Cracking the Code on Complex Proteins: Your Development and Manufacturing Questions Answered

Key Takeaways: 

• Start cell line development strategy as early as possible. Sequence risks, vector design, and the end therapeutic goal should all shape decisions from day one.
• The highest-titer clone isn't always the best choice. Scoring clones on titer, viability, and targeted product quality data together reduces late-stage risk.
• Stability data needs to reflect real manufacturing conditions, including longer-than-expected low pH holds and storage relative to glass transition temperature.
• Complex molecules need tailored purification, such as alternative affinity resins and added polishing steps, rather than a standard mAb platform.

What makes bispecific antibodies and other complex proteins challenging to manufacture?

Bispecific antibodies, trispecific antibodies, fusion proteins, and other atypical formats are opening new therapeutic possibilities, but they rarely follow the development and manufacturing strategies used for standard monoclonal antibodies. And no two molecules are exactly alike. Chains can mispair, chain ratios must be precise, linkers can clip, and high-expressing cells can struggle to fold and modify proteins correctly. Add compressed timelines, and complex protein manufacturing calls for earlier, more data-driven decisions at every stage.

During our recent panel webinar, Cracking the Code on Complex Proteins: A Discussion on Process Innovation & Scalable Production, attendees asked how to approach these challenges from cell line development through purification. Here, we provide insights on the strategies we use at each stage of the process.

When should cell line development strategy start shaping decisions for complex proteins?

As early as possible. Each sequence carries its own risk factors, so effective cell line development for complex proteins starts with a close look at the amino acid sequence and its variants. Vector design choices, including signal peptides, chain ratios, and promoters, should then be made with the end therapeutic application in mind so nothing has to be redone mid-program.

Sponsors can help by sharing what they already know about manufacturability. If a molecule is unstable at low pH or needs a minimum salt concentration, that data shapes the downstream process from day one. If those limits are unknown, saying so is valuable too, because it tells us where to look first.

How do different molecular architectures affect expression stability and scalability?

Format matters at every step of bispecific antibody development. Whether a molecule is symmetric or asymmetric, how its mechanism of action works, and how its Fc region functions all influence downstream design and scale-up strategy. Assembly is another concern in atypical protein expression: a molecule held together by non-covalent interactions can be expressed and still fail to form its functional configuration. Linkers between domains can also be prone to proteolysis, so clipping needs monitoring. At the cell line level, inserting the full gene cassette as a single unit through transposase-based integration helps maintain the correct chain ratios these molecules need to assemble properly.

How does high-throughput screening change clone selection in cell line development?

High-throughput screening lets teams assess productivity and quality together, reducing late-stage surprises and protecting compressed timelines. The key is intentionally selecting analytics that accurately identify risk. Titer alone can be misleading. A hypothetical clone producing 10 g/L with 50% aggregate delivers far less useful product and adds downstream risk. Combining titer, viability, and targeted product quality data into one scoring system gives a fuller picture, and the highest-titer clone is not always the one that advances.

Automated purification and analysis make it possible to evaluate dozens of clones quickly and handle more projects overall. In addition, AI-assisted analysis of these growing datasets is expected to make clone selection increasingly predictive.

How can analytical data support downstream purification of complex proteins?

It starts with knowing the molecule. Complex proteins can behave very differently under each process condition, and low pH steps such as viral inactivation are a frequent pain point. We focus on collaboration to make strategic decisions. Analytical teams help downstream colleagues understand what the molecule looks like at each step, using size exclusion chromatography and, increasingly, mass spectrometry. High-throughput methods already built for cell line development can be carried forward to give downstream teams more data, faster, throughout process development.

How can early formulation and stability work prevent late-stage setbacks?

In one program we recently completed, a molecule showed reversible aggregation at low pH, severe enough to threaten the purification strategy. Identifying the ionic strength needed to break up those interactions made a low pH step workable.

Storage matters too. Every drug substance has a glass transition temperature, and storage should sit well away from it. In one case, a molecule stored right at that threshold dipped below it each time the freezer was opened, and repeated excursions caused problems later traced back to storage. Because glass transition depends on both the molecule and its formulation, it should be measured for each program. This an example of the level of detail needed in our work to ensure we are keeping a project on track

What are the most common purification pitfalls with complex proteins, and how can you avoid them?

The first is incomplete stability data. At manufacturing scale, a viral inactivation step with a stated 60 to 90 minute hold can take three to four hours once titration, transfer, and neutralization are included. Stability data needs to cover that full window at the operating pH.

The second is treating a complex molecule like a standard monoclonal antibody. In bispecific antibody manufacturing, platform Protein A capture may not separate mispaired species from the target. Screening alternative affinity resins, such as CH1-targeted options, and adding tailored polishing steps can close that gap, while process modeling helps narrow conditions with fewer experiments.

Want the full discussion?

Watch the complete panel on demand to see how our teams approach complex protein manufacturing, from transposase-based cell line development and rapid analytics to tailored downstream purification.