The most effective medicines in the world are of limited value if they can’t be produced in bulk, and at a reasonable cost.  Therapeutic antibodies take years to develop. Without good oversight, each stage of development risks operating in a vacuum, with teams concentrating solely on their task before passing the candidate antibody onto the next team. An antibody developed with this “pass the parcel” approach may tick all the boxes for moving into clinical trials; it may even ace clinical trials. But if production cannot be scaled up for bulk manufacturing at a reasonable cost, the antibody is simply not commercially viable. Early engagement with manufacturing and commercialization experts and a helicopter view of the whole project from start to finish keeps the focus on developing an antibody that’s fit for purpose.

Scaling up determines cost of goods

Developers should look further than affinity alone when selecting a lead antibody. It would be a cruel twist of irony to develop a safe and effective antibody that cannot be manufactured in sufficient supply to treat all patients. Therapeutic antibodies are administered in high quantities to achieve the therapeutic effect – this can be in the range of milligrams to grams per patient per treatment cycle. For example, patients treated with the therapeutic antibody adalimumab (Humira™) take 80 mg on day one and then 40 mg every other week. The manufacturing process must keep up with the demand created by such dosing requirements.

Production is a key factor determining the ultimate cost of goods. Antibodies are churned out by genetically modified mammalian cells, grown in suspension in bioreactors. The antibody sequence is a critical “part” for the recombinant cell line “machinery”. As any mechanic will tell you, simple robust parts allow the machinery to run smoothly. In this case, well designed antibody sequence “parts” are easier for the cellular “machinery” to churn out in quantity.

Ideally, affinity maturation and sequence optimisation should optimise the antibody for ease of expression by mammalian cells. The yield, or titre, of antibody produced by these cells determines the ease of manufacturing and therefore the cost of goods.  A yield of 2 g of antibody per litre of cell suspension is the threshold for a commercially viable antibody (1). Titre up to 4-5 g/L and higher have been reported by some companies.

The titre determines how many cells are needed to harvest enough antibody – this in turn determines the size and number of bioreactors needed and the size of the factory needed to house them. Bioreactors typically range from containing 1,000 litres of cell culture up to 20,000 litres. The cost and time required to purify the antibody out of the cell suspension is another factor in scaling up production, and the ultimate cost of goods.

Production for clinical trials versus the market

Drug development is a race against the clock. Time and investment pressure means that antibodies are first produced in small batches for clinical trials before, or alongside, technology transfer, production scale-up and stability studies. Ideally the small batch production for clinical trials should form the basis for the scaled up bulk manufacturing. For example, changing the cell line to achieve higher titres for bulk production would require validation in additional clinical trials, setting timelines back by years. Similarly, a shelf life of one year may be sufficient for clinical trials, but stability of 3 years is needed for the market. Drug development timelines don’t always allow for a 3-year stability study before clinical trials – but doing things the other way around can lead to a commercially unusable antibody, even if efficacy and safety are good.

Development strategies

One of the main business decisions in antibody development is whether to make or buy, i.e. whether to perform all steps of the process in-house, or outsource certain tasks to specialist providers. A third option is to partner with providers, in a risk-sharing agreement. Whatever route is chosen, teams should start seeking and integrating expert advice about scale-up and commercialisation from the early days of the drug development process.

RAMP™, our rational affinity maturation platform, accelerates and optimizes selection of lead antibody candidates that are “pre-screened” for manufacturing suitability. Our proprietary rational library design introduces mutations in both the CDR and framework regions, inspired by how B cells use somatic hypermutation to generate antibody diversity. In silico modelling then screens out variants with sequence liabilities known to compromise expression and stability.

Contact us to see how RAMPTM can generate lead candidates that are manufacture-ready

References

1            https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759494/

Therapeutic antibodies have revolutionized the treatment of numerous diseases, but not every antibody has what it takes to become a licensed medicine. Finding a high-affinity antibody that binds to your target is only the first step. The ideal therapeutic antibody must have good efficacy, safety, pharmacokinetics (PK) and stability, and in addition, be easy to manufacture to ensure commercial viability.

