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Protein Sciences

Uniting Drug Development Expertise with Antibody & Protein Production Services

Micro Developability

Early-Stage Assessment of Your Antibody Candidates

 

Most drug candidates are initially screened and selected based on affinity and functionality. Less attention is paid to other developability-related properties, such as the pharmacokinetics (PK) profile, biophysical properties, and immunogenicity risk. As a result, during the discovery stage, we have established a Micro Developability service that evaluates developability properties via a fast high-throughput system that requires only small amounts of material making it efficient and cost-effective.

One-stop solution for early-stage antibody drug developability assessment using <1mg antibody. No need to outsource to multiple vendors.

Our Micro Developability Services Include:

  • In silico analysis
  • Pharmacokinetics prediction
  • Biophysical characterization via mass spec analysis

 

Most of the assays in these studies are completed within 2 weeks using <1 mg antibody. All of these tests are conducted by our experienced in-house staff, offering streamlined and efficient testing without the need to outsource to multiple vendors. This means you can evaluate the developability potential of your targets and avoid developing undruggable targets.

Our Micro Developability services cover in silico analysis, PK prediction, biophysical characterization via mass spec analysis, and early-stage product stability.

Pharmacokinetics (PK)-predicting Assays

Our PK-prediction panel consists of HTP, sensitive, and robust assays that examine the physicochemical properties of monoclonal antibodies (mAbs) to help you predict the PK profile of your lead molecules.

 

  • AC-SINS/HIC: Evaluation of self-association tendency
  • Baculovirus/DNA/Insulin ELISA: Assessment of charge-based/non-specific binding
  • FcRn Affinity: Evaluation of clearance
  • Serum Stability: Assessment of half-life

AC-SINS (Affinity-Capture Self-Interacting Nanoparticle Spectroscopy)

The AC-SINS assay evaluates the propensity of antibody self-association and aggregation.

 

  • HTP, low concentration, and purity requirement
  • Assessment of antibody’s propensity of self-association/aggregation, viscosity, etc.

Figure A: Our high-throughput AC-SINS assay exploits the colloidal properties of Au nanoparticles by using anti-human Fc antibodies coated on Au nanoparticles to capture mAbs.

anti-human Fc antibodies coated on Au nanoparticles to capture monoclonal antibodies in AC-SINS assay to evaluate antibody self-association and aggregation.

Figure B: Data generated using the AC-SINS assay demonstrates comparability with previously published data and confirms AC-SINS is a powerful tool for predicting aggregation and PK risk of early-stage biotherapeutic molecules.

Our AC-SINS data is consistent with the published data for clinical monoclonal antibodies.

AC-SINS HIC Correlation

The following graphs present AC-SINS and HIC analysis of conformational changes in native forms of proteins.

Figure A: Shows an example of self-associating antibody, where the absorbance peak exhibits a red shift in absorbance wavelength.

 

Self-associating antibody vs. non-self-associating mAbs in the AC-SINS assay.

Self-associating antibody vs. non-self-associating mAbs in the HIC assay.

Figure B: HIC offers one type of assessment of mAbs for their robustness during manufacturing, long-term storage, and delivery. The HIC results here showed that the mAbs peak shape of self-association shifted posteriorly, and there were tailing peaks.

Figure C: Further selection could been made through the combination of AC-SINS and HIC assay results plotted on the same graph. Molecules showing less wavelength shift and shorter retention time suggest better PK profiles.

Combine AC-SINS and HIC assay results to predict PK profile.

Baculovirus/DNA/Insulin ELISA

These HTP assays offer robust, cost-effective assessments of the non-specific binding of mAbs.

 

  • Reliable indicator for non-specific binding of mAbs
  • Most sensitive assays for measuring low-affinity, charge-based interactions of mAbs
  • High throughput, low cost, and robust

Figure A: mAb3 shows little poly-reactivity, suggesting a good PK profile. Alternatively, mAb1 and mAb2 show significant concentration-dependent binding to BVP, DNA and insulin, suggesting high risk and poor PK profiles.

Using Baculovirus, DNA and Insulin ELISA for the assessing the non-specific binding of mAbs.

A panel of clinical mAbs were tested in BVP/DNA/insulin ELISA showing diverse profiles.

Figure B: A panel of clinical mAbs were tested in BVP/DNA/insulin ELISA showing diverse profiles.

