Challenges of Manufacturing the Expanding Range of Bi/Multispecific Modalities

The pharmaceutical industry is increasingly focused on the development of next-generation drug products that allow the targeted delivery of active ingredients with multiple functionalities, increasing both efficacy and safety. Bi- and multispecific antibodies are prime examples of new drug substances with the potential to offer higher potency combined with new mechanisms of action while also reducing the cost and time for drug development.

Market Growth Reflects Increasing Interest

Currently, two U.S. FDA-approved bispecific antibody (BsAb) products are on the market: Removab (catumaxomab) from Fresenius Biotech and Trion Pharma for the treatment of patients with malignant ascites and Blincyto (blinatumomab for injection) from Amgen for the treatment of relapsed/refractory acute lymphoblastic leukemia. There are more than 85 BsAbs in clinical development today, and this number is expected to increase.1

Researchers from academia and industry, including big pharma and small, specialty biotech firms, are advancing new bi/multispecific modalities, and this is happening at a much faster rate than standard mAbs.2 Overall, the pipeline of new molecular formats is predicted to grow at up to three times the rate of standard mAbs through to 2025, with the majority of BsAbs targeting cancer indications.3 By 2025, the value of the global market for BsAbs will surpass $8 billion as new molecules are introduced to the market and commercial products gain approvals for additional indications.4

Many Benefits of Bi/Multi Modalities

The interest in bispecific antibodies is driven by their potential to be both more precisely targeted and more potent than conventional mAbs.5 BsAbs are designed to bind to two separate antigens or different epitopes of the same antigen. The close proximity of the binding/interaction sites can lead to the formation of new protein complexes and trigger new cellular contacts.6 In many cases, the enhancements are greater than if two individual drugs were administered as a combination therapy. BsAbs may also provide access to new therapeutic targets or combinations of targets not possible with mAbs.

With two or more sites for interacting with the target cell, more targeted binding can be achieved and additional immune responses can be activated via the redirection of cytotoxic immune effector cells. such as T cells and natural killer (NK) cells, leading to significantly greater targeted cytotoxic effects.7 Alternatively, BsAbs can act as inhibitors of two proteins within a single disease pathway or from different signaling cascades simultaneously. The involvement of multiple binding sites and different pathways may also reduce the potential for development of resistance.8

Multispecific antibodies designed as T cell engagers may also offer advantages over autologous chimeric antigen receptor (CAR) T cell therapies, as they would be available off the shelf but not carry the potential for immunogenic responses associated with allogeneic cell therapies.9

As of mid-2019, more than 20 commercialized technology platforms were available for BsAb development and production.

Zoo of Molecules

Advances in protein engineering have led to the development of many different bi/multispecific modalities: a zoo of molecules made through the combination of different numbers and formats of heavy and light chains. BsAbs are generally categorized on the basis of whether they contain a Fragment crystallizable (Fc) region, and the pharmacokinetics, half-life, Fc receptor-mediated function (if applicable) and biological activity can vary significantly depending on the structural details.

Some bi/multispecific Abs are designed to improve the effector function or extend the half-lives so that they are comparable to conventional mAbs. These are interesting as second-generation products but do not provide access to new biological targets. A second group with additional binding sites for greater targeting specificity may enable the realization of new mechanisms of action.9 Others are based on antibody fragments or other protein scaffolds that can be linked together, and a fourth group includes conjugates of antibodies with other molecules, such as antibody–drug conjugates.

BsAbs can also be generated by fusing different antigen-binding moieties, such as scFv or Fab, to other protein domains, enabling further functionalization.6 Most candidates in the clinic today are either BiTEs, DARTs, homodimeric “knob-in-hole” Abs or trifunctional BsAbs.8


Various Manufacturing Approaches

As of mid-2019, more than 20 commercialized technology platforms were available for BsAb development and production.1 Platforms under development by companies such as Amunix, Invenra, Glycotope, Xencor, Novartis, Daiichi Sankyo and Roche are intended to streamline bi/multispecific antibody development, increase patient safety and enhance efficacy.9

Production platforms are predominantly mammalian, but for smaller (<50 kD), simpler BsAbs, such as Fab fragments that do not involve the Fc region or require glycosylation, production in Escherichia coli might be the best route. Manufacture of BsAbs in yeast, notably Pichia, is also attracting attention, particular for slightly larger molecules that do not require posttranslational modifications (PTMs).