Safety first

Safety is the first requirement for any medicine, and this is no different for therapeutic antibodies. Perhaps most importantly, a good therapeutic antibody should bind to its target with high specificity. For example, an antibody that is designed to target tumour cells should ignore healthy cells, as off-target binding might lead to unexpected side effects.

It’s also crucial to fine-tune the antibody’s affinity – how tightly it binds to its target. If the affinity is too low and the antibody binds its target only weakly, the therapeutic effect may not be achieved. Conversely, if the affinity is too high, the antibody dose could be “used up” too quickly, and the likelihood of off-target activity might increase.

Another important step is to minimise the risk of the antibody generating an immunogenic reaction. If an antibody is unstable, it can aggregate, misfold or even give rise to potentially dangerous metabolites such as charge variants (which occur when an amino acid has been oxidised or deaminated). These seemingly minor changes can render an antibody immunogenic, as well as reducing its efficacy.

Optimising efficacy and PK

When it comes to optimising the efficacy of an antibody, one of the major challenges is fine-tuning its affinity. The antibody needs to have sufficiently high affinity to ensure that it works as intended, but not high enough that it binds to off-target molecules and causes unintended side effects. In some cases, reducing the antibody’s affinity might actually increase its functionality, particularly in the case of multi-specific antibodies that bind two or more antigens at once (1).

Therapeutic antibodies also need to be delivered efficiently to where they’re needed in the body, and hang around for long enough to have an effect. At the same time, there should be a focus on minimising the number of doses the patient requires. The PK of the antibody can be optimised by careful screening for liabilities in its sequence that could, for example, make it unstable, meaning greater and more frequent doses are required.

Cost of goods

With increasing numbers of approvals, the therapeutic antibody market is getting crowded and competitive. Healthcare payers need to control costs, a pressure that is passed onto manufacturers. Thus, cost control should be part of every antibody development programme from the beginning.

Optimising antibody expression and considering the “manufacturability” of antibody sequences is fundamental to developing any therapeutic antibody, and ultimately also has an impact on cost. A functionally perfect antibody that becomes overly modified or degraded during large scale manufacture will not make it to the clinic. For example, manufacturability can be affected by free cysteines, formation of interchain disulphide bond formation and aggregation. Less dramatic self-interaction may still only be evident at the high concentrations required for a clinical production batch.

Stability is another cost factor. Optimizing the antibody for longer shelf life and reducing the need for cold storage can help to keep costs down.

Help from a trusted partner

Fusion Antibodies’ rational affinity maturation platform – RAMPTM – produces functional antibodies that are optimised to jump the hurdles littering the path to the clinic, helping to control costs along the way. Rational library design reduces the risk of sequence liabilities and the downstream risks of aggregation and immunogenicity (2). This can increase yield, optimise affinity and promote stability. The best candidates are screened out in silico from the library and expressed in mammalian cells for further characterisation – including manufacturability.

References

1 Mazor, Y., Sachsenmeier, K., Yang, C. et al. Enhanced tumor-targeting selectivity by modulating bispecific antibody binding affinity and format valence. Sci. Rep. 7, 40098 (2017).

2 Tabasinezhad, M., Talebkhan, Y., Wenzel, W. et al. Trends in therapeutic antibody affinity maturation: From in-vitro towards next-generation sequencing approaches. Immunol. Lett.212, 106–113 (2019).

Phage display has had a good run. After bursting onto the antibody engineering scene in the 1990s, phage display rapidly disrupted the field. The in vitro high throughput screening and selection offered by phage display hit the jackpot in 2002 with the approval of adalimumab (Humira®), which we now know as the world’s best-selling therapeutic antibody.  

However phage display’s crown has been slowly slipping. Despite early promise, only 10 of the 100 or so FDA-licensed monoclonal antibodies were developed using phage display (1, 2). So what are the pitfalls of phage?

