Recombinant Antibody Production FAQs

Yes, when starting from gene synthesis/molecular construction, codon optimization for mammalian expression is included. There is no extra fee for this service.

For secreted proteins, Curia has a signal sequence to recommend. The signal peptide has been built into Curia’s vector and it has been used regularly without issues. Alternatively, Curia can use the client’s preferred sequence with no extra charge.

No, it doesn’t matter what vector the template plasmids are in. Please provide the nucleotide sequences of the target insert so that the team can PCR amplify and clone into Curia’s proprietary vector.

5-10 µg minimum.

5-10 µg is sufficient to allow Curia to scale up and keep an aliquot for potential troubleshooting of the original sample if needed.

The standard length contains 10 amino acids.

Yes, there are mutations on the backbone of the antibody that may enhance ADCC activity. Please inquire for more details.

HEK293 cells are human-derived cells that are easy to transfect and ideal for rapid research-scale protein expression, whereas CHO cells are hamster-derived cells optimized for high-yield, scalable manufacturing and are the industry standard for therapeutic biologics due to their well-characterized regulatory history and product quality. HEK293 cells and CHO cells can give different post-translational modifications (PTMs) on the protein. However, the bioactivity of the protein is usually not affected. If it is a concern, Curia suggests performing a small-scale pilot first, to obtain materials from CHO and HEK293 cells so that a comparison can be drawn between the quality of the protein produced in CHO (TunaCHO^®) vs. 293 (Tuna293^®).

“Start in CHO, stay in CHO” is recommended since client may eventually plan to move the molecule to the clinic and will need a CHO stable cell line for commercialization.

In terms of antibody productivity, our Curia’s TunaCHO^® platform often has a higher production titer than Tuna293^®. TunaCHO™ also offers both a 7-day and 14-day production duration for flexibility in timeline and maximal yield performance.

For well-behaved human IgG1s, the range is ~100 – 900 mg/L with a large percent of the constructs expressing within the ~200-300 mg/L range.

The range for Curia Tuna293^® process is ~50 – 400 mg/L with a large percent of the constructs expressing within the ~100-150 mg/L range.

The TunaCHO^® 14-day process is not expected to create more aggregates than the 7-day process, however this is not guaranteed and is also construct dependent.

The expression level of Fab is usually similar to its IgG counterpart.

The yields for scFvs can be difficult to predict, but Curia would expect yields to be not more  than a well-behaved hIgG1 antibody.

We use both shake flasks and WAVE bags. Typically we use shake flasks for production volumes ≤10 L and WAVE bags for >10 L.

Typically, the purity (measured by CE-SDS) and monomer % (measured by SE-UPLC) are >95% for well-behaved antibodies in optimal formulation buffer after Protein A purification. If polishing is performed, >99% is achievable.

Yes, Curia can purify and characterize both IgGs and Fabs. As part of the production package, Curia can determine the concentration and provide reducing CE-SDS analysis for standard IgGs and reducing and non-reducing CE-SDS analysis for non-standard IgGs and proteins. Intact mass analysis is complimentary for standard human and mouse IgGs only, not Fabs.

For an additional fee, we can also measure endotoxin levels. However, if there is an endotoxin level you are targeting, it is best to have an endotoxin requirement rather than measuring and then removing endotoxin later.

Endotoxin removal could result in a loss of yield. Endotoxin Removal alone is more expensive than quoting for an Endotoxin Guarantee from the beginning.

All final products are filtered through a 0.2 µm sterile filter in a biosafety cabinet prior to aliquoting.

Freezing in PBS buffer is not recommended because it may cause precipitation during freeze/thaw cycles. Curia can provide buffer evaluations to determine the protein stability under freeze/thaw cycles. Please inquire.

In a standard purification, Curia includes a Certification of Analysis with CE-SDS analysis (reducing for standard antibody, reducing and non-reducing otherwise), concentration measured via NanoDrop, endotoxin levels (if included in the scope) and complimentary intact mass QC (for standard human and mouse IgGs only).

Mass spectrometry (MS) QC analysis confirms the molecular mass of proteins present in a sample, verifying that the observed mass matches the expected mass of the recombinant product and identifying major variants or impurities.

MS QC provides molecular weight information rather than direct sequence information. For standard recombinant antibodies, it confirms that the observed heavy- and light-chain masses match the expected values, providing confidence that the correct DNA constructs were expressed and assembled into the intended antibody.