Micro Developability Service Details:

Service Item Description Duration Request A Quote
DSF for Tm

1. Sample requirements: protein samples amount >25 μg; concentration >0.1 mg/mL

 

2. Deliverables: protein Tm

For small scale:
≤20 samples: 2 weeks;
20-50 samples: 3-4 weeks

 

For high throughput: 2-3 weeks

 Request A Quote
Polyreactivity study-AC-SINS for self-association

1. Sample requirements: antibody samples or rProtein amount >20 μg; concentration >0.5 mg/mL

 

2. Deliverables: wavelength shift and AC-SINS score
Polyreactivity study-Baculovirus particle (BVP) ELISA

1. Sample requirement: antibody samples amount >250 μg; concentration >0.5 mg/mL

 

2. Deliverables: titration curve and BVP binding score
Polyreactivity study-DNA ELISA

1. Sample requirement: antibody samples amount >250 μg; concentration >0.5 mg/mL

 

2. Deliverables: titration curve and DNA binding score
Polyreactivity study-Insulin ELISA

1. Sample requirement: antibody samples amount >250 μg; concentration >0.5 mg/mL

 

2. Deliverables: titration curve and Insulin binding score

Hydrophobicity-HIC HPLC

1. Sample requirement: protein samples amount >50 μg; concentration >1.0 mg/mL

 

2. Deliverables: hydrophobicity score
Particle size by DLS (DLS-Radius)

1. Sample requirement: protein samples purity >95%, concentration >1.5 mg/mL, 100 μL

 

2. Deliverables: particle size distribution
Solubility check and prediction (DLS-kD & Appearance inspection)

1. Sample requirement: protein samples purity >95%, amount >4 mg; concentration >0.5 mg/mL; 50 mL formulation buffer is required

 

2. Deliverables: kD
High concentration study (A280 and viscosity)

1. Sample requirement: >200 mg of protein sample is required; concentration based on needs

 

2. Deliverables: A280 and viscosity at designated concentrations, and recovery rate
Viscosity

1. Sample requirement: >200 μL of 0.2 μm filtered protein sample

 

2. Deliverables: viscosity at designated temperature
FcRn binding assay

1. Sample requirement: antibody samples amount >100 μg

 

2. Deliverables: KD value
PK predicting-Serum cleavage assay

1. Sample requirement: antibody samples amount >100 μg; concentration >0.5 mg/mL

 

2. Deliverables: protein cleavage percentage and/or cleavage sites
PK predicting-PTM change in serum stability assay

1. Sample requirement: antibody samples amount >50 μg; concentration >0.5 mg/mL

2. Sample requirement: antibody samples amount >100 μg; concentration >0.5 mg/mL

 

 TBD

Note:
1. Intact mass analysis and certain purity tests offer high-throughput capability. Please consult your Business Development (BD) representative for details.
2. For other special requirements, please consult your BD representative.

Preliminary Stability Assessment Service Details:

(Example stability protocol)

  Thermal Stress Study Low pH Stress Freeze/Thaw
  T0 END T0 END T0 END
iCIEF Yes Yes Refer to T0 in thermal stress study NT Refer to T0 in thermal stress study NT
SEC Yes Yes Yes Yes
Caliper NR Yes Yes NT NT
Caliper R Yes Yes NT NT
CE-SDS NR Yes Yes NT NT
CE-SDS R Yes Yes NT NT
Peptide Mapping (optional) Yes Yes NT NT
FcRn affinity at pH 6.0 (optional) Yes Yes Yes NT
SPR (affinity, optional) TBD TBD TBD TBD
Intact Mass (optional) Yes Yes NT NT
UV280 (optional) Yes Yes Yes Yes
Appearance (color & clarity, optional) Yes Yes Yes Yes
Particulates by MFI (optional) Yes Yes Yes Yes
DLS (optional) Yes NT NT Yes
CE-MS (optional) TBD TBD NT NT

Note:
1. NT stands for Not Tested, and TBD stands for To Be Determined.
2. Optional testing services (high pH stress, oxidative stress, and agitation) are available upon request.

 

Frequently Asked Questions for our Micro Developability Services

Q: How much antibody is needed for each protein characterization assay?

A: We only require a small amount of antibody for each assay. Approximately 1 mg of antibody is sufficient for most assays except for forced degradation and stability studies. Specific requirements needed for each assay are detailed in the above table.

