TRACE IMPURITIES
Overnight enrichment of trace impurities for ICH Q3A/Q6B reference standards, charge variants, glycoforms, and more. Across published cases: 100–1,000× Enrichment factor | 80–95% Purity (from <20% by batch) | 10 h vs. 300 h analytical HPLC | 69× Less solvent
Sources: Bigelow et al. (BMS), J. Chromatogr. A 2021; Weldon & Müller-Späth, J. Chromatogr. A 2022
Regulatory agencies require isolated, pure impurity reference standards — not just detected peaks. ICH Q3A(R2) mandates structural identification above 0.10% and toxicological qualification above 0.15% for certain classes of molecules. ICH Q6B demands comprehensive characterization of all product-related variants for biologics. A common FDA citation deficiency: “failure to provide a sample of a critical impurity.”
The problem: trace impurities co-elute with a dominant main peak. Conventional batch preparative HPLC requires hundreds of injections, produces dilute, low-purity fractions, and may take 30+ days per isoform. Custom synthesis costs $5,000–$50,000+ per compound — and is simply impossible for biologic impurities like charge variants, glycoforms, or aggregates that only exist as product-related species.
N-Rich® is YMC ChromaCon’s automated enrichment process for isolating trace impurities from complex mixtures. Two identical columns cyclically accumulate the target impurity on-column across repeated load–recycle–wash cycles — progressively depleting the dominant main peak. The result: 100- to 1000-fold enrichment in an overnight run using conventional resin (IEX, HIC, RP-C8).
ChromIQ® software’s N-Rich® Process Wizard configures the entire run from a single batch chromatogram: Once configured, N-Rich® runs fully unattended.
Same resin. Your existing batch conditions are the starting point. N-Rich works with the IEX, RP, HIC, and other standard stationary phases and mobile phases already used in your existing analytical or batch preparative method. The ChromIQ® N-Rich® Process Wizard builds the initial enrichment method parameters from a single batch chromatogram — minimal chromatographic expertise required.
Fully Automated Runs
Purity Achieved
Enrichment Factor
vs. 30+ Days (Biosimilar mAb)
Productivity Gain (Peptide)
Faster than Batch (mAb BMS)
| Parameter | Conventional Batch HPLC | N-Rich® Twin-Column Process |
|---|---|---|
| Impurity starting abundance | 0.1–5% of feed | 0.1–5% of feed |
| Achievable purity | <20% for co-eluting trace species | 80–95%+ |
| Enrichment factor | ~1–2× | 100–1000× |
| Time to isolate | 30+ days (hundreds of injections) | Overnight (1 automated run) |
| Automation | Semi-automated fraction collection | Fully automated via ChromIQ® |
| Yield | ~5% for trace species | 15–86% |
| Solvent consumption | Very high (hundreds of injections) | Up to 69× reduction |
| Resin required | 1 Large column (low load) | 2 Smaller columns |
| Custom synthesis alternative | $5K–$50K+, weeks–months | N/A — isolates directly from mixture |
| Applicable to biologics | Limited — extremely low productivity | Yes — charge variants, glycoforms, capsids |
N-1 deletions, deamidation products, and oxidation variants co-elute with the parent peptide and resist single-pass batch isolation. N-Rich® enriches them for NMR characterization, toxicological qualification, and ICH Q3A(R2) reference standard submission in a single overnight run. 88% purity, 79× productivity gain vs. analytical HPLC (Weldon & Müller-Späth 2022)
Shortmers, failure sequences, PS→PO variants, and deamination products co-elute with full-length oligonucleotide and resist conventional LC. N-Rich® accumulates several impurity species simultaneously in a single automated overnight run. 15× purity increase, 20× mass enrichment vs. conventional isolation (Lievore et al. 2022)
Acidic and basic charge variants require high-purity isolation for forced degradation studies, potency assays, and biosimilar comparability. N-Rich® completes the task in hours rather than weeks of repetitive batch preparative IEC. 95% purity in 10 hours vs. 300 hours by analytical HPLC (Bigelow et al./BMS 2021); ~100% purity at 86% yield (Jing et al. 2021)
Minor bioactives from plant or fermentation extracts — crocin variants, flavonoid aglycones, alkaloid side-products — co-elute with dominant matrix compounds and resist batch isolation. N-Rich® concentrates them for, identification, characterization and natural product reference standard preparation. Applicable – <5% minor co-eluting species in complex natural matrices; saffron crocin fractionation demonstrated (Hooshyari Ardakani et al. 2024)
Odd-DAR variants (DAR 1, 3, 5, 7) appear as minor HIC shoulders on dominant even-DAR peaks and are inaccessible for individual PK/PD or toxicology studies. N-Rich® enriches these rare species from your existing HIC method.
Empty and partial capsid species co-elute with the full capsid peak on AEX. N-Rich® isolates each population — empty, partial, and full — for independent characterisation and reference standard preparation.
Dimers, trimers, and higher-order aggregates at 0.5–5% abundance co-elute as leading SEC shoulders with the target monomer. N-Rich® isolates them for immunogenicity testing and in vivo safety studies at significantly higher productivity than preparative SEC.
Ready to Isolate Your Critical Impurities?
A twin-column continuous chromatography method enables simultaneous separation and collection of both acidic and basic mAb charge variants in a single continuous operation.
N-Rich applied for simultaneous isolation of multiple low-concentration dsRNA impurities enables efficient characterization of oligonucleotide process-related substances.
N-Rich demonstrated for peptide impurity isolation with up to 15-fold purity improvements and 20-fold mass enrichment compared to conventional methods.
N-Rich continuous chromatography for mAb charge variant enrichment achieves 95% purity in 10 hours versus 78-87% purity in 48-300 hours with conventional methods.
N-Rich applied for mAb acidic variant enrichment achieves 86% yield at nearly 100% purity compared to 59% yield with scaled-up batch chromatography.
Clear answers to your most common questions about oligonucleotide purification and continuous chromatography.
