MOLECULAR CONSTRUCTION/DNA SCALE-UP
Q1: Does LakePharma do codon optimization? How much would it be?
Yes, when starting from gene synthesis/molecular construction, codon optimization for mammalian expression is included. There is no extra fee for this service.
Q2: Does LakePharma have a preference with signal peptides (leader sequence, secretion sequence)?
For secreted proteins, LakePharma has a signal sequence to recommend. The signal peptide has been built into LakePharma’s vector and it has been used regularly without issues. Alternatively, LakePharma can use client’s preferred sequence with no extra charge.
Q3: Does it matter which vector the template plasmids are in?
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 LakePharma’s proprietary vector.
Q4: How much DNA does the client need to send LakePharma for molecular construction (template provided by client)?
5-10 µg minimum.
Q5: How much DNA does client need to send LakePharma for DNA scale-up?
5-10 µg should be sufficient to allow LakePharma to scale up and keep an aliquot for potential troubleshooting of the original sample if needed.
Q6: What is the standard His tag length?
The standard length contains 10 amino acids.
Q7: Can LakePharma ADCC enhance proteins?
Yes, there are mutations on the backbone of the antibody that may do that. Please inquire for more details.
Q1: What is the difference between HEK293 and CHO?
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, LakePharma would suggest performing a small scale pilot first, to obtain materials from CHO and 293 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.
Q2: If LakePharma were to produce the same constructs from Tuna293™ and TunaCHO™, how would the yields compare?
In terms of antibody productivity, our LakePharma’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.
Q3: What is the range and median of expression observed in this TunaCHO™ 14-day system for IgG1 antibodies?
For well-behaved human IgG1s, the range is ~100 - 900 mg/L with a large percent of the constructs expressing at the ~200-300 mg/L range.
Q4: What is the range and median of expression observed in Tuna293 system for IgG1 antibodies?
The range for LakePharma Tuna293™ process is ~50 - 400 mg/L with large percent of the constructs expressing at the ~100-150 mg/L range.
Q5: Will the TunaCHO™ 14-day process create aggregates?
The TunaCHO™ 14-day process is not expected to create more aggregates, however this is not guaranteed and is also construct dependent.
Q6: What levels of expression of Fabs have you seen with the 14-day TunaCHO™?
The expression level of Fab is usually similar to its IgG counterpart.
Q7: What yield is expected for scFvs?
The yields for scFvs are difficult to predict but LakePharma would expect less than the average of a well-behaved hIgG1 antibody.
Q8: For large-scale production, what system, shake flasks or WAVE bags, do you use?
We use both shake flasks and WAVE bags. Typically we use shake flasks for production volumes ≤10 L and WAVE bags for >10 L.
Q1: Does LakePharma have any data on the average purity and % monomer for well-behaved antibodies?
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.
Q2: For the pilot scale experiment, do you also purify and characterize the IgGs and Fabs?
Yes, LakePharma can purify and characterize both IgGs and Fabs. As part of the production package, LakePharma 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 then removing endotoxins later.
Q3: Why is it better to have Endotoxin Guarantee vs. Endotoxin Measurement + optional endotoxin removal?
Endotoxin removal could result in a loss of yield. Endotoxin Removal alone is more expensive than quoting for Endotoxin Guarantee from the beginning.
Q4: Can we include sterile filtration of the product?
All the final products LakePharma produced are filtered through a 0.2 µm sterile filter prior to aliquoting. Both steps are performed in a biosafety hood.
Q5: Can PBS buffer be frozen?
Freezing PBS is not recommended because it may cause precipitation during freeze/thaw cycle. LakePharma can provide buffer evaluations to determine the protein stability under freeze/thaw cycles. Please inquire.
Q1: What standard QC does LakePharma include in a production/purification?
In a standard purification, LakePharma 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)
Q2: I noticed that there is an MS QC document in the project page. Can you tell me what information this analysis provides?
Mass spec QC analysis aims to identify the molecular weight contents in a protein solution. It identifies the molecular weight contents following a recombinant production to determine if the observed molecular weight matches the calculated molecular weight of a given sequence.
Q3: Does MS QC give you any information about the sequence of the antibodies or just the molecular weight?
The complementary MS QC of standard human / mouse antibody following a production service confirms the molecular weight of the heavy chain and light chain of each Ab to make sure the correct DNA pairs are transfected.