Antibodies on display in phage

Phage, or bacteriophage to give them their full name, selectively infect bacteria and hitchhike the bacterial machinery to replicate themselves. Biologists in turn have hijacked phage by inserting genetic code into the phage which is then manufactured into a protein by the bacterial hosts’ protein expression system and displayed on the phage outside coat. This link between the genotype (the genetic sequence inserted into the phage) and the phenotype (the antibody manufactured based on the code) is the most important principle of phage display.

Antibody engineers exploit this system for antibody discovery and for affinity maturation of lead antibody candidates. The genetic sequence of the starting antibody is diversified into a library of variants. Each genetic variant is inserted into a phage, in turn inserted into a bacterial factory that pumps out phage replicas bearing the antibody variant in protein format.

In a high-throughput process called biopanning, the antibody target (the epitope) is immobilised and a sample of each variant in the library is washed over the target. Any variants that bind to the target are considered hits.

Phage display libraries – big but have they delivered?

The “display” principle linking genotype and phenotype isn’t limited to phage. Numerous display systems exist, where the antibody of interest is displayed on ribosomes, or on the cell surface of bacteria, yeast or mammalian cells. Each system has its pros and cons, but phage display has several advantages. First the bacterial libraries hosting the phage are relatively fast, cheap and easy to grow up, compared to slower growing yeast and mammalian cells. Phage display has another big advantage – and that is big, big libraries of up to 10^12 variants.

However these large phage libraries come with a price, favouring quantity over quality. While fast, synthetic and semi-synthetic methods of creating library diversity create artificial variants that would never be generated by natural B cell diversification. In addition, synthetic random libraries can be skewed with certain bases showing up too often (or not often enough) at certain positions in the DNA sequence (3). The knock-on effect is the presence of amino acids in “unnatural” spots in the antibody. Such variants may have good enough affinity to register as hits in phage display biopanning, but these “hits” may turn out to have folding or expression problems.

Express yourself

Expression bias and folding errors are two of the biggest drawbacks of phage display. The prokaryote machinery of bacteria is simply not equipped to fold complex human antibodies and cannot make crucial post-translational modifications such as forming disulphide bonds. E.coli, the most popular phage host, can be picky about which antibodies it makes, preferring to express proteins rich in certain amino acids such as methionine and lysine. Phage display handles expression and folding of smaller proteins such as functional snippets of antibodies much better. Single-chain variable fragments (scFv) of antibodies are most suited to phage display, followed by antigen-binding fragments (Fab) (4). However even scFv expression is not without its problems. For example, these antibody snippets are prone to aggregation which can cause false negatives or positives. Another limitation with antibody fragments such as scFv occurs if they need to be reformatted to full length IgG or other formats.   After the high-throughput screening phase of phage display, reformatting promising scFv variants can bring an antibody development project skidding back to a crawl. Plus, a promising scFv can turn out to have disappointing affinity once the whole antibody is expressed and folded properly in a mammalian cell.

The eukaryote protein expression and post-translational machinery of yeast display can overcome some of the expression problems of prokaryote phage display. But amongst the trade-offs are smaller library sizes and the larger volumes needed for growing enough yeast cells (5).

Alternatives on display

Some of phage display’s limitations can be overcome by pairing phage display with other technologies. Some groups pair phage display with yeast display (6), or screen phage display hits for functionality with flow cytometry (7) while others batch reformat scFv to IgG before further functionality analysis. But all of these approaches take time, money and resources.  

Another option is to bypass phage display altogether. Here at Fusion Antibodies, our rational affinity maturation platform (RAMPTM) sidesteps most of the issues with phage display. Our rationally designed in silico libraries are magnitudes larger than phage libraries, with around 10^25 variants. Diversity is introduced rationally, following the example of somatic hypermutation, which minimises the risk of finding amino acids in unexpected places. Rapid in silico screening of the library yields a micro-library of the strongest candidates which are then expressed directly as full length IgGs in mammalian CHO cells – neatly avoiding expression bias, folding problems and the need to reformat fragments to full IgGs. RAMP™ can also be used in combination with phage display. For example for performing affinity maturation of a phage-derived lead antibody candidate, or for sequence optimisation to improve expression and stability in CHO cells.

