AAE-MS™ as an Orthogonal Method in Host Cell Protein Analytics

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Host cell proteins (HCPs) co-purify with biological drug substances (DS) and pose potential risks for both patients and drug manufacturers. While many HCPs are benign, some are immunogenic, others may interact with the drug substance (DS) and impact its efficacy, and others can interfere with DS stability. Thus, the quantity and nature of residual HCPs in DS are generally considered a critical quality attribute (CQA) constituting a significant component of a biopharmaceutical drug developer’s overall risk-management strategy (1-3).

Identification and quantification of HCPs by mass spectrometry (MS) is a powerful complementary method to demonstrate the suitability of a HCP ELISA. When combined with antibody affinity extraction (AAE™), MS can be used to demonstrate that an HCP ELISA is suitable for the purpose of monitoring purification process consistency and product lot release.AAE-MS™ can also be used to identify and quantify HCPs in in-process and DS samples.

Antibody Affinity Extraction

Antibody Affinity Extraction (AAE™), a type of immunoaffinity chromatography, is an advanced orthogonal method designed to assess coverage of a polyclonal antibody to an array of HCPs present in a given process, as well as HCP antibody reactivity to downstream, process-specific HCPs that may co-purify with a drug substance. Developed by Cygnus Technologies in 2013, this method overcomes the analytical deficiencies of the traditional 2D Western Blot (2D WB) and two-dimensional differential in blot electrophoresis (2D-DIBE) orthogonal methods used to assess coverage of polyclonal antibodies to total host cell protein. To perform AAE, the polyclonal HCP antibody is covalently immobilized on a chromatography support. The column is then conditioned to prevent significant leaching of the antibody and to minimize any non-specific binding. The HCP sample in its native, undenatured state is passed over the column for binding and then eluted with acid. The HCP sample is again cycled over the column by binding and elution until no additional HCP is bound. All HCP elution fractions are pooled, buffer exchanged, and concentrated back to the original sample volume. The final sample is then separated by 2D PAGE and analyzed by either a comparison to a silver stain of starting, unextracted sample or by differential gel electrophoresis (DIGE) using Cy3 and Cy5 to label the extracted and starting, unextracted samples (Figure 1). The coverage is assessed by the number of HCPs in the AAE elution fraction (silver stain or Cy3 labeled) compared to those seen in the 2D PAGE of the starting antigen sample (silver stain or Cy5 labeled). Alternatively, HCPs in the Pre-AAE starting material and Post-AAE elution fraction are analyzed by LC-MS.

HCP antibody coverage analysis by AAE-MS

Combining AAE with MS for HCP coverage analysis provides both the % coverage and identifies HCPs in the harvest material and HCPs reactive with the antibody.In the example below, coverage analysis was performed on the CHO HCP sample using the CHO HCP Antibody AAE column. LC-MS detected 1673 CHO HCPs in the sample. 1639 HCPs spots were detected in the AAE elution fraction representing 97 - 98% coverage (Table 1). The HCPs identified in the Pre- and Post-AAE samples in the virtual two-dimensional gels of the sample were within the same range of molecular weight (MW) and isoelectric point (pI) and covered the major range of the CHO proteome (Table 1, Figure 2).

Figure 1: Antibody coverage by AAE with 2D PAGE or MS analysis options.

Table 1: Anti-CHO HCP 3G (Cat. # 3G-0016-1-AF, F550-1) pAb Coverage

Figure 2: Virtual 2D Gel of anti-CHO HCP 3G pAb Coverage (Cygnus Technologies, Cat #: 3G-0016-1-AF, F550-1 CHO HCP ELISA, 3G)

LC-MS and ELISA are confirmatory orthogonal methods through the downstream purification process.

Cygnus Technologies’ LC-MS and CHO 3G ELISA can be used to quantify CHO HCPs in a purification process. As presented in Figure 3, HCPs were quantified in ng/mL and the level of HCPs were confirmed with the two separate methods. HCPs were quantified in samples from purification steps 1-2, and in different lots of DS. LC-MS and ELISA confirmed a breakdown of the purification process of lot DS #2 and warranted further investigation.

Figure 3: ELISA and LC-MS are Confirmatory Orthogonal Methods

Identification of HCPs in a DS by AAE-MS

Identification and quantification of HCPs by MS is a powerful complementary method to ELISA; however, drug products often mask HCPs by a factor of 104-106. AAE is highly effective at enriching HCPs and depleting DS. Figure 4a shows the relative abundance of DS and HCPs according to their extracted ion chromatograms that were normalized to one and graphed in a stacked bar chart. Pre-AAE (left) shows the DS heavy chain (DS HC) in orange, the DS light chain (DS LC) in light blue and HCPs in assorted colors beneath demonstrating that most peptides in the Pre-AAE samples belong to DS. Following AAE enrichment (Post-AAE, right), however, the relative abundance of the HCPs dramatically increased and that of the DS HC and LC decreased.

MS identifications of HCPs in DS provides predictive MW and pI. These data can be graphed into a “virtual 2D gel” as shown in Figure 4b. These visual representations of the HCPs present in DS samples can provide strategies for DSP purification groups. By focusing on a “potentially problematic HCP” in a quadrant of the virtual 2D gel, chromatography groups can employ column chemistries to eliminate HCPs of interest.

Fig 4:

4a: AAE Enriches HCPs and Depletes DS

4b: Virtual Two-Dimensional Gel of CHO HCPs in a DS Sample: MW vs. pI.


MS plays an important role in HCP analytics from IND through post-marketing, when evaluating the impacts of a process change, risk assessment, and characterizing reagent changes. And although complete characterization of downstream HCPs is not part of the current regulatory guidelines, the value of this information to biopharmaceutical companies to better assure safety and efficacy are recognized as value added data by proactive manufacturers and regulators.

Cygnus Technologies, part of Maravai LifeSciences, offers generic HCP ELISA Kits for 23 different expression platforms, advanced orthogonal antibody coverage analysis services, HCP identification in process samples and drug substances by AAE-MS™, generic assay qualification services, and expert process-specific antibody and assay development services. For more information, visit us at:



  1. Durocher, V. et al. J. of Biotech. 2017
  2. Levy, N. et al. Biotechnol Bioeng. 2014
  3. Vanderlaan, M. et al Biotechnol. Prog. 2018