The first BsAbs were produced via the production of a quadroma through the fusion of two different hybridoma cell lines, but this process resulted in random association of heavy and light chains, with the desirable combination produced as only a fraction of the resulting mixture.7 The hurdle of heavy chain mispairing was overcome with knobs-in-holes technology, which enforces heterodimeric formation by introducing a “bulky knob-like structure” on one arm and a “hole-like structure on the other.”

Today, most BsAbs are generated via chemical conjugation/covalent bonding using crosslinkers, fusion of hybridoma lines into quadromas or genetic engineering to produce recombinant bispecific antibody fragments comprising the VH and VL domains of the parental mAbs. Depending on the components and the technology used, they vary in the number of antigen-binding sites, geometry, half-life in the blood serum and effector functions.

Many Manufacturing Challenges

The complexity of bi/multispecific antibody formats creates manufacturing challenges both from an expression and a process point of view. These include low titers, variability in product quality and aggregation.

Lonza has developed a full expression toolbox designed to overcome the challenges associated with stable, efficient expression of increasingly complex protein formats. A combination of optimized mammalian and microbial expression systems combined with vectors tailored for multichain proteins delivers high titers and stable, scalable production.

The latest version of our GS Gene Expression System — GS Xceed® — is designed with future manufacturability in mind. It produces high product titers in a robust, chemically defined, animal component–free (CDACF) environment and requires no MSX for stability.

For production of multichain (3 or 4) proteins, including BsAbs, the pXC Multigene vector allows assembly of either a double, triple or quadruple product in a single vector using type IIs restriction enzymes. Site-specific conjugation (SSC) vectors are also available to simplify production of conjugation-competent antibodies.

Recently, we also added the proven transposon-based technology piggyBac™ to this toolbox. PiggyBac preferentially targets large gene cargos to stable regions of the genome associated with high expression. Lonza is also evaluating additional synthetic promoters that will enable fine-tuning of expression of different components (VH and VL) of the molecule. By modulating expression of the components, the increase in the proportion of correctly paired chains will help overcome some of the purification and analytical issues outlined below.

For certain new-molecular formats or components, microbial systems may provide a faster and more cost-efficient solution. Lonza’s XS™ Pichia Expression Systems allow rapid combinatorial screening and straightforward fermentation regimes that yield high titers (up to 10 g/L) in short total fermentation times. Gene optimization and synthesis, primary and secondary screening and the production of milligram quantities of the target BsAb are achieved in just 10–12 weeks.

For all downstream processes, the use of platform solutions is more challenging and process optimization must be achieved based on the specific characteristics of the BsAb.10 Alternative resins (rather than protein A) are often needed for some BsAbs due to absence of the Fc region. Resin screening is therefore typically required, and much more time and effort must be invested in finding the right resins for purification of these BsAbs.

As many of the candidate BsAbs move through the clinic, challenges with scale-up of manufacturing processes are a growing concern. Many of the current technologies used in the laboratory are not scalable, and significant development efforts are underway to identify practical commercial-scale solutions.

Finally, alternative delivery solutions for BsAbs may be necessary due to their higher potency, such as micro delivery systems.5

At Lonza, we are building capabilities to meet the manufacturing needs of next-generation protein therapeutics, including bi-specific antibodies.

Analytical Issues

Product-related impurities, such as aggregates and variants, are generally present at a higher concentration than in a monoclonal antibody process. These impurities can have very similar physicochemical characteristics to the product itself, such as hydrophobicity and net surface charge. To remove these impurities, extra chromatography steps are often required, but, given the similarities between the molecules, the resolution for these chromatography steps is generally lower than in a mAb process. High 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.

