TRACE IMPURITIES

Isolate What’s Barely There. Trace Impurity Enrichment with N-Rich®.

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

Talk to Our Impurity Isolation Specialists

N-Rich Tutorial Paper

Talk to Our Impurity Isolation Specialists

N-Rich Tutorial Paper

The Impurity Isolation Bottleneck

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.

The Solution: N-Rich® Twin-Column Enrichment Chromatography

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.

Learn About N-Rich® with AutoPeak®

Proven Results: N-Rich® vs. Conventional Isolation

Overnight

Fully Automated Runs

>80–95%

Purity Achieved

100–1000×

Enrichment Factor

3 days

vs. 30+ Days (Biosimilar mAb)

>50×

Productivity Gain (Peptide)

>10×

Faster than Batch (mAb BMS)

Case Study 1: mAb Charge Variant Isolation — Bristol Myers Squibb

Molecule: Monoclonal antibody acidic and basic charge variants Equipment: Contichrom CUBE 30 Mode: CEX and AEX; both salt gradient and pH gradient evaluated Validation Partner: Bristol Myers Squibb

Method	Purity	Time
Analytical HPLC fraction collection	87%	300 hours
FPLC preparative	78%	48 hours
N-Rich®	95%	10 hours

Key finding: N-Rich® achieved the highest purity (95%) in the shortest time (10 hours) — 30× faster than analytical HPLC fraction collection and at higher purity than all other methods tested. Both CEX and AEX resins were evaluated, confirming N-Rich® applicability across ion exchange modes for mAb charge variant characterisation.

Bigelow, C.E. et al. (Bristol Myers Squibb). J. Chromatogr. A, 1643, 462008 (2021). DOI: 10.1016/j.chroma.2021.462008

Case Study 2: mAb Charge Variants — Near-100% Purity at 86% Yield

Molecule: IgG1 mAb acidic charge variants Equipment: Contichrom CUBE 100 — two 3.93 mL columns, 22 accumulation cycles Mode: CEX (cation exchange chromatography)

Parameter	Batch CEX	N-Rich®
Purity	Not reported	~100%
Yield	59.18%	86.21% (+1.46× improvement)
Column volume required	88.2 mL	2 × 3.93 mL

Key finding: N-Rich® achieved near-100% purity at 86.21% yield for IgG1 acidic variants — a 1.46× yield improvement over batch CEX. Critically, the result was achieved with two 3.93 mL columns versus the 88.2 mL column required by batch, demonstrating that N-Rich® miniaturises the footprint of charge variant characterisation work while delivering superior purity and yield.

Jing, Y. et al. (Zhejiang University). Eng. Life Sci., 21(6), 382–391 (2021). DOI: 10.1002/elsc.202000087

Case Study 3: Biosimilar mAb — 9 Isoforms Isolated in 3 Days

Molecule: Biosimilar monoclonal antibody; 9 charge variant isoforms (1.5–56% of feed, including low-abundance variants <5%) Equipment: Contichrom CUBE 30 Mode: IEC (ion exchange chromatography)

Metric	Conventional Batch	N-Rich®
Total process time	>30 days	3 days
Analytical samples required	>600	50
Single-isoform isolation time	96 h / 32 batch runs	24 h / 1 N-Rich run
Yield	~5%	15%
Loading concentration	5 g/L	10 g/L
Enrichment factor (1.5% isoform)	~1×	Up to 69×

Key finding: All 9 biosimilar mAb isoforms — including variants present at <5% of feed — were isolated in 3 days versus >30 days by conventional batch. A single N-Rich® run replaced 32 batch runs for individual isoform isolation, with 3× higher yield and up to 69× enrichment for the lowest-abundance species.

ChromaCon/YMC. Biosimilar mAb Charge Variant Isolation by N-Rich®. Application Note (2022). chromacon.com

Case Study 4: Peptide Impurity Isolation — Angiotensin II

Molecule: Angiotensin II with microgram-level co-eluting peptide impurities Equipment: Contichrom CUBE 30 Mode: RP-HPLC; two N-Rich® modes evaluated (simultaneous multi-impurity vs. targeted single-impurity)

Parameter	Analytical HPLC fraction collection	N-Rich®
Productivity (multi-impurity mode)	Baseline	>9× higher
Productivity (targeted 1 mg, 88% purity)	Baseline	79× higher
Solvent consumption	Baseline	69× lower

Key finding: Two complementary N-Rich® modes were demonstrated: simultaneous isolation of multiple microgram-level impurities in a single run (>9× productivity gain), and targeted isolation of 1 mg of a critical impurity at 88% purity with a 79-fold productivity increase and 69-fold reduction in solvent consumption.

Weldon, R.; Müller-Späth, T. J. Chromatogr. A, 1667, 462894 (2022). DOI: 10.1016/j.chroma.2022.462894. Open access.

