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Liquid Chromatography in the Biopharmaceutical Industry

A hand wearing a blue glove adjust a liquid chromatography machine.
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The search for drugs to treat and prevent COVID-19, has led to the repurposing of available small-molecule pharmaceuticals such as remdesivir, and the development of several new drugs and vaccines.1 Many of the new therapeutics are biopharmaceuticals, a term coined in 1982.2 These biologics or “large molecule drugs”, such as therapeutic proteins, monoclonal antibodies, oligonucleotides and antibody-drug conjugates, have become invaluable tools for the treatment of a variety of diseases. This includes cancers, rheumatoid arthritis and psoriasis, for which effective small molecule therapeutics are limited or not available.

Unlike small molecule pharmaceuticals that are manufactured chemically, biopharmaceuticals are derived from living cells (microbes, animal, plant or human). Complex manufacturing processes can result in heterogeneity or variations in the structure of the target molecule which could impact the efficacy, safety and stability of the molecule. Consequently, to ensure that the right compound is formed with the desired purity, constant monitoring of the processes as well as the quality of the product is essential. This involves in-depth characterization of variant(s) obtained from the process. In addition, contamination of drug products and/or formulations with host cell proteins during manufacture, or with molecular fragments or aggregates of the target molecule formed during storage or transport necessitates periodic analysis.

Moreover, large numbers of “affordable” biosimilars, which are expected to be identical to the innovators’ products, also require testing to evaluate their similarity to the original or the reference product, quality and efficacy. The various routes of administration of biopharmaceuticals impact their pharmacokinetics, as a result of which therapeutic drug monitoring in body fluids becomes significant.

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How is liquid chromatography used in biopharma?

While liquid chromatography (LC) coupled with mass spectrometry (MS) (LC-MS) is a powerful and commonly used technique for the characterization of biopharmaceuticals during drug development, LC coupled with ultraviolet-visible (UV-Vis)/photodiode array (PDA), fluorescence and light-scattering detectors is used for routine analysis. A variety of LC methods have been developed to assess the critical quality attributes of these molecules, such as purity and chemical modifications to biotherapeutic molecules, aggregation and charge variants, as well as to quantify them in complex matrices and ensure the absence of impurities. Some examples of such methods are listed below:

  • Peptide mapping is a well-established technique for identifying a protein, monitoring its structural integrity, and determining any amino acid modifications in the primary structure. It involves digestion of a protein with enzymes such as trypsin, followed by separation and identification of its fragments. A rapid peptide mapping method using reverse phase (RP) chromatography, or RP-high performance liquid chromatography (HPLC) with UV detection has been developed for estimating site-specific oxidation in monoclonal antibodies.3
  • Size-exclusion chromatography (SEC) is used to separate molecules in a solution based on their size or molecular weight. SEC with UV or light scattering detection is the technique of choice to study the aggregation in proteins and antibodies, or disintegration of the molecule during manufacturing or subsequent handling. An SE-HPLC method has been developed for the quality control of a viral vaccine and virus-like particles (VLPs).4
  • Ion-exchange chromatography (IEX) with UV detection is amenable for detecting the presence of charge-variants. IEX also helps scientists understand protein-protein interactions in native states.5
  • Hydrophobic interaction chromatography (HIC) is used to purify biomolecules by isolating them using their hydrophobicity. This technique has been applied to separate pharmaceutical-grade supercoiled plasmid DNA from other isoforms.6 HIC in conjunction with RP-HPLC has been used for analyzing antibody-drug conjugates.7
  • LC techniques such as RPLC, HIC, SEC and multidimensional separation have been employed for determining drug-to-antibody ratios, evaluating drug load distribution and analyzing process-related impurities in antibody-drug conjugates.8
  • The glycans or sugars attached to monoclonal antibodies are characterized by fluorescence or MS analysis post-LC separation.9
  • LC-MS is commonly used to analyze post-translational modifications, such as methionine oxidation or deamidations, in biologics. A recent study has used an untargeted LC-high-resolution (HR) MS to detect global transformations to a monoclonal antibody by differential analysis.10
  • Affinity chromatography, which is based on specific interactions between the biomolecules and their ligands, is used for their isolation and titer (concentration) determination.11
  • HPLC has been shown to be suitable for routine compliance testing of biosimilars.12
  • Besides one-(1D-LC) and two-dimensional LC (2D-LC), multi-dimensional LC (mD-LC) that combines online sample preparation with multi-level assays using the same LC system are being developed for biopharmaceutical analysis.13

Scientists examining samples and biotherapeutic medications with molecular structures.