It is also critical to be able to identify these product-related impurities and clearly understand which molecules are present in the supernatant to develop effective purification processes.

Given the multiple heavy and light chains, simple gel analyses are insufficient. Mass spectrometry is thus an essential tool. The higher potency of BsAbs also demands the development of analytical methods with much higher sensitivities.5

While methods must be tailored, high-throughput analytic capabilities and access to rapid analytical methods are also essential. The goal is to establish platform solutions for aspects of analytical work that can be applied across different bi/multispecific antibody modalities.


At Lonza, we are building capabilities to meet the manufacturing needs of next-generation protein therapeutics, including bi-specific antibodies. Through our work with companies in preclinical development, we see that around two-thirds of the early pipeline consists of non-standard antibodies. Supporting these therapies with rapid and scalable manufacturing for drug substance and drug product will be vital in offering better outcomes for patients.

In addition, many of the companies developing these new molecular formats are small, even virtual biotechs with a strategy of commercializing their molecule. Many have little interest in building in-house manufacturing capacity and are looking for partners who can not only provide the right technical solutions but can also de-risk their path to market and ensure they are set up to scale when needed.

As more complex proteins make it through approval and the number of bispecifics grows, advanced molecular biology and flexible manufacturing systems will be essential to making them a commercial reality.


  1. Labrijn, Aran F. , Maarten L. Janmaat, Janice M. Reichert and Paul W. H. I. Paren. “Bispecific antibodies: a mechanistic review of the pipeline.” Nature Reviews Drug Discovery. 18: 585–608 (2019).

  2. Global Bispecific Antibody Market to 2025: Drug Sales & Clinical Pipeline Insights with an $8 Billion Market Opportunity. Research and Markets. 18 Jan. 2019. Web.

  3. Bispecific Antibodies Market - Global Industry Insights, Trends, Outlook, and Opportunity Analysis, 2018-2026. Rep. Coherent Market Insights. Oct. 2019. Web.

  4. Bispecific Antibodies Market By Type (Immunoglobulin G (IgG) Like Molecule, Non Immunoglobulin G (IgG) Like Molecule), by Application (Oncology, Autoimmune Disease, Others) - Growth, Future Prospects & Competitive Analysis, 2018 – 2026. Rep. Credence Research. Dec. 2018. Web.

  5. Challener, Cynthia A. “Witnessing Major Growth in Next-Gen Antibodies.” BioPharm International. 30: 14–19 (2017).

  6. Kontermann, Roland E. and Ulrich Brinkmann. “Bispecific antibodies.” Drug Discovery Today. 20: 838–847 (2015).

  7. Carrao, Catarina. “The emergence and benefits of bispecific antibodies.” Knect 3 Jul. 2019. Web.

  8. Sedykh, Sergey E., Victor V. Prinz, Valentina N. Buneva and Georgy A. Nevinsky. ”Bispecific antibodies: design, therapy, perspectives.” Drug Des. Devel. Ther. 12: 195–208 (2018).

  9. Clift, Ian C. “Bispecific, Multispecific Antibodies Grapple with Cancer.” Genetic and Engineering News. 7 Feb. 2019. Web.

  10. Scanlan, Claire, Ruta Waghmare, Elina Gousseinov and Alejendro Becerra-Artega. “Challenges and Strategies for the Downstream Processing of BiSpecific Antibodies (BsAbs).” ADC Review. 6 Jun. 2014. Web.

Bernie Sweeney

A highly experienced cell and molecular biologist, Dr. Sweeney has responsibility for developing and applying novel and innovative technologies to Lonza’s GS expression systems. Part of Lonza’s R&D network, Dr. Sweeney’s team is currently focused on improving the manufacturability of increasingly complex protein therapeutics, such as bi-specifics and other new molecular formats, in both mammalian and microbial systems. Prior to joining Lonza, Dr. Sweeney led multi-disciplinary teams developing expression systems at UCB-Celltech.