The Threshold System

Applications

• Assay development
• Fermentation monitoring
• Process development
• Quality control
• Clinical assays

assay development

• Rapidly develop sensitive immunoassays using your antibodies
• Assay development time is reduced
• Flexible assay format—sandwich or competitive
• Liquid-phase binding
• Less antibody per test than ELISAs
• Wide dynamic range

fermentation monitoring

• Maximize product yield by determining optional harvest time
• Detect onset of protein expression
• Monitor product degradation
• May detect conformational changes
• Fast results in less than an hour
• More sensitive than HPLC

		Figure 1. Fermentation profile. The speed and sensitivity of the Threshold system allow monitoring of the early stages of protein production during fermentation. Since it is an immunoassay, protein degradation and confirmational changes may be detected by the Threshold system assay.

process development

• Improve media selection and optimize purification conditions
• Rapid sample analysis streamlines development
• Increased assay sensitivity allows dilution to minimize sample interference
• Wide, dynamic range means one assay method from purification to QC
• No radioactivity or harmful chemicals

	Protein Load (mg/mL)	Percentage of Product Recovered	DNA (pg/Dose)
Original Buffer Conditions	5	63%	18
	14	81%	60,000
	16	98%	300,000
New Conditions	5	98%	< 2

Table 1: Optimization of an anion exchange column. The Threshold system has been used to optimize the buffer conditions used during chromatography, maximizing product yield and contaminant removal. Using the original buffer, it was shown that increasing the protein load increased the product yield. However, the Threshold system data shows the column was increasingly ineffective at DNA removal under the original conditions as protein load increased. A new protocol was developed that provides excellent product yield and DNA clearance.

quality control

A validatable method for accurately and reproducibly quantitating contaminant levels in the final product without radioactivity.

• Precision and reproducibility, typically < 10%
• Validatable assays with quality controlled reagents and software
• Transferable between operators
• Replaces radioimmunoassays

		Figure 2. The standard curve for the Threshold Total DNA Assay ranges from 2 pg to 200 pg per test, with precision and reproducibility typically less than 10%.

Standard Curve	2 to 260 ng/ml
Intra-Day Precision	3.0% CV (N=6)
Inter-Day Precision	8.4% CV (N=69)
Limit of Quantitation	4 ppm (4 ng host cell protein/mg product)

Table 2: E. Coli host cell protein assay. The liquid-phase binding, reproducibility, and sensitivity of the Threshold Immuno-Ligand Assay has allowed rapid development and validation of a rugged process-specific host cell protein assay.

clinical assays

Evaluate drug clearance and immune response in clinical samples.

• High sensitivity—equal to or better than RIA%
• Perform pharmacokinetic studies
• Evaluate immunogenicity

		Figure 3. Pharmacokinetics of human IgG1 monoclonal antibodies to Pseudomonas aeruginosa type A flagellae in phase 1 patient sera. Dr. William Landsperger, Baxter Healthcare Corporation. The Threshold system has been used to monitor the concentration of a therapeutic antibody in human serum over a 6 week period with reproducibility and sensitivity as low as 1.2 µg/mL in serum.