Q: What does a change in iCIEF percentage indicate for the 40°C sample?

A: The change in iCIEF percentage shows alterations in the distribution of main, acidic, and basic protein species. High-temperature stress causes these changes mainly due to post-translational modifications (PTMs) and chemical alterations, like deamidation and oxidation, which can alter the charge properties of these protein species.

Q: Can you briefly introduce your in silico analysis service?

A: Our in silico analysis includes three main categories: sequence liability, aggregation propensity, and immunogenicity prediction. Sequence liability uses an open-source algorithm to predict PTMs that might affect antigen-binding in the complementarity-determining regions (CDRs). Aggregation propensity is assessed based on the solvent-accessible surface area of hydrophobic residues and the presence of free cysteine, categorizing potential aggregations as high, medium, or low. It’s important to note that the formation of protein aggregates is affected by several factors, including the solvent micro-environment, concentration, storage time, and storage conditions. So, our predictions only reflect the potential impacts of hydrophobicity and unwanted disulfide bonds. Lastly, our immunogenicity prediction demonstrates approximately a 0.6 correlation between the generation of anti-drug antibodies (ADA) and our predictions, which are based on publicly available data.

Q: What in silico tools do you use for aggregation profiling? Is this open source or proprietary?

A: We use a proprietary algorithm for aggregation profiling. It analyzes the solvent-accessible surface region of hydrophobic residues and the presence of free cysteine from structural information.

Q: Do you conduct high pH hold for forced degradation studies?

A: Yes, we can perform high pH hold conditions upon request for forced degradation studies.

Q: How do you determine if observed degradation in serum stability assays is due to incubation in serum or stress from capture/elute post-incubation?

A: We use Herceptin as a control in serum stability assays. By comparing the percent intensity of each sample at 48 hours and 96 hours to Herceptin’s percent intensity, we determine if the degradation is systematic or specific to the sample.

Q: What is the throughput of your Micro Developability platform?

A: Our Micro Developability platform typically handles about 100 molecules but can easily accommodate several hundred if necessary.

Q: What is the typical turnaround time for your full Micro Developability package?

A: We can complete most of our pharmacokinetic-predicting assays within two weeks. The duration for forced degradation studies varies based on the conditions requested, and it typically takes an additional 1-2 weeks to generate the final report after experiments conclude.

Q: What instruments do you use for TM measurements?

A: For TM measurements, we have two main options: Differential Scanning Fluorimetry (DSF) and Differential Scanning Calorimetry (DSC). DSF is a more cost-effective choice while DSC is more expensive. The selection depends on your specific needs.

Q: For AC-SINS assays, do you only test antibodies with human Fc domain?

A: Most of the samples tested are human antibodies, and we have a default stock of AuNP for human antibody measurements. We can also adapt to customized species, such as mouse or canine. Please inform us in advance so that we can prepare the corresponding reagents to produce customized AuNP.

Q: Why are AC-SINS results related to pharmacokinetics (PK) predictions?

A: Molecules prone to self-association or aggregation suggests fast clearance in vivo and can be predicted through AC-SINS.

Q: Why are Baculovirus (BV), DNA, and Insulin chosen? How is the ELISA score evaluated?

A: DNA and Insulin: These were adapted from assays identifying autoreactive antibodies in lupus patients and are highly sensitive in detecting charge-based, low-affinity, or above-background off-target binding (DOI: 10.1080/19420862.2017.1417718 and 10.1080/19420862.2015.1016696).

 

BV Virions: These stable particles mimic infected cell surfaces, presenting a complex mixture of phospholipids, carbohydrates, glycoproteins, extracellular matrix and nucleic acids, as well as the viral capsid. This allows for the detection of various interactions, such as electrostatic and hydrophobic interactions.

 

The ELISA score of each sample is the percentage referenced to our positive control mAb, which has fast clearance in actual PK studies. Thresholds for low, medium and high risk are set based on publications from pharmaceutical companies, such as Pfizer and Genentech as well as our own experience.

Q: Why is mouse serum selected for serum stability studies?

A: Mouse serum is used because it aligns with animal study results. The components in the mouse matrix are different from human antibody Fc-domains, which is beneficial for target antibody enrichment in the mouse serum. However, IgG depleted human serum will be up and running very soon!

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