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Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

Bomans K, Lang A, Roedl V, Adolf L, Kyriosoglou K, Diepold K, Eberl G, Mølhøj M, Strauss U, Schmalz C, Vogel R, Reusch D, Wegele H, Wiedmann M, Bulau P - PLoS ONE (2013)

Bottom Line: A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low 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.

View Article: PubMed Central - PubMed

Affiliation: Pharma Development, Roche Diagnostics GmbH, Penzberg, Germany.

ABSTRACT
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 E. coli 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.

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Related in: MedlinePlus

SDS-PAGE analysis of RP-HPLC fractions.(a) Mark12™ Standard; (b) Reference material, drug substance level; (c) RP-HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 13 ppm; (d) RP‑HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 35 ppm.
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pone-0081639-g003: SDS-PAGE analysis of RP-HPLC fractions.(a) Mark12™ Standard; (b) Reference material, drug substance level; (c) RP-HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 13 ppm; (d) RP‑HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 35 ppm.

Mentions: In general, no significant differences in product variants where observed for batches with elevated HCP levels at purification step 1 level. However, a slight but distinct increase in peak intensity of the product variant with a retention time of 25 min could be observed for batches with elevated HCP levels (Figure 2B; marked by an arrow). The existence of elevated product variants or HCP levels was further suggested by SDS-PAGE analysis of the respective HPLC fractions (retention time window: 24-26 min), in which an increased content of a protein species with a molecular weight of around 23 kDa was observed for batches with elevated HCP levels (Figure 3). The unknown protein was identified as E. coli Alkyl hydroperoxide reductase subunit C (see Table 1) by nano-ESI-MS peptide mapping after in tryptic-gel digestion combined with database searching (mass spectrometric data not shown). Additionally, various potential host cell proteins, not detected by RP-HPLC analysis, were visualized by SDS-PAGE analysis of total purification step 1 elution pool (Figure 4). The dominant HCP with a molecular weight of around 50 kDa was identified as Bacterial Alkaline Phosphatase (BAP; see Table 1) by the procedure as described above (data not shown) and located in the injection peak of the RP-HPLC chromatogram by HPLC fractionation (retention time window: 2-4 min, Figure 2B). The SDS-PAGE analysis of purification step 1-3 elution pools suggest a complete HCP removal by the applied purification process (Figure 4). Nevertheless, tryptic peptide mapping combined with comprehensive online liquid chromatography mass spectrometry (LC-MS/MS) was utilized to identify low abundant HCPs of the different elution pools. Protein identification by database searching was successful to identify various bacterial proteins present in the elution pools of the purifications steps 1 and 2. The identified HCPs are summarized in Table 1. Although the analysis was performed with a highly sensitive LTQ Orbitrap Velos electrospray mass spectrometer, only one bacterial protein (BAP) was verified in the elution pool of purification step 3 and no HCPs were detected at the final drug substance level (Table 1). Since the deployed process-specific ELISA system does demonstrate low abundant HCP levels at final bulk stage (Figure 2), we introduced HCP enrichment by affinity chromatography using the ELISAs polyclonal antibodies in our sample preparation protocol to detect bacterial proteins of low abundance (see details in materials and methods). In order to judge if the HCP enrichment step does significantly enrich or deplete individual HCPs, purification step 1 elution pool was again analyzed with and without applying the affinity enrichment procedure. The identified proteins (identification score ≥ 20; peptides with an FDR <1%) are listed in Table 2 (Repeatability results are summarized in Table S1). All abundant bacterial proteins of the purification step 1 elution pool were also detected after applying the HCP enrichment step. Although the identification scores of some bacterial proteins do suggest alterations of the relative protein abundances only minor effects on protein sequence coverage were observed. In addition, an increasing number of identified HCPs was demonstrated as a consequence of the affinity enrichment procedure. Thus, from a qualitative point of view, the results achieved do not suggest significant alterations of the HCP profile related to the affinity enrichment step.


Identification and monitoring of host cell proteins by mass spectrometry combined with high performance immunochemistry testing.

