Ensuring Renewable Antibody Production
An important aspect of using monoclonal antibodies is that you should be able to go back and make them as often as you want - they should not be a limited resource. This is true for both hybridomas and recombinant antibodies.
Challenges in Storing Hybridoma Stocks
Hybridomas are a physical material, so the hybridoma itself has to be stored, usually in liquid nitrogen or vapor phase nitrogen. In order to maintain those stocks, they often have to be thawed, expanded, and then refrozen.
Anyone who has done a lot of tissue culture or dealt with liquid nitrogen freezers may shudder a little at the thought of this. You’ve probably seen boxes with the edges missing and tubes floating in tanks. You certainly need to be very careful with hybridomas to maintain these stocks to make sure that you don't lose that physical material.
Advantages of Recombinant Antibodies in Data Archiving
With recombinant antibodies, on the other hand, while there is a physical material—generally in the form of a glycerol stock of the plasmid expressing the heavy chain and the plasmid expressing the light chain—you also have the sequence as data. That data can be archived in a secure fashion with backups. There's really no reason, with off-site backups and cloud availability, that you would ever lose the ability to make that antibody.
The Importance of Reproducibility in Antibody Production
There is a great need for antibodies to be reproducible in the way that they behave. This can be more of an issue with polyclonal antibodies than with monoclonals, since polyclonals are, at some level, different every time you make them, since you go back to a different set of animals and attempt to recreate the polyclonal that was made originally.
However, even with monoclonals, there's a growing drive to make sure that these are recombinant, so that they're all derived from a defined sequence. Having that sequence available as data means that you can protect against loss of that antibody. You don't have to worry about your hybridoma becoming contaminated or genetic drift of the hybridoma cell line—the accumulation of mutations through repeated cell culture. These are all things that can affect reproducibility in research and when you think about disease diagnosis and therapy, obviously the stakes are even higher than for basic research, and the reproducibility bar that much higher.
Scaling Antibody Production to Meet Demand
In order to take advantage of an antibody in some applications, it's important to be able to make a lot of that antibody. That means you will have to be able to robustly scale antibody production to meet your needs. One issue with hybridomas is that they are often finicky, so each hybridoma is its own cell line. Even hybridomas from the same fusion will not behave in the same way. Often, they will not expand in a predictable fashion. Sometimes you will expand a hybridoma, and it will not grow past a certain point. Or it may continue to grow so you can scale up the cell volume, but the level of antibody it produces may decrease over time.
The problem with hybridomas in this situation is that your options may be limited; it is an intrinsic property of the hybridoma. Recombinant antibodies, on the other hand, can be produced at gram scales by transient transfection and at kilogram scales by amplified stable cell lines. To reiterate, the advantage of hybridomas is that they secrete the selected antibody; there's nothing else to make—you already have a way to produce the antibody. Recombinant antibodies, on the other hand, require a heterologous expression system.
Advantages of Recombinant Antibodies in Expression Systems
Small-scale transient expression involves preparing DNA in the amount needed for that scale of transfection. For larger scales, we would make a stable cell line. Those are expensive to make; it takes between three to six months to make them, and there's various technologies involved in making those cell lines, which may require outsourcing or even licensing.
However, the advantage of stable cell lines over hybridomas is that you've separated out the selection of the antibody from the selection of the expression vehicle, so you're not stuck with a low-producing hybridoma. With a recombinant antibody, you can drive the expression in the way that you need to. Typically, that's done using a host cell line such as CHO cells, for which there's an extensive track record in the production of antibody, and especially in the field of clinical materials.
Clinical Production of Recombinant Monoclonal Antibodies
Of the top-selling prescription antibodies in 2018, seven out of the ten were produced in CHO. There are many advantages of using cell lines such as CHO, which includes their traceable provenance, so it's clear how that cell line came to produce the antibody that is going to end up in human patients. There's also a huge amount of knowledge around how to push good levels of antibody expression from CHO cells.
Regulatory Acceptance and Trust in Hybridoma Clones
Hybridoma-derived antibodies are very well established in the diagnostic space. There are many go-to, widely accepted hybridoma clones for common diagnostic targets. There's a level of trust of those hybridoma clones that is difficult to overcome because of the years for which they've been used, and there's generally not a problem producing the amounts of material that are required in diagnostic applications from hybridomas.
However, recombinant antibodies are making inroads in the diagnostic sphere. Many newer targets do not have established hybridoma clones, so they're in need of new antibody clones. There are also opportunities to improve upon existing hybridoma clones. Diagnostic manufacturers—as the number of those manufacturers increase and they're competing for access to clones—have a desire to secure and own their own supplies. Because of this, we're starting to see recombinant antibodies contribute more in the diagnostic space.
Shift to Recombinant Antibodies in Therapeutics
When it comes to therapeutics, there are over a hundred therapeutic antibody drugs so far that have received FDA approval since the first one in 1986. Upwards of eighty percent of those hundred-plus drugs were developed using hybridoma technology, but only one of them is currently manufactured using hybridoma technology; all of the others are produced recombinantly.
Hybridomas generated from wild-type mice yield mouse antibodies. Because they are mouse proteins, when they're injected into people as drugs, they have strong immunogenicity, which limits their effectiveness, their long-term use, and how safe they are. This is why only one currently of the 100-plus drugs is actually produced from a mouse hybridoma.
Advantages of Humanized Mice in Recombinant Antibody Production
There are now humanized mice; these were developed in the 1990s. They produce fully human antibodies. Those fully human antibodies are minimally immunogenic, but you're still stuck with the manufacturing limitations of hybridomas in terms of their scalability and the long-term safety of those hybridomas as a renewable resource.
Driving Innovation with Recombinants
In summary, recombinant monoclonal antibodies address many limitations associated with hybridoma-derived antibodies. They offer enhanced reproducibility due to their defined sequences and eliminate concerns like genetic drift and contamination. Their scalability allows for robust production at gram to kilogram scales, meeting the demands of various applications.
Additionally, recombinant antibodies are making significant inroads in both diagnostic and therapeutic fields, overcoming manufacturing limitations and reducing immunogenicity issues seen with hybridomas. As technology advances, recombinant antibodies are poised to become the primary choice for antibody production, driving innovation in research, diagnostics, and patient care.
