Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS

Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection is frequently used to separate and quantify mAb variants in routine quality control (QC). However, characterization of the chromatographic peaks resulting from an IEC separation is an extremely time-consuming process, involving many cumbersome steps. Presented here is an online four-dimensional high performance liquid chromatography–mass spectrometry (4D HPLC/MS) approach, developed to circumvent these limitations. To achieve this, a 2D HPLC system was extended through the introduction of additional modules, hence enabling fully automated bioseparation of mAbs, fractionation of peaks, reduction, tryptic digestion, and reversed-phase (RP) separation of resulting peptides followed by MS detection. The entire separation and analytical process for an unknown peak is performed in less than 1.5 h, leading to a significant time savings, with comparable sequence coverage. To show the comparability with the traditional offline process, a proof of concept study with a previously characterized mAb is presented in this paper

Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

<div><p>Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various <i>E. coli</i> host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.</p> </div

Monitoring of product variants (*) by RP-HPLC.

<p>Batches with different HCP content at drug substance level (A) and the product elution pool after metal affinity purification step 1 (B). Batch differences are marked by an arrow.</p

SDS-PAGE analysis of in-process controls.

<p>(a) Mark12™ Standard; (b) Purification step 1 elution pool; (c) Purification step 2 elution pool; (d) Purification step 3 elution pool; (e) Drug substance, HCP content: 30 ppm.</p

SDS-PAGE analysis of RP-HPLC fractions.

<p>(a) Mark12™ Standard; (b) Reference material, drug substance level; (c) RP-HPLC fraction (24-26 min, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081639#pone-0081639-g002" target="_blank">Figure 2B</a>) of purification step 1 elution pool, HCP content at drug substance level: 13 ppm; (d) RP‑HPLC fraction (24-26 min, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081639#pone-0081639-g002" target="_blank">Figure 2B</a>) of purification step 1 elution pool, HCP content at drug substance level: 35 ppm.</p