In the control strategy for process related impurities in biopharmaceuticals the enzyme linked immunosorbent assay (ELISA) is the method of choice for the quantification of host cell proteins (HCP). Besides two dimensional – western blots (2D-WB), the coverage of ELISA antibodies is increasingly evaluated by affinity purification based liquid chromatography-tandem mass spectrometry (AP-MS) methods. However, all these methods face the problem of unspecific binding issues between antibodies and the matrix, involving the application of arbitrarily defined thresholds during data evaluation. To solve this, a new approach (optimized AP-MS) was developed in this study, for which a cleavable linker was conjugated to the ELISA antibodies enabling the subsequent isolation of specifically interacting HCPs.
By comparing both approaches in terms of method variability and the number of false positive or negative hits, we could demonstrate that the optimized AP-MS method is very reproducible and superior in the identification of antibody detection gaps, while previously described strategies suffered from over- or underestimating the coverage. As only antibody associated HCPs were identified, we demonstrated that the method is beneficial for hitchhiker analysis. Overall, the method described herein has proven as a powerful tool for reliable coverage determination of ELISA antibodies, without the need to arbitrarily exclude HCPs during the coverage evaluation.
Direct Atomic-Scale Observation of Ultrasmall Ag Nanowires that Exhibit fcc, bcc, and hcp Structures under Bending
Metals usually have three crystal structures: face-centered cubic (fcc), body-centered cubic (bcc), and hexagonal-close packed (hcp) structures. Typically, metals exhibit only one of these structures at room temperature. Mechanical processing can cause phase transition in metals, however, metals that exhibit all the three crystal structures have rarely been approached, even when hydrostatic pressure or shock conditions are applied.
Here, through in situ observation of the atomic-scale bending and tensile process of ∼5 nm-sized Ag nanowires (NWs), we show that bending is an effective method to facilitate fcc-structured Ag to access all the above-mentioned structures. The process of transitioning the fcc structure into a bcc structure, then into an hcp structure, and finally into a re-oriented fcc structure under bending has been witnessed in its entirety. This re-oriented fcc structure is twin-related to the matrix, which leads to twin nucleation without the need for partial dislocation activities. The results of this study advance our understanding of the deformation mechanism of small-sized fcc metals.
An Effective Strategy to Maintain the CALPHAD Atomic Mobility Database of Multicomponent Systems and Its Application to Hcp Mg-Al-Zn-Sn Alloys
In this paper, a general and effective strategy was first developed to maintain the CALPHAD atomic mobility database of multicomponent systems, based on the pragmatic numerical method and freely accessible HitDIC software, and then applied to update the atomic mobility descriptions of the hcp Mg-Al-Zn, Mg-Al-Sn, and Mg-Al-Zn-Sn systems. A set of the self-consistent atomic mobility database of the hcp Mg-Al-Zn-Sn system was established following the new strategy presented.
A comprehensive comparison between the model-predicted composition-distance profiles/inter-diffusivities in the hcp Mg-Al-Zn, Mg-Al-Sn, and Mg-Al-Zn-Sn systems from the presently updated atomic mobilities and those from the previous ones that used the traditional method indicated that significant improvement can be achieved utilizing the new strategy, especially in the cases with sufficient experimental composition-distance profiles and/or in higher-order systems. Furthermore, it is anticipated that the proposed strategy can serve as a standard for maintaining the CALPHAD atomic mobility database in different multicomponent systems.
l-Asparaginase and HCP quantification by SWATH LC-MS/MS for new and improved purification step in Erwinia chrysanthemil-asparaginase manufacture
- Erwinase® or Erwinaze® are the proprietary names for the L-asparaginase enzyme derived from Erwinia chrysanthemi.L-asparaginase is an integral part of the treatment of Acute Lymphoblastic Leukaemia (ALL) in children and adolescents. E. chrysanthemiL-asparaginase was first developed in the early 1970s at Porton Down and is currently manufactured by Porton Biopharma Ltd. One of the early purification steps during E. chrysanthemiL-asparaginase manufacture, involves use of batch cation exchange carboxymethyl resin, and alternatives to this older technology are currently under investigation using mass spectrometry to understand the impact of resin changes on the impurity profile.
- In this study, a novel SWATH library was developed for E. chrysanthemi proteome and used to evaluate this potential process change on product yield and host cell protein (HCP) profile and clearance. An ELISA assay is currently used as a quality control release test for quantifying HCPs at the Drug Substance (DS) stage, but these early extract samples are too crude for interference-free analysis by ELISA. Given that ELISA assay could not be used in the assessment of new resin options, SWATH LC-MS/MS analysis proved to be pivotal in selecting a resin for further scale-up and implementation.
