HCP Analysis: DS and In-Process Samples
A major challenge in HCP analysis is the presence of varying drug substance (DS) levels in Harvest or in-process samples, which interferes with HCP profiling and HCP antibody coverage.
Our platforms resolve this issue by locating, tagging, and removing all DS spots from the harvest 2D image. The report includes standard analysis containing all spots and advanced analysis removing DS spots.
A typical HCP analysis with DS removal contains two components:
- HCP Image Analysis: Direct visualization and comparison of in-process & DS profiles, allowing the accurate location of all DS spots that no other platform can achieve.
- HCP Quantitative Analysis: Quantitation of spot counting, similarity, and spot overlapping with & without DS, achieving accurate tracking of HCP removal in the presence of DS.
A. HCP Image Analysis
Gel images of each sample and overlay of two sample images will be generated. In the overlay images, spots with green, red and yellow color indicate decreased, increased and similar expression, respectively.
Example: Image analysis of CHO HCP Standard, CHO Harvest and DS (mAb). These 3 samples are labeled with different CyDyes and run on a single 2D DIGE gel, allowing accurate location of DS spots in the Harvest samples.
Spot counts are shown before DS spot removal, and the DS spots are circled in blue.
CHO HCP Standard: 2516 Spots
CHO Harvest: 1594 Spots
DS
CHO HCP Standard / CHO Harvest
CHO Harvest / DS
B. HCP Quantitative Analysis
The following HCP quantification analysis is performed with and without DS:
- Spot Counting for each sample
- Protein level comparison (protein ratio) between samples: Using our standard or customer set cutoffs, % spots increased, decreased or similarly expressed between HCP samples.
- Spot overlapping between samples: % spots overlapped between HCP samples
Example: Quantification analysis of CHO HCP Standard and CHO Harvest with and without DS
B1. Spot Counting for each sample: Number of detected spots for each sample, before and after DS spot removal
- CHO HCP Standard: 2516 total spots
- CHO Harvest: 1594 total spots / 1465 spots excluding DS
B2. Protein ratios between samples: The table below shows the % spot numbers in each category using 2-fold and 5-fold of protein ratios as a cutoff. When excluding the interference of DS spots, the similarity between CHO Standard and Harvest is increased.
| CHO Harvest / CHO HCP Std | Protein Ratio: 2 Fold Cutoff | Protein Ratio: 5 Fold Cutoff | ||||
|---|---|---|---|---|---|---|
| Decreased % | Similar % | Increased % | Decreased % | Similar % | Increased % | |
| All Spots | 1.1% | 78.8% | 20.1% | 0.0% | 91.6% | 8.4% |
| No DS | 1.2% | 82.8% | 16.1% | 0.0% | 96.1% | 3.9% |
Below is spot map showing spot distribution with 2-fold cutoff: Red, green and yellow indicate spots with increased>2-fold, decreased>2-fold and within 2-fold, respectively.
CHO Harvest / CHO HCP Standard
CHO Harvest / CHO HCP Standard / DS Spots
B3. Spot Overlapping between samples: The table below shows the overlapping spot count between CHO HCP Standard and CHO Harvest, with and without DS. The percent coverage of one sample by another is also shown. After DS spot removal, the Harvest sample had a lower % spot coverage of the HCP Standard sample, accurately reflecting the removal of HCP spots in Harvest samples.
| Comparison | CHO HCP Std | CHO Harvest | Overlapping Spots | % HCP Std spots covered by Harvest | % Harvest spots covered by HCP Std |
|---|---|---|---|---|---|
| All Spots | 2516 | 1594 | 1552 | 61.7% | 97.4% |
| No DS | 2516 | 1465 | 1425 | 56.6% | 97.3% |
Sample Info
Please follow these general principles regarding biohazardous materials and buffer condition in getting your samples ready.
Protein amount for each gel: 20-50 µg of total protein from each sample.
Protein concentration: 5-20 mg/mL
Buffer: Please feel free to submit samples in whichever buffer that you would normally use. The sample preparation including protein extraction (if applicable), sample cleaning and measuring protein concentration is not covered by the 2D DIGE gel cost. Additional charge may apply on samples with concentration less than 4 mg/mL, or require significant amount of the extra work such as protein eluting, sample pooling, extensive buffer exchange or serum abundant protein depletion.
Sample type: Please inform our scientists about your sample type. We will send you additional tips for each sample type.
Pricing
| Services Description | Academic/Governmental Labs | Industrial/Commercial Labs | ||
|---|---|---|---|---|
| Code | Price | Code | Price | |
| 2D DIGE Analytical Gel (2 CyDye labeling) | 101A2 | $1259 | 101N2 | $1569 |
| 2D DIGE Analytical Gel (3 CyDye labeling) | 101A3 | $1399 | 101N3 | $1769 |
| HCP Quantitative Analysis | 202A-HCP | call for price | 202N-HCP | call for price |
Price covers:
- Experimental design
- Measure protein concentration
- Fluorescence dye labeling using CyDye (2 samples for 2 CyDye labeling; 3 samples for 3 CyDye labeling)
- 1st dimension IEF and 2nd dimension SDS-PAGE
- Gel image scan using Typhoon scanner
- Two hour data analysis covering image and DeCyder analysis
- Data report