Bio-conjugates Columns and Kits

Bio-conjugates Columns and Kits

Rely on Waters as your single source solution for bio-conjugate and antibody drug conjugate (ADC) columns and kits across discovery, development, and manufacturing applications.

Rely on Waters as your single source solution for bio-conjugate and antibody drug conjugate (ADC) columns and kits across discovery, development, and manufacturing applications.
Facilitating the assessment of many important Critical Quality Attributes (CQAs).
Facilitating the assessment of many important Critical Quality Attributes (CQAs).

Overview

As biopharmaceutical organizations explore the use of bio-conjugates and ADCs as a class of cancer therapies, significant challenges are found in analyzing their complex, heterogeneous structures. Waters provides solutions for these unique analytical challenges by facilitating assessment of many important Critical Quality Attributes (CQAs) including determination of drug-to-antibody ratios (DAR) even at low levels, using Reversed Phase (RP) or Hydrophobic Interaction Chromatography (HIC) columns, aggregation and fragmentation using Size Exclusion (SEC) columns, and sites of conjugation using PeptideWorks Tryptic Protein Digestion Kits for peptide mapping among others.

Our comprehensive column offerings include:

  • Reversed-Phase Columns
  • HIC Columns
  • SEC Columns
  • Ion Exchange Columns
  • Glycoprotein Columns

Whether you are analyzing lysine-conjugated ADCs seeking to minimize secondary interactions between ADCs and column hardware, or looking to overcome other data quality challenges, Waters has the right column technology to consistently and reliably perform your bioseparations.

Reducing non-desired hydrophobic interactions with MaxPeak Premier High-Performance Surfaces (HPS)

Reducing non-desired hydrophobic interactions with MaxPeak Premier High-Performance Surfaces (HPS)

Waters MaxPeak Premier Columns enable scientists to have more control over their chromatographic separations with the inclusion of MaxPeak High Performance Surfaces (HPS) Technology. As an example, when separation of proteins occurs by size in solution, secondary interactions can occur causing sample loss, broader peaks, and inaccurate quantitation of aggregates. MaxPeak High Performance Surface Technology was developed utilizing Ethylene Bridged Hybrid (BEH) SEC particles with high coverage hydroxy-terminated polyethylene oxide (PEO) and are exceptionally bioinert for more accurate data. These minimize hydrophobic interactions that cause secondary interactions providing outstanding peak shape and consistent quantification.

Available in 1.7 µm (ACQUITY Premier Columns), 2.5 µm (XBridge Premier Columns, XSelect Premier Columns), and select BioResolve Premier Columns ≤ 3 µm particle sizes and formats gain the expected the chromatographic performance, flexibility, and assurance from Waters trusted particle technologies.

Solutions

Reversed-phase columns for DAR Determination & Drug Distribution 

Reversed-phase columns for DAR Determination & Drug Distribution 
Reversed-Phase Columns

Reversed-Phase Columns for bio-conjugates and Antibody Drug Conjugates with MaxPeak HPS such as the ACQUITY and XBridge Premier Protein BEH C4 Columns are uniquely designed to minimize secondary interactions between ADCs and column hardware and suitable for the analysis of lysine-conjugated ADCs. Unlike cysteine-conjugated ADCs, the intra-chain disulfide bonds that maintain linkages between the heavy and light chains of the mAb for the lysine-conjugated ADCs are intact. Therefore, reversed-phase chromatography is suitable for the analysis of lysine-conjugated ADCs when linker chemistry is not labile at acidic pH. In addition, Waters offers Reversed-Phase Columns with solid-core particle technology, such as BioResolve RP mAb Polyphenyl for intact and subunit analysis to determine the localization of linked drugs at the domain level. 

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  • Reversed-Phase Analysis

Hydrophobic Interaction Chromatography (HIC) for DAR Determination

Hydrophobic Interaction Chromatography (HIC) for DAR Determination
HIC Columns

Waters Protein-Pak Hi Res HIC Columns with non-porous particles contain a low density of butyl ligands for outstanding resolution in less time compared to use of many traditional porous HIC offerings, ensuring consistent batch-to-batch performance by quality control testing with defined protein standards.