Case Study 5: Peptide Impurity Isolation — Fibrinopeptide A

Molecule: Fibrinopeptide A; target impurity ~1% of feed; dominant main compound ~30% Equipment: Contichrom CUBE 30 Mode: IEX (N-Rich® cyclic accumulation)

Parameter	Conventional Batch HPLC	N-Rich®
Achieved purity	<20%	>80%
Concentration improvement	1×	10×
Enrichment factor (vs. main compound)	~1×	>600×
Time to isolate	Weeks	Overnight

Key finding: N-Rich® achieved >80% impurity purity from a feed where the target comprised only ~1% — a >600× enrichment relative to the dominant main compound. Conventional batch HPLC could not concentrate the impurity at all. The entire isolation was completed in a single overnight automated run.

ChromaCon/YMC. Fibrinopeptide A Impurity Isolation by N-Rich®. Application Note (2022). chromacon.com

Case Study 6: Oligonucleotide Impurity Isolation — dsRNA Therapeutic

Molecule: Double-stranded RNA (dsRNA) therapeutic produced by solid-phase synthesis; multiple co-eluting impurity species Equipment: Contichrom CUBE 30 Mode: Ion-pair RP-HPLC Collaboration: University of Ferrara and YMC ChromaCon

Parameter	Conventional Isolation	N-Rich®
Purity improvement	Baseline	Up to 15× higher
Mass enrichment	Baseline	Up to 20× higher
Impurities isolated per run	One at a time	Several simultaneously
Processing time / handling / waste	High	Drastically reduced

Key finding: First published application of N-Rich® to oligonucleotide impurities. Multiple impurity species were accumulated simultaneously in a single automated run — delivering up to 15-fold purity improvement and 20-fold mass enrichment versus conventional isolation, with substantially less processing time, manual handling, and waste.

Lievore, G.; Weldon, R. et al. J. Chromatogr. B, 1209, 123439 (2022). DOI: 10.1016/j.jchromb.2022.123439. Open access.

N-Rich® Purification Comparison Table:

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

The Contichrom® Platform: CUBE, PILOT 300X, and TWIN HPLC

N-Rich® runs on the Contichrom CUBE. For high-pressure applications (peptides, oligonucleotides, small molecules), N-Rich® can also be scaled to the PILOT 300X and TWIN HPLC.

Systems Overview

Contichrom® CUBE

The essential platform for chromatography process development. This versatile 100-bar benchtop FPLC system runs batch, integrated batch, and continuous chromatography — including MCSGP and CaptureSMB — out of the box. Develop purification processes for mAbs, peptides, oligonucleotides, ADCs, and viral vectors, with verified scale-up at over 100× to pilot and production systems.

Contichrom® PILOT

Scale peptide and oligonucleotide purification to pilot and clinical GMP manufacturing. This 100-bar preparative HPLC system features MCSGP with AutoPeak® to eliminate the yield-purity trade-off — delivering +10–50% absolute yield increase at target purity and significantly less solvent than batch HPLC. ATEX Zone 2 / Class I Div 2 certified for organic solvents. Flow rates up to 300 mL/min.

Contichrom® TWIN HPLC

Produce peptides and oligonucleotides at commercial GMP scale. This 80-bar industrial HPLC system uses MCSGP with AutoPeak® to eliminate the yield-purity trade-off — increasing recovery to >90% while reducing solvent consumption compared to batch. ATEX Zone 2 / Class I Div 2 certified for organic solvents, and flow rates up to 36 L/min.

Systems Overview

N-Rich® Applications Across Modalities

Use Case 1: Peptide Impurity Reference Standards

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)

Use Case 2: Oligonucleotide Impurity Isolation

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)

Use Case 3: mAb Charge Variant Characterisation

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)

Use Case 4: Low-Abundance Bioactives from Natural Extracts

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)

N-Rich® Applications Across Modalities Continued….

Use Case 5: ADC Drug-Antibody Ratio (DAR) Variant Isolation

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.

Use Case 6: AAV Capsid Species & Gene Therapy Vectors

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.

Use Case 7: Protein Aggregates & Degradation Products

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.

Getting Started with N-Rich® Impurity Isolation

There are two ways to work with N-Rich: use YMC ChromaCon’s purification service — where you ship material and we return isolated impurity fractions — or run N-Rich® in-house on a Contichrom CUBE. Both journeys start the same way: a free demo and expert chromatogram review, followed by optional computational modeling and a feasibility study that generates the performance data you need to choose your path.

See It First — Demo/Webinar & Application Review

Not yet familiar with N-Rich® or the Contichrom® platform? Start here. ChromaCon offers no-charge demonstrations and introductory webinars tailored to your team. Share your analytical chromatogram and method conditions and our application scientists will assess N-Rich® suitability for your isolation goal — no starting material required.

