The present disclosure relates to the use of host cell protein biomarkers to assess disulfide bond reduction in compositions comprising a protein of interest. In some embodiments, the disclosure relates to methods of predicting the occurrence of disulfide bond reduction or low molecular weight protein species in compositions comprising a protein of interest, wherein the expression or activity level of at least one host cell protein is measured and provides a benchmark value associated with the occurrence of disulfide bond reduction or low molecular weight species of said protein of interest. In some embodiments, the disclosure relates to methods of producing a protein of interest, wherein host cells capable of producing the protein of interest are cultured, the expression or activity level of at least one host cell protein is measured, and downstream isolation of the protein of interest is informed by the host cell protein measurements.

In accordance with some embodiments herein, methods of determining signatures of HMGB1 isoforms in a subject are provided. In some embodiments, antibodies that bind specifically to HMGB1 isoforms are provided. In some embodiments, immunoassay kits are provided.

Embodiments described herein relate to devices, and methods for quantifying thiol content in a sample containing a mixture of proteins or protein isoforms. The method includes conjugating a portion of the sample with free thiol detection binders, separating the contents in the portion of the sample into separated protein isoforms, detecting fluorescence signals associated with each separated protein isoform, and quantifying, based on the fluorescence signals, a relative amount of free thiol associated with each separated protein isoform. In some instances, the method includes quantifying the amount of each separated protein isoform based on absorbance signals associated with each separated protein isoform. In some instances, the fluorescence and/or absorbance signals associated with protein isoforms conjugated with detection binders can be compared with the corresponding signals associated with unconjugated protein isoforms. In some instances, the method further includes applying a reducing agent and quantifying total-thiol content in the sample.

Methods for peptide and/or protein quantification by mass spectrometry using labeled peptides, wherein multiple labels lead to distinct fragments for the labeled peptides and their unlabeled variant, thus facilitating data analysis and enhancing the potential for quantification. Methods for selecting the label and label position are further given, as well as sets of labeled peptides resulting from or for use in the above-mentioned methods. The methods and substances are especially useful for data-independent or multiplexed parallel reaction monitoring proteomics applications involving peptide quantification.

The application discloses compositions, methods, systems, and apparatuses for rapid sequence analysis of proteins, including location of post-translational modifications and disulfide bonds. Limited digestion of fully denatured antibody occurs in seconds by flowing sample in 8M urea at constant pressure through a micro column reactor containing immobilized aspergillopepsin I, resulting in a product mixture containing 3-10 kDa peptides, which is then fractionated by capillary column chromatography and analyzed by both electron transfer dissociation (ETD) and collision activated dissociation mass spectrometry. This method provides 95% sequence coverage of a mAb and detects numerous post-translational modifications. For disulfide bond location, native mAb is subjected to longer digestion times. Release of disulfide containing peptides from accessible regions of the folded protein occurs with short digestion times. The identity of peptides connected by a disulfide bond is determined using ETD and ion-ion proton transfer chemistry.

The present invention relates to a method for analysing protein variants of a recombinant protein of interest, such as antibodies or Fc-fusion proteins, in a liquid sample of a mammal. Specifically the method comprises a step of affinity purifying the recombinant protein of interest from the sample together with an internal standard, and analyzing the protein variants using an analytic separating method such as HPLC, capillary electrophoresis or MS. This method is particularly suited to measure pharmacokinetic parameters of a recombinant protein of interest, such as a biopharmaceutical, in a mammal in clinical or pre-clinical studies. It allows for the use of a small sample volume and the possibility to operate with high throughput, such as in a 96-well plate sample preparation. It also provides high sensitivity and allows analysis of protein variants individually.

Embodiments described herein relate to devices, and methods for quantifying thiol content in a sample containing a mixture of proteins or protein isoforms. The method includes conjugating a portion of the sample with free thiol detection binders, separating the contents in the portion of the sample into separated protein isoforms, detecting fluorescence signals associated with each separated protein isoform, and quantifying, based on the fluorescence signals, a relative amount of free thiol associated with each separated protein isoform. In some instances, the method includes quantifying the amount of each separated protein isoform based on absorbance signals associated with each separated protein isoform. In some instances, the fluorescence and/or absorbance signals associated with protein isoforms conjugated with detection binders can be compared with the corresponding signals associated with unconjugated protein isoforms. In some instances, the method further includes applying a reducing agent and quantifying total-thiol content in the sample.

Methods and system that determines disulfide and trisulfide linkages within analytes (e.g., polypeptides) is described. In certain aspects. a sample comprising polypeptides (such as an antibody) may be subjected to dissociation using an electron activated dissociation (which can include electron capture dissociation and electron transfer dissociation) and the fragmentated portions are analyzed using a mass spectrometer to produce a spectrum. The spectrum is analyzed by a processor to identify peaks from the spectrum that are related to one another in the spectra by a separation of 32 mass units. In identifying an antibody comprising peptide segments linked via a trisulfide bond. for example, four different peaks representing two different peptides are searched for and identified representing a first peptide portion having mass/charge of A and A+32 and a second peptide having mass/charge of B and B+32.

The technology described herein is directed to agents that reduce the level of oxidant-modified ET-B Cys405, Cys403, or Cys402 and the identification and use of such agents for, e.g. to treat hypertension.

The present invention is directed to methods for determining the relative amount of wrongly disulphide bridged TNFR2:Fc in a sample of TNFR2:Fc, a fusion protein which is used in a variety of therapeutic applications. In addition, the invention pertains to a method for purifying TNFR2:Fc using said method for determining the percentage of wrongly disulphide bridged TNFR2:Fc, and to TNFR2:Fc compositions obtained thereby.