can be well applied to bsAb processes as well. Specifically, EMD Millipore’s new ‘D’ screen device allows for successful formulation step while remaining within customer’s designated manufacturing process pressure range. Cellulosic-based membranes are often used at this step for their low binding characteristics.
7.0 ADC Considerations
Bispecific antibodies are prevalent in the area of cancer immunotherapy. This is particularly relevant for bispecifics engineered from scFv (single chain variable fragments). To increase drug targeting and in-vivo half-life and decrease side effects, the BsAb can be coupled with a cancer drug (cytotoxin), which then binds to antibodies in the body and attaches to the surface of cancer cells. The cytotoxic drug is then released and can attack the cancer cells. This realm of biotherapetuics is increasing and to date there are 3 ADC’s that have received market approval and many more in company’s drug pipelines.
There are critical process considerations necessary in the production of ADC’s. Post production (as outlined in this paper), the bispecific antibody is conjugated with the cytotoxin and requires further purification (either through chromatography or Ultrafiltration/diafiltration). After conjugation, the process needs to be closed and is regulated by CDER (unlike other protein conjugations). At this stage in the process, the batch sizes are small so some manufacturing can be done in a hood, but this can be cumbersome. The use of disposable technology and closed purification systems (TFF and chromatography) is recommended. Due to the high level of toxicity of the chemicals used in these processes, it is recommended to check the chemical compatibility and leachables/extractables profile of the disposable technologies used (particularly the films).
Modified Mabs such as BsAb and ADC molecules are generating increased interest as the demand for target therapeutics with improved efficacy continues to grow. These molecules present some challenges that are different than the “traditional” processes used for the development and manufacture of monoclonal antibodies.
The the BsAb can also be coupled with a cancer drug (cytotoxin), which then binds to antibodies in the body and attaches to the surface of cancer cells (ADC). This assists with further increasing the drug targeting.
For BsAbs, the general purification process is similar to Mabs. However there are some unique challenges. Each of the steps in recovery and purification must be optimized based on the process requirements and the molecule characteristics in order to ensure a robust, stable, and scalable BsAb production process.
Acknowledgments: The authors would like to thanks Sladjana Tomic, David Beattie, Martin Zillman, and Mark Wagner for their help.
June 6, 2014 | Claire Scanlan, Elina Gousseinov, Alejandro Becerra-Artega, Ph.D, Ruta Waghmare, Ph.D | Corresponding Author Ruta Waghmare, Ph.D; [email protected] | doi: 10.14229/jadc.2014.6.6.001
Received May 8, 2014 | Accepted May 29, 2014 | Published online June 6, 2014