Liquid chromatography finds numerous, diverse applications in the biopharmaceutical industry. Credit: Technology Networks

In addition to characterizing and quantifying biotherapeutics, LC has also been applied for the analysis of excipients,14 impurities such as leached silicone oil in therapeutic protein formulations collected from pre-filled syringes,15 as well as for purification of biologics.16

What is the significance of chromatography in pharmaceutical analysis?

During production, biopharmaceuticals are susceptible to alterations in their structures. For instance, during cell culture, protein structure can be modified by:

  • methylation, phosphorylation or glycosylation of amino acid residues
  • disulfide scrambling
  • deamidation
  • C-terminal lysine or arginine cleavage

This necessitates separation of the variants from one another as well as the media and impurities. LC is an effective tool for this purpose. Moreover, most biopharmaceuticals are UV active or possess chromophores which make them amenable to detection by UV-Vis/PDA detectors. Using RP-HPLC to determine the primary structure of proteins eliminates the need to perform the time-consuming Edman degradation to determine the amino acid sequence. Compared to the conventional techniques for analysis of antibodies such as enzyme-linked immunosorbent assay (ELISA), HPLC provides several advantages such as lower detection limits, suitability for quantitative analysis and better reproducibility. Furthermore, there is no dependency on the availability of antibodies as in the case of the ELISA. Characterization of vaccines by LC is a viable alternative to expensive and time-consuming animal testing, ELISA or analytical ultracentrifugation.

The availability of HPLC instruments in most labs, ease of operation and maintenance make chromatography an ideal technique for routine analysis. The range of forms of LC mentioned above makes it suitable for characterizing different aspects of biopharmaceuticals. With ultra-high-performance liquid chromatography (UHPLC), the speed of analysis can be improved; further availability of
bio-inert versions of UHPLC components makes it ideal for rapid and routine analysis of biopharmaceuticals.

What makes biopharma samples challenging for LC?

The analysis of biopharmaceuticals poses unique challenges due to their:

a) relatively larger size as compared with small molecule drugs

b) physical characteristics, such as multiple charge states in the case of protein and peptide molecules

c) micro-heterogeneity

This necessitates the use of multiple orthogonal techniques for complete characterization. Some structural variants that could impact the safety and potency of the drug may be present at low concentrations. As a result, sample preparation to isolate the low-abundance proteins is a crucial step in biopharmaceutical analysis. At times, novel biopharmaceutical drugs may need to be evaluated from the ground up as very little information may be available in the literature. This can require the use of universal detectors such as mass spectrometers for initial assessment. Additionally, complementary techniques may be necessary to detect non-UV-absorbing modifications, impurities or excipients.

Although HPLC is ideal for routine analysis of well-established biopharmaceuticals, some factors have to be borne in mind to ensure better chromatography. To minimize or eliminate adhesion of the biomolecules to the flow path and interaction with the metal ions, specifically iron or steel, periodic passivation of the flow path, as per the standard operating procedure (SOP) or use of bio-inert HPLC systems is recommended. In addition to preserving the integrity of the molecule, use of a bio-inert system eliminates corrosion of the metallic components in the sample flow path resulting from long-term exposure to buffers with high salt concentrations or high pH. These systems have sample flow-paths made of inert materials such as titanium, PEEK (polyetheretherketone, an engineering plastic) or ceramic that are resistant to oxidation and corrosion.

For biopharmaceutical analysis, columns with appropriate chemistries and dimensions are required for different types of analyses. RP columns with larger pore sizes and short alkyl chains are recommended for intact protein analysis, while SEC and IEX columns are used for size-exclusion and ion-exchange chromatography applications respectively. Hydrophilic interaction liquid chromatography (HILIC) columns are preferred for glycan analysis, and peptide mapping is done using C18-RP columns. For almost all of these applications, bio-inert columns can be used to minimize non-specific binding and improve reproducibility.

Chromatographic parameters such as flow rate are optimized to achieve good resolution and sensitivity; adjusting the ionic strength and pH of the mobile phases ensures analyte solubility, minimizes their interaction with the free silanol groups on the column and maintains the activity of the biomolecules. Maintaining optimal column temperature is important as biomolecules are susceptible to denaturation at elevated temperatures. SEC columns have to be calibrated with suitable standards of known size and weight. Sensitive detectors are required to detect and identify low-abundance proteins and minor modifications.