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  • HIC Analysis

Achieve Consistent Aggregation Accuracy with SEC Columns

Achieve Consistent Aggregation Accuracy with SEC Columns
SEC Columns

Eliminate the need to create multiple SEC Eluent mobile phases with different proteins by using a platform method with Phosphate Buffered Saline (i.e., PBS) for consistently accurate aggregate, monomer, and fragmented data with Waters SEC Columns. In addition, for bio-conjugates which can be hydrophobic creating unwanted secondary interactions, there is no need to add organic solvent with MaxPeak Premier Protein SEC Columns and it’s PEO bonded BEH particles.

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  • SEC Analysis

Characterize and Monitor Charge Variants with Cation Exchange Chromatography

Characterize and Monitor Charge Variants with Cation Exchange Chromatography
SCX Columns

Accurately measure charge variant profiles of cysteine-linked and lysine-linked ADCs with the BioResolve SCX mAb Column and BioResolve CX pH Buffer Concentrates for salt or pH gradients with minimal secondary interactions in optical detection. For structural insights with mass spectrometry in ion exchange, our MS-compatible buffers, IonHance CX-MS pH Buffer Concentrates, make online IEX-MS a reality with high-purity, volatile buffers suitable for mass spectrometry.

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  • Charged Variant Analysis
  • Cation-Exchange Chromatography

Measure Oligonucleotide Conjugation with Anion Exchange Columns

Measure Oligonucleotide Conjugation with Anion Exchange Columns
AEX Columns

Antibody oligonucleotide conjugates (AOCs) are commonly used in protein diagnostics and are a promising therapeutic for targeted cell therapy. The net negative charge of an AOC expands with each additional conjugation site, resulting in increased retention on an anion exchange resin. The GenPak FAX Column contains a weak anion exchange sorbent that exhibits controlled retentivity and a unique selectivity for protein and oligonucleotide samples. Recent application work demonstrates that AEX can be coupled with MALS to generate absolute molecular weight information.

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  • AEX OIigonucleotide Conjugate Analysis

Precisely locate glycans within a protein structure with glycoprotein columns

Precisely locate glycans within a protein structure with glycoprotein columns
Glycoprotein Columns

Hydrophillic Interaction Chromatography (HILIC) has been widely used to separate small polar compounds, yet its application to large biomolecules, other than released glycans, has been surprisingly limited. With Waters widepore Glycoprotein Columns, it is now possible to use HILIC to glean previously unattainable information from intact proteins (with or without glycosylation), protein fragments, and complex, released glycans.

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  • Hydrophillic Interaction Chromatography (HILIC)
  • Released Glycans

Rapid, automatable, and robust protein digestion workflow

Rapid, automatable, and robust protein digestion workflow
PeptideWorks Tryptic Protein Digestion Kits

Waters PeptideWorks Tryptic Protein Digestion Kits for manual or automated workflows uniquely provide your lab with high efficiency, reproducible peptide maps in under 2.5 hours—4x faster than average home-brew methods. Achieve 30-minute digestions with a 1:5 enzyme:peptide ratio, 78% reduction in missed cleavages, and a 98% reduction in contaminated autolysis peaks. PeptideWorks is ideal for drug conjugation site determination, protein structural characterization, protein identification, and protein modification monitoring including post-translation modifications.

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  • Peptides
  • Peptide Mapping
  • Protein Digestion

Glycan analysis just got easier and more sensitive

Glycan analysis just got easier and more sensitive
GlycoWorks RapiFluor-MS Released N-Glycan Kits

Re-imagine a streamlined, fast, and highly sensitive way to profile N-glycans on ADCs with the GlycoWorks Kit. Achieve unprecedented fluorescent and mass spectrometric performance for released N-glycan analysis while maximizing productivity with a solution that is automatable on Waters Andrew+ Pipetting Robot. With just 3 easy steps in as little as 30 minutes, the GlycoWorks RapiFluor-MS N-Glycan Kit reduces complicated, time-consuming sample preparation.