On-site in Zurich (half day, 2–4 hours): see the CUBE in person, see ChromIQ® software and the N-Rich® wizard demonstrated by our application scientists
Remote webinar (1–3 hours): interactive live session covering N-Rich® principles, ChromIQ® software, the N-Rich® wizard interface, and the full Contichrom® ecosystem
In both formats, our specialists can review your analytical chromatogram and method conditions — column, stationary phase, mobile phase, gradient, and impurity abundance — and advise on suitability and any adaptations needed
Sessions can focus on N-Rich® for your molecule class — peptides, oligonucleotides, proteins, or small molecules and natural extracts

→ Request a free demo or introductory webinar

N-Rich® Modeling Assessment

For applications where you want a more quantitative estimate of N-Rich® performance before committing material or making a purchase decision, YMC ChromaCon can build a calibrated mechanistic model from your chromatographic conditions to predict enrichment factor, cycle requirements, and expected output purity. This step is optional — many customers proceed directly to the feasibility study.

Submit your chromatographic method details, column dimensions, and fraction analysis data if available — from any HPLC or UPLC system; no Contichrom® required
YMC ChromaCon estimates achievable enrichment factor and impurity purity across different N-Rich® operating points, and identifies any chromatographic conditions that would improve performance
Typical timeline: 3–5 weeks from receipt of data; no material shipment required

→ N-Rich® Process Modeling Service

Feasibility Study — Performance Data on Your Compound

Ship your starting material to YMC ChromaCon’s Zurich facility. We run a batch experiment on the CUBE to establish N-Rich® starting conditions using the ChromIQ® wizard, then run enrichment cycles targeting your impurity. The results give you the performance evidence needed to decide your next step — whether that is using YMC ChromaCon as your ongoing N-Rich® service provider or purchasing a CUBE for in-house work.

Deliverable: enrichment factor, isolated impurity fraction purity, and a recommended workflow for your compound and resin
You are welcome to visit our Zurich facility during the study and receive progress updates by remote webinar
Typical timeline: 3–6 weeks after receipt of starting material and purchase order

→ YMC ChromaCon Feasibility Studies

Choose Your Path — N-Rich® Service or In-House CUBE

Once you have feasibility data, you have two options. Both are fully supported by YMC ChromaCon.

N-Rich® purification service: ship starting material to YMC ChromaCon on an ongoing basis — we run N-Rich® on your compound and return isolated impurity fractions with full analytical data, ready for NMR, LC-MS/MS, ICH Q3A/Q3B qualification, or regulatory reference standard preparation. No capital investment required.
Purchase or rent a Contichrom CUBE: run N-Rich® in-house using ChromIQ® software and the N-Rich® wizard. Rental packages include application scientist setup support for your first run. CUBE purchasers can expand to the Contichrom PILOT or TWIN for higher-throughput isolation of target molecules after establishing their in-house workflow.

→ N-Rich® Custom Purification Service

→ Contichrom® CUBE Rental Program

→ Request a CUBE Quote

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Ready to Isolate Your Critical Impurities?

What is N-Rich® and how does it differ from standard preparative HPLC?

N-Rich® is a twin-column continuous chromatography process that cyclically accumulates trace impurities on-column across repeated load–recycle–wash cycles — concentrating species that are too dilute or too co-eluted for single-pass preparative isolation. Batch HPLC requires hundreds of injections and achieves <20% purity for co-eluting traces; N-Rich® achieves 80–95%+ overnight.

Learn More

How does N-Rich® compare to MCSGP?

Both are YMC ChromaCon twin-column processes, but they serve opposite goals. MCSGP purifies the main product by recycling impure side-fractions — maximizing yield of the dominant component. N-Rich® isolates the minor impurities by accumulating trace species while depleting the main peak. MCSGP is a manufacturing purification tool; N-Rich® is an enrichment and characterization tool.

What types of samples can N-Rich® process?

Peptides, oligonucleotides (including dsRNA, ASOs, siRNAs), monoclonal antibodies and biosimilars (charge variants, isoforms), ADC DAR variants, viral vector capsid species (AAV, lentiviral), protein aggregates and fragments, natural products, and small molecule pharmaceutical impurities. If your target has partial chromatographic resolution from the main peak, N-Rich® can enrich it.

How much starting material is needed?

N-Rich® is designed for the Contichrom® CUBE lab-scale columns (0.1–100 mL/min flow). Practical input is gram-to-tens-of-gram quantities of crude mixture, yielding milligram to low-gram quantities of purified impurity — sufficient for NMR, MS/MS characterization, and regulatory reference standards.

What resins and mobile phases does N-Rich® support?

Any conventional chromatographic resin — IEX (CEX, AEX), HIC, RP-C8/C18, and other standard stationary phases. N-Rich® uses the same mobile phases as your existing analytical or batch preparative method. No affinity step or custom resin is required.

How is N-Rich® configured — does it require chromatographic expertise?

The ChromIQ® N-Rich® Process Wizard guides setup in four steps from a single batch chromatogram: load chromatogram → set column size → set washing/cleaning protocol → set cycles and fractionation. Once configured, the process runs fully unattended overnight with no operator intervention required.

Is N-Rich® available as a contract service?

Yes. YMC offers N-Rich® as a contract service (“N-Rich® Impurity Insight Service”) with turnaround of several weeks, available at labs in Zurich, Devens MA (USA), Japan, India, Korea, China, Singapore, and Taiwan.