- The data quantified that L-asparaginase from the new process step was 2.28-fold higher in concentration than in legacy-process samples. The new step, using a modern ion exchanger, was at least equivalent and in some cases outperformed the legacy resin step in terms of HCP clearance for 78.2% of total HCPs (528 of 675 total proteins).
HcP-H |
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| T32049-10mg | TargetMol Chemicals | 10mg | Ask for price |
HcP-H |
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| T32049-1g | TargetMol Chemicals | 1g | Ask for price |
HcP-H |
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| T32049-1mg | TargetMol Chemicals | 1mg | Ask for price |
HcP-H |
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| T32049-50mg | TargetMol Chemicals | 50mg | Ask for price |
HcP-H |
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| T32049-5mg | TargetMol Chemicals | 5mg | Ask for price |
HcP-H |
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| MBS5781632-INQUIRE | MyBiosource | INQUIRE | Ask for price |
HCP protein |
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| 30R-3273 | Fitzgerald | 50 ug | 308.4 EUR |
Coelenterazine HCP |
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| 10113 | Biotium | 50uG | 80 EUR |
Coelenterazine HCP |
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| 10113-1 | Biotium | 1MG | 80 EUR |
Coelenterazine HCP |
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| 10113-1-1 | Biotium | EA | 831 EUR |
Coelenterazine HCP |
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| 10113-2 | Biotium | 250uG | 257 EUR |
Coelenterazine HCP |
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| 10113-2-1 | Biotium | EA | 257 EUR |
Coelenterazine hcp |
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| T36847-10mg | TargetMol Chemicals | 10mg | Ask for price |
Coelenterazine hcp |
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| T36847-1g | TargetMol Chemicals | 1g | Ask for price |
Coelenterazine hcp |
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| T36847-1mg | TargetMol Chemicals | 1mg | Ask for price |
Coelenterazine hcp |
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| T36847-50mg | TargetMol Chemicals | 50mg | Ask for price |
Coelenterazine hcp |
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| T36847-5mg | TargetMol Chemicals | 5mg | Ask for price |
Coelenterazine hcp |
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| MBS5797882-INQUIRE | MyBiosource | INQUIRE | Ask for price |
HCP-3 Antibody |
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| SAB493 | SAB | 100ul | 319 EUR |
HCP-3 Antibody |
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| MBS9458580-005mL | MyBiosource | 0.05mL | 245 EUR |
HCP-3 Antibody |
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| MBS9458580-01mL | MyBiosource | 0.1mL | 305 EUR |
HCP-3 Antibody |
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| MBS9458580-5x01mL | MyBiosource | 5x0.1mL | 1230 EUR |
Anti-CHO HCP |
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| 3G-0016 | Cygnus Technologies | 1 ml | 400.8 EUR |
Anti-CHO HCP |
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| 3G-0016-AF | Cygnus Technologies | 1 mg | 2295.6 EUR |
Anti-CHO HCP |
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| 3G-0016-AFB | Cygnus Technologies | 1 mg | 4143.6 EUR |
Anti-CHO HCP |
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| 3G-0016-PA | Cygnus Technologies | 1 ml | 585.6 EUR |
Versatile LC-MS-Based Workflow with Robust 0.1 ppm Sensitivity for Identifying Residual HCPs in Biotherapeutic Products
Residual host cell proteins (HCPs) in the drug product can affect product quality, stability, and/or safety. In particular, highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) due to their high dynamic range between HCPs and biotherapeutic proteins. We employed new strategies to address the challenge: (1) native digest at a high protein concentration; (2) sodium deoxycholate added during the reduction step to minimize the inadvertent omission of HCPs observed with native digestion; and (3) solid phase extraction with 50% MeCN elution prior to LC-MS/MS analysis to ensure effective mAb removal. A 50 cm long nanoflow charged surface hybrid column was also packed to allow for higher sample load for increased sensitivity.
Our workflow has increased the sensitivity for HCP identification by 10- to 100-fold over previous reports and showed the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by consistently identifying >85% of 48 UPS-1 proteins (0.10 to 1.34 ppm, 6.3 to 82.9 kDa MW) in a monoclonal antibody (mAb) and the largest number (746) of mouse proteins from NIST mAb reported to date by a single analysis. Our work has filled a significant gap in HCP analysis for detecting and demonstrating HCP clearance, in particular, extremely low-level hydrolases in drug process development.