  1. https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/30516432/
  2. https://en.wikipedia.org/wiki/Monoclonal_antibody_therapy#FDA-approved_therapeutic_antibodies
  3. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193332
  4. https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/28613102/
  5. https://academic.oup.com/peds/article/24/9/711/1558039
  6. https://academic.oup.com/peds/article/24/9/711/1558039
  7. https://www.sciencedirect.com/science/article/pii/S1871678417302121

Learn how RAMPTM can boost or replace phage display for affinity maturation and sequence optimisation of your antibody

Today, therapeutic antibodies are reaching the clinic in unprecedented numbers. However the path from laboratory to the clinic is far from smooth. Antibodies face a range of obstacles during development, from insufficient efficacy, to manufacturing difficulties to immunogenicity – any of which can spell the costly end of an antibody development program.

Since 2012, Fusion Antibodies has delivered over 160 successful antibody humanization projects to customers worldwide. For many of these projects, we and our customers observed that our humanized antibodies retained and even improved antigen affinity, compared to the parent antibody.

Antibody candidates originate from many sources, using ever-improving discovery technologies. However, achieving a balanced antibody profile can be challenging. Our customers need to identify and eliminate those initially promising antibodies that bind tightly, but later turn out to have problems with manufacturing, stability or immunogenicity. They need to be sure they have the right antibody before establishing a stable cell line, and be confident that their lead candidate can get through CMC testing and make it to clinic.

RAMP™ – a 2 step affinity maturation platform

To help customers face these challenges, we have poured our expertise into creating RAMP™ – our rational affinity maturation platform designed to accelerate and optimize selection of your lead antibody candidate.

RAMP™ combines innovative library design with stringent in silico screening of variants. This sieves out the strongest candidates into a micro-library that can be expressed in mammalian cells for further characterization.

Rational library design

Taking the parent antibody, we create a massive library of around 10^20 variants. Our proprietary rational design approach takes a leaf from nature, inspired by how B cells use somatic hypermutation to generate antibody diversity.

RAMP™ introduces mutations in both the CDR and framework regions to create diversity, allowing only amino acids that can naturally occur at each position in the human antibody sequence. This “natural” approach reduces the likelihood of hydrophobic patches of amino acids and the downstream risks of aggregation and immunogenicity.

At the same time, strict sequence checks are applied to screen out primary sequence liabilities such as deamidation sites, cleavage sites and free cysteines. These checks and balances help create a library of variants that are “pre-screened” for manufacturing and clinical use.

In silico refinement

The library is then refined using in silico software that rapidly models variant-antigen binding and predicts affinity and stability. Over the 3-week in silico phase, the initial library is funneled down into a micro-library of the 100 strongest variants. At this stage we either express the micro-library as full length IgGs in CHO mammalian cells or hand the micro-library back to the client for further in-house testing.

RAMP™ up your chances of getting to the clinic

RAMP™ for affinity maturation is a fast, reliable method for improving affinity and selecting your lead antibody candidate. In a promising performance test, RAMP™ improved the affinity of the best-selling breast cancer drug trastuzumab in silico and we’re currently validating the best variants experimentally.

RAMP™ can also be applied to “rescue” molecules, with promising functional activity but poor developability profiles, where finessing of the sequence is required. Our novel library design approach can also open up new sequence space to potentially build on your patent family and increase the value of your program.

Want to select the best possible antibody for the clinic?

Design, development and clinical testing of therapeutic antibodies is a race against the clock, and against competitors. Companies are increasingly turning to in silico approaches to turbo boost the process. At Fusion Antibodies, we were early adopters of in silico techniques. We’ve seen first hand how embracing this technology accelerates antibody design and development, which reduces costs.

AI and machine learning

Despite the buzz, the holy grail of true artificial intelligence (AI) – machines that can “think” and reason like humans do – remains elusive. In the meantime, deep machine learning is the established star of the in silico show. Machines are “trained” by feeding them large sets of experimental data and teaching them (via algorithms) how to perform a task. With each repetition of the task, the machine adds the experience to their knowledge bank, improves performance and comes up with results that humans didn’t necessarily expect. So how do in silico techniques, including machine learning, fit in to antibody development?