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  • n-glycan analysis
  • glycosylation

Take the complexity out of protein quantification

Take the complexity out of protein quantification
ProteinWorks Sample Prep Kits

Simplify and accelerate protein digestion with ProteinWorks eXpress Digest Kits. Pre-measured, lot-traceable reagents and standardized protocols for accurate, precise, and sensitive LC-MS quantification of proteins, including ADCs, in plasma and serum. Achieve unmatched performance and flexibility for reproducible protein digestion with kits optimized for various workflows, including protein bioanalysis/DMPK.

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  • Proteins
  • Protein bioanalysis

Reduce the cost of SEC analyses without compromise 

Reduce the cost of SEC analyses without compromise 
MaxPeak Premier Protein SEC 250Å Guards

Use of available and cost effective MaxPeak Premier Protein SEC 250 Å Guards help extend your analytical SEC column life without degrading desired separated component resolution or compromising required size variant separations.  

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  • SEC Columns
  • Proteins

Applications

Streamline data acquisition, processing and reporting for ADC analysis by LC-UV or LC-MS. Learn more with these application notes

Streamline data acquisition, processing and reporting for ADC analysis by LC-UV or LC-MS. Learn more with these application notes

Application Notes

Reliable, reproducible determination of DAR based on drug load distribution on cysteine-conjugated ADCs through peak areas is consistently achieved with Protein-Pak Hi Res Hydrophobic interaction chromatography (HIC) column using optical detection. Learn more with these application notes.

Reliable, reproducible determination of DAR based on drug load distribution on cysteine-conjugated ADCs through peak areas is consistently achieved with Protein-Pak Hi Res Hydrophobic interaction chromatography (HIC) column using optical detection. Learn more with these application notes.

Application Notes

Size-exclusion chromatography (SEC) determines peptide and protein aggregates, monomers, and fragments in research, development, and manufacturing of ADCs with molecular weights from 100,000 – 650,000 Daltons. Maintain high productivity in ADC characterization with our BEH-based SEC columns are quality-controlled with MassPrep Peptides and Proteins Standards for separation consistency across the SEC-MS workflow. Learn more with these application notes.

Size-exclusion chromatography (SEC) determines peptide and protein aggregates, monomers, and fragments in research, development, and manufacturing of ADCs with molecular weights from 100,000 – 650,000 Daltons. Maintain high productivity in ADC characterization with our BEH-based SEC columns are quality-controlled with MassPrep Peptides and Proteins Standards for separation consistency across the SEC-MS workflow. Learn more with these application notes.

Application Notes

Charge variant analysis for lysine- and cysteine-linked ADCs with salt and pH gradient elution mechanisms is routinely achievable with Waters SCX Columns. These corrosion–resistant columns contain non-porous, polymer-based particles grafted with a rigorously-optimized, multi-component network of negatively charged sulfonic acid ligands – for reproducible, high-resolution, charged-based separations of mAbs and other proteins in LC and LC-MS applications. Learn more with these application notes.

Charge variant analysis for lysine- and cysteine-linked ADCs with salt and pH gradient elution mechanisms is routinely achievable with Waters SCX Columns. These corrosion–resistant columns contain non-porous, polymer-based particles grafted with a rigorously-optimized, multi-component network of negatively charged sulfonic acid ligands – for reproducible, high-resolution, charged-based separations of mAbs and other proteins in LC and LC-MS applications. Learn more with these application notes.

Application Notes

Separating intact glycoproteins, glycoprotein fragments, and glycopeptides can be challenging. HILIC columns can yield higher recovery of particularly difficult mAb and ADC subunits compared to common RPLC methods while maintaining good separation. HILIC of mAb subunits can also be used to assess N-glycosylation profiles. With Waters HILIC columns, each batch of Glycoprotein BEH Amide 300 Å, 1.7 µm material is quality control tested with Waters Glycoprotein Performance Test Standard for batch-to-batch consistency. Learn more with these application notes.

