Waving goodbye to antibody engineering and moving your lead candidates into transient protein expression is a big milestone in therapeutic antibody development. Many developers are in a rush at this stage. Time is money, there’s pressure coming from upper management and it’s all hands-on deck to move the project forward as quickly as possible. But beware. Breakneck development can trip up drug developers, who are left with costly setbacks to fix before re-joining the race. Wise developers budget a few weeks into their timelines to invest in a small-scale pilot project before scaling up. Pilot cultures of around 50 mL can yield milligrams of antibody in just a few weeks, validating procedures and giving an important sneak peek into antibody behavior.
In our experience, you can’t afford to skip a protein expression pilot. The risks of an underperforming protocol or a misbehaving antibody are just too high. From experience, we can tell you that pilots are faster and cheaper than troubleshooting and rescuing another company’s failed large-scale transient protein expression. Our pilot phase also gives an important holistic preview into how the antibody is behaving and helps eliminate dead-end molecules – avoiding a costly failed scale-up. Begin with the end in mind and scale up the right antibody candidate.
Here are two scenarios you could face if you overlook a transient protein expression pilot:
A developer goes straight to large scale expression, harvesting 10 L of supernatant. Downstream purification and analyses reveal that most of the target antibody is in an aggregated state. The developer is forced is go back to the engineering stage, where a rescue project screens the lead antibody candidate for liabilities in silico, identifying an aggregation hotspot in the antibody 3D structure. This is engineered out and the protein expression is repeated, at a considerable hit to budget and timelines.
A developer goes straight to large scale expression. After protein purification, unexpected bands are seen on gels, due to glycosylation issues. The problem is solved by going back and re-optimizing the protein expression protocol to achieve acceptable glycosylation. The developer loses a month by skipping the pilot
Transient protein expression
Transient protein expression uses plasmids to transfect the antibody DNA sequence into mammalian expression systems such as CHO or HEK cells. The cells manufacture the antibody, fold it into the correct functional structure, and perform post-translational modifications such as glycosylation.
After a few days of culture, the cells are harvested, and the antibody is purified. However, the antibody sequence does not integrate into the genome and is eventually lost as the cells replicate – this is why expression is termed “transient”.
Here’s our top five tips for getting pilot projects right.
1. Engineer your best chance of success
Are you really ready to scale up to full preclinical development? Are you sure you’re finished with engineering? Have you scrutinized your sequences for all liabilities and optimized for manufacturability? If not, we strongly advise going back to check. It’s better to discover that your antibody has aggregation issues in silico or at the pilot scale, rather than attempting to purify a few milligrams of aggregate from a 10 L expression culture.
Before even the pilot phase, make sure you have given yourself the best shot at success with a thorough engineering process for antibody humanisation and affinity maturation.
2. Optimize the transient protein expression protocol
A well-planned pilot will optimize all aspects of the procedure, from the ratio of DNA plasmids, cell feeding procedure, culture temperatures and times. All these variables are iteratively tweaked until the isolated antibody has high purity, low aggregation, ultra-low endotoxin, ultra-low host cell protein and ultra-low nucleic acid contamination. Optimizing the expression protocols will make sure that your antibody is a close representative of an early-stage stable clone to take forward for manufacturing.
This pilot phase has become even more important with the advent of non-standard molecules such as bispecifics and multispecifics, which account for a growing slice of therapeutic antibodies in development. For example, in 2018, bispecifics accounted for 25% of therapeutic antibodies in development, up 150% from the early 2010s, with a continued upward trajectory. Multispecific proteins are much more problematic when it comes to expression and purification.
Fusion Antibodies is experienced at mitigating these issues associated with non-native formats thanks in part to our systematic optimization during expression pilots.
3. Quality control as you mean to continue
Quality and quantity are not mutually exclusive, but if forced to choose, we recommend going for quality every time. Anything else is false economy, and seemingly small errors or liabilities at the pilot phase will only amplify as the candidate antibody moves closer to the clinic. Quality control (QC) is a task that’s best started with the end in mind. From the pilot onwards, we recommend using the same QC protocols and procedures that would be used later in large scale manufacturing from stable cell lines. Scalable QC protocols give you added confidence at the pilot phase and can translate into downstream time savings. Problems such as low monodispersity, as well as purification issues are spotted at the pilot phase, with time to troubleshoot before scale-up or moving to stable cell line development. Robust QC also helps establish batch consistency.
4. Pilot your stability strategy
Imagine purifying grams of antibody from litres of culture supernatant only to find that it degrades in a matter of weeks, or to discover it doesn’t like the freezer… after freezing it for transport. A pilot gives you vital information about how your antibody will withstand the shelf or storage. This is crucial for establishing realistic timelines for downstream analysis and development. Plus, it gives you confidence in any downstream analysis of therapeutic activity.
5. Don’t over-work your cells
We favour quality over quantity. This is partly because when high-yielding mammalian expression systems are pushed to the extreme, the quality of the antibody protein produced suffers. Side effects of over-worked cells include glycosylation issues, yellow discoloration, and unstable proteins. A pilot picks up all these issues, avoiding over stressed cells and disappointing antibodies in larger-scale phases
Finding a balance
Therapeutic antibody development is all about balance. Making time for a transient protein expression pilot helps strike that balance and find the sweet spot between yield, quality, cost, and speed. Skipping the small-scale pilot in the pursuit of speed is ultimately a false economy.