The variety of LC formats and column properties (including RP, SEC, IEX, HILIC, HIC, affinity or preparative) and ability to combine with various detectors, such as UV-Vis, fluorescence, light-scattering and MS, help to make LC an ideal tool for the routine analysis of the critical quality attributes of biopharmaceuticals.


Majumder J, Minko T. Recent developments on therapeutic and diagnostic approaches for COVID-19. AAPS J. 2021;23(1):14. doi: 10.1208/s12248-020-00532-2
2.    Kesik-Brodacka, M. Progress in biopharmaceutical development. Biotechnol. Appl. Biochem. 2018;65 (3): 306-322. doi:10.1002/bab.1617
3.    Li X, Xu W, Wang Y, et al. High throughput peptide mapping method for analysis of site specific monoclonal antibody oxidation. J. Chromatogr. A, 2016;1460:51-60. doi:10.1016/j.chroma.2016.06.085
4.    Yang Y, Li H, Li Z, et al. Size-exclusion HPLC provides a simple, rapid, and versatile alternative method for quality control of vaccines by characterizing the assembly of antigens. Vaccine, 2015;33(9):1143-1150. doi:10.1016/j.vaccine.2015.01.031
5.    Spanov B, Olaleye O, Lingg N, et al. Change of charge variant composition of trastuzumab upon stressing at physiological conditions, J. Chromatogr. A, 2021;1655: 462506. doi:10.1016/j.chroma.2021.462506
6.    Bo H, Wang J, Chen Q, Shen H, Wu F, Shao H, Huang S. Using a single hydrophobic-interaction chromatography to purify pharmaceutical-grade supercoiled plasmid DNA from other isoforms. Pharm Biol. 2013;51(1):42-8. doi: 10.3109/13880209.2012.703678
7.    Ouyang J. Drug-to-antibody ratio (DAR) and drug load distribution by hydrophobic interaction chromatography and reversed phase high-performance liquid chromatography. Methods Mol Biol. 2013;1045:275-83. doi: 10.1007/978-1-62703-541-5_17
8.    Bobály B, Fleury-Souverain S, Beck A, Veuthey JL, Guillarme D, Fekete S. Current possibilities of liquid chromatography for the characterization of antibody-drug conjugates. J Pharm Biomed Anal. 2018;147:493-505. doi: 10.1016/j.jpba.2017.06.022
9.    Kinoshita M, Saito A, Yamamoto S, Suzuki S. A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis. J Pharm Biomed Anal. 2020;186:113267. doi: 10.1016/j.jpba.2020.113267
10.  Yao M, Chen B, Zhao W, Mehl JT, Li L, Zhu M. LC-MS differential analysis for fast and sensitive determination of biotransformation of therapeutic proteins. Drug Metab Dispos. 2018;46(4):451-457. doi: 10.1124/dmd.117.077792
11.  Dunn ZD, Desai J, Leme GM, Stoll DR, Richardson DD. Rapid two-dimensional Protein-A size exclusion chromatography of monoclonal antibodies for titer and aggregation measurements from harvested cell culture fluid samples. MAbs. 2020;12(1):1702263. doi: 10.1080/19420862.2019.1702263
12.  Huo Y, He J, Li F.  Sialic acids content analysis of the innovator and biosimilar darbepoetin alfa by fluorometric HPLC assay. Current Pharm. Anal.2019;15(4):333-337. doi: 10.2174/1573412914666180427160327
13.  Hebbi V, Chattopadhyay S, Rathore AS. High performance liquid chromatography (HPLC) based direct and simultaneous estimation of excipients in biopharmaceutical products. J Chromatogr B Analyt Technol Biomed Life Sci. 2019;1117:118-126. doi: 10.1016/j.jchromb.2019.04.022
14.  Camperi J, Goyon A, Guillarme D, Zhang K, Stella C. Multi-dimensional LC-MS: the next generation characterization of antibody-based therapeutics by unified online bottom-up, middle-up and intact approaches. Analyst. 2021;146:747-769. doi: 10.1039/D0AN01963A
15.  Liu J, Ronk M, Fujimori K, Lee H, Nashed-Samuel Y. Analysis of silicone oil in prefilled syringes and biopharmaceutical drug products using high-performance liquid chromatography. AAPS PharmSciTech. 2021;22:75. doi:10.1208/s12249-021-01947-6
16.  De Luca C, Lievore G, Bozza D, et al. Downstream processing of therapeutic peptides by means of preparative liquid chromatography. Molecules. 2021;26(15):4688. Published 2021 Aug 3. doi:10.3390/molecules26154688