Bomans K, Lang A, Roedl V, Adolf L, Kyriosoglou K, Diepold K, Eberl G, Mølhøj M, Strauss U, Schmalz C, Vogel R, Reusch D, Wegele H, Wiedmann M, Bulau P - PLoS ONE (2013)

SDS-PAGE analysis of RP-HPLC fractions.(a) Mark12™ Standard; (b) Reference material, drug substance level; (c) RP-HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 13 ppm; (d) RP‑HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 35 ppm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842259&req=5

pone-0081639-g003: SDS-PAGE analysis of RP-HPLC fractions.(a) Mark12™ Standard; (b) Reference material, drug substance level; (c) RP-HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 13 ppm; (d) RP‑HPLC fraction (24-26 min, Figure 2B) of purification step 1 elution pool, HCP content at drug substance level: 35 ppm.
Mentions: In general, no significant differences in product variants where observed for batches with elevated HCP levels at purification step 1 level. However, a slight but distinct increase in peak intensity of the product variant with a retention time of 25 min could be observed for batches with elevated HCP levels (Figure 2B; marked by an arrow). The existence of elevated product variants or HCP levels was further suggested by SDS-PAGE analysis of the respective HPLC fractions (retention time window: 24-26 min), in which an increased content of a protein species with a molecular weight of around 23 kDa was observed for batches with elevated HCP levels (Figure 3). The unknown protein was identified as E. coli Alkyl hydroperoxide reductase subunit C (see Table 1) by nano-ESI-MS peptide mapping after in tryptic-gel digestion combined with database searching (mass spectrometric data not shown). Additionally, various potential host cell proteins, not detected by RP-HPLC analysis, were visualized by SDS-PAGE analysis of total purification step 1 elution pool (Figure 4). The dominant HCP with a molecular weight of around 50 kDa was identified as Bacterial Alkaline Phosphatase (BAP; see Table 1) by the procedure as described above (data not shown) and located in the injection peak of the RP-HPLC chromatogram by HPLC fractionation (retention time window: 2-4 min, Figure 2B). The SDS-PAGE analysis of purification step 1-3 elution pools suggest a complete HCP removal by the applied purification process (Figure 4). Nevertheless, tryptic peptide mapping combined with comprehensive online liquid chromatography mass spectrometry (LC-MS/MS) was utilized to identify low abundant HCPs of the different elution pools. Protein identification by database searching was successful to identify various bacterial proteins present in the elution pools of the purifications steps 1 and 2. The identified HCPs are summarized in Table 1. Although the analysis was performed with a highly sensitive LTQ Orbitrap Velos electrospray mass spectrometer, only one bacterial protein (BAP) was verified in the elution pool of purification step 3 and no HCPs were detected at the final drug substance level (Table 1). Since the deployed process-specific ELISA system does demonstrate low abundant HCP levels at final bulk stage (Figure 2), we introduced HCP enrichment by affinity chromatography using the ELISAs polyclonal antibodies in our sample preparation protocol to detect bacterial proteins of low abundance (see details in materials and methods). In order to judge if the HCP enrichment step does significantly enrich or deplete individual HCPs, purification step 1 elution pool was again analyzed with and without applying the affinity enrichment procedure. The identified proteins (identification score ≥ 20; peptides with an FDR <1%) are listed in Table 2 (Repeatability results are summarized in Table S1). All abundant bacterial proteins of the purification step 1 elution pool were also detected after applying the HCP enrichment step. Although the identification scores of some bacterial proteins do suggest alterations of the relative protein abundances only minor effects on protein sequence coverage were observed. In addition, an increasing number of identified HCPs was demonstrated as a consequence of the affinity enrichment procedure. Thus, from a qualitative point of view, the results achieved do not suggest significant alterations of the HCP profile related to the affinity enrichment step.

Bottom Line: A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low 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.

View Article: PubMed Central - PubMed

Affiliation: Pharma Development, Roche Diagnostics GmbH, Penzberg, Germany.

ABSTRACT
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 E. coli 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.

Show MeSH
Related in: MedlinePlus