Separating intact glycoproteins, glycoprotein fragments, and glycopeptides can be challenging. HILIC columns can yield higher recovery of particularly difficult mAb and ADC subunits compared to common RPLC methods while maintaining good separation. HILIC of mAb subunits can also be used to assess N-glycosylation profiles. With Waters HILIC columns, each batch of Glycoprotein BEH Amide 300 Å, 1.7 µm material is quality control tested with Waters Glycoprotein Performance Test Standard for batch-to-batch consistency. Learn more with these application notes.

Application Notes

The data speaks for itself

The data speaks for itself
Deconvoluted intact mass spectra for cysteine-conjugated ADCs from native SEC-LC-MS after deglycoslytation. Drug distribution was compared for three different cysteine-conjugated ADC samples with increasing drug load. 
Charge-variant profile of a discontinued, cysteine-linked antibody drug conjugate from Pfizer as obtained with ion-exchange chromatography. (A) UV chromatograms obtained with a salt gradient method of 20 mM MES (pH 5.4) and a linear increase of sodium chloride concentration from 140–220 mM in 30 minutes at 0.80 mL/min on a BioResolve SCX mAb, 4.6 x 100 mm Column. UV chromatograms obtained with pH gradient methods on a BioResolve SCX mAb, 4.6 x 50 mm Column using BioResolve CX pH Concentrates and mobile phases without (B), with 5% (C), or with 10% acetonitrile (D) and a linear increase of mobile phase B percentage from 0–100% in 30 minutes at 1.00 mL/min. (E) Overlay of UV chromatograms obtained with pH gradient methods on a BioResolve SCX mAb, 4.6 x 50 mm Column using BioResolve CX pH Concentrates and mobile phases without, with 5%, and with 10% acetonitrile. UV detection was at 280 nm.
Hydrophilic Interaction Chromatography of a mAb versus its ADC. Numbers represent how many linker/payloads are present in the conjugate, where a and b denote positional isomers. *Denotes linker/payload hydrolysis products. #Denotes trace level oxidation. Glycosylation is labeled according to Oxford notation. F denotes fucose, A2 represents that the structures are biantennary, and G stands for galactose (FA2 = G0F, FA2G1 = G1F, and FA2G2 = G2F).
AEX-MALS of an AOC sample as obtained with a Waters Gen-Pak FAX Column. A UV 280 nm chromatogram is shown with the molar mass overlaid. Peak boundaries are indicated by the vertical dashed lines.
Peptide Mapping (UV chromatograms) comparison of a conjugated and unconjugated Trastuzumab
Top: Trastuzumab biosimilar tryptic digestion.
Bottom: Trastuzumab emtansine (Kadcyla), antibody drug conjugate tryptic digestion.
Both tryptic digestions utilized RapiZyme Trypsin and PeptideWorks Tryptic Protein Digestion Kit with 100 µL injection (~ 10 µg) on XSelect Premier Peptide CSH C18, 2.5 µm, 2.1 x 150 mm column. Additional peaks observed in Kadcyla chromatogram (bottom) compared to unconjugated trastuzumab (top). 
Comparison of hydrophobic secondary interactions performance using the ADC ado-trastuzumab emtansine. Upon increase of organic concentration, the degree of change observed for the XBridge Premier Protein SEC 250 Å, 2.5 µm Column is again negligible, with outstanding peak shape from 0 to 15% MeCN. Hydrophobic secondary interactions from have been largely mitigated through use high coverage hydroxy-terminated PEO packing material bonding.
Cysteine conjugated ADC analysis using HIC. Drug distribution was determined for three different cysteine-conjugated ADC samples with increasing drug load.

Webinars and Resources

  • Article Reprint

High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing
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  • Poster

Enhancing subunit level profiling of mAbs and ADCs with MS-Quality Difluoroacetic Acid

Enhancing subunit level profiling of mAbs and ADCs with MS-Quality Difluoroacetic Acid
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Related

Bio-conjugates
Streamline the discovery and development of bio-conjugates with Waters fit-for-purpose sample preparation, columns, UPLC, MS, Light Scattering, and informatics solutions.

Learn more about Bio-conjugates and Antibody Drug Conjugates (ADC) Columns.

Learn more about Bio-conjugates and Antibody Drug Conjugates (ADC) Columns.
Facilitating the assessment of many important Critical Quality Attributes (CQAs).
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