In silico antibody development

Antibody engineers have a plethora of options in their in silico toolkit for optimising antibodies. These bioinformatics tools include homology modelling to predict antibody structure, molecular docking to identify antibody-antigen interactions and algorithms to calculate energy changes in mutated versions of the antibody. Each of these processes can benefit from machine learning to speed up predictions and improve decision making. Stitching these processes together into an automating streamlined workflow saves even more valuable time.

Focus on libraries and screening

In silico techniques in antibody development have been described as third generation, following second generation in vitro and first generation in vivo methods1. Affinity maturation is a good example of how throughput has soared with in silico methodology. In silico libraries of around 10^25 variants have smashed through the experimental library size ceilings of mammalian display (10^10 variants) and phage display (10^12 variants).

Similarly, the time needed to screen through these libraries for the best sequence has decreased drastically from 3 months with mammalian cells or phage display, down to just 3-4 weeks in silico.

An added bonus is that in silico libraries and screening avoid the potential expression and biophysical issues related with phage display systems, while leaving the door open to promising variants that may not have expressed well in phage.

Challenges remain

The in silico approaches currently used in antibody design remain overwhelmingly knowledge-based. However, machine learning is only as good as the data you train it with. A current challenge is the scarcity of robust experimental open-access datasets, and a lack of widely accepted standards for validating data quality.

Another challenge is the changing skillsets needed by today’s biologists. Data scientists, programmers and technologists are now staple members of biology teams, and everyone needs to learn to speak a common language. Training university students in such interdisciplinary working will ensure the teams of tomorrow are well placed to harness the power of machine learning and in silico techniques.

Future applications

An exciting application of in silico technologies would be to open avenues of investigation previously hampered by experimental roadblocks. For example, G-protein coupled receptors (GPCRs) are a rational antibody target for many disease processes, but are notoriously difficult proteins to isolate out of the cell membrane where they are firmly embedded. In silico modelling could sidestep the difficulty posed by purification and antibody development. This means that proteins that were previously very difficult to raise antibodies against can now be targeted.

RAMP™Rational Affinity Maturation Platform

At Fusion Antibodies, we are firm believers in integrating bioinformatics and in silico techniques to accelerate our workflows and therefore your journey to the clinic. That’s why we developed RAMP™ – our rational affinity maturation platform. It takes RAMP™ just 3 weeks to create a massive library of around 10^25 variants of the parent antibody and to select out the best candidates using rapid in silico screening. The result is a micro-library of the 100 best candidates, selected for binding potential and stability.

Harness the power of our in silico RAMP™ technology to optimise your lead antibody faster.

1              https://www.ncbi.nlm.nih.gov/pubmed/30298157

Fusion Antibodies are working with a number of clients on pilot projects for their latest technology – RAMP™ – Rational Affinity Maturation Platform.

RAMP™ was launched at the end of 2018 and clients have been coming on board to benefit from the innovative in silico approach to Affinity Maturation, particularly for time sensitive projects.

The range of clients includes one of the world’s top ten pharmaceutical companies, multiple biotech companies in Asia and Europe, and a UK based academic research institution.

Dr. Paul Kerr, CEO, Fusion Antibodies, said, “Having presented the technology platform at Antibody Engineering and Therapeutics, San Diego at the end of 2018, we are delighted that a number of projects have now commenced with us. We welcome the opportunity to help these clients address difficult roadblocks in their antibody development projects.”

Chief Technology Officer, Dr. Richard Buick commented, “We have already tested the power of our platform on one of the world’s bestselling drugs, Trastuzumab, and significantly improved its affinity. We are excited for the opportunity to demonstrate the power that lies in our approach.”

RAMP™, a Rational Affinity Maturation Platform, is designed to improve the ability of an antibody to bind without damaging the overall profile, has delivered results beyond affinity maturation. Factors that can be improved using the platform include stability, aggregation, yield, specificity and cross-reactivity, which are key considerations for clients who want to get their antibody to the clinic and market approval.