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 can be conducted to prevent disulfide scrambling in liquid chromatography-mass spectrometry analysis of a protein of interest. In particular, methods can be conducted by employing a NEM analog, such as maleimide, and a heavy isotope counterpart of the NEM analog, such as a heavy isotope substituted maleimide, to alkylate a protein of interest and to alkylate a peptide digest of the protein of interest under conditions that prevent disulfide scrambling and subjecting the samples to a liquid chromatography-mass spectrometry analysis.

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.

The present invention is generally directed to methods for measuring Insulin Resistance (IR) and for modulation of Type 2 Diabetes (T2D) onset treatment. In particular, diagnosis of IR is based on detection of different forms of disulfide bonds pairs that may form in insulin, affecting the normal insulin structure. Abnormal insulin is defined as any insulin that contains at least one of the forms of abnormal disulfide bond pairs. The methods herein measure human insulin isomers' concentration in a sample by utilizing HPLC or a monoclonal/polyclonal antibody to human insulin that contains normal or one or more abnormal disulfide bonds combinations. Some aspects of the invention also provide kits adapted for use in such methods. The human insulin isomers' concentration results are fed into a system to regulate the level of exogenous insulin needed to augment the level of normal insulin detected by the methods for a specific patient, or effectively regulate the amount of metformin, sulfonylureas, thiazolidinediones (PPARy agonists; glitazones), GLP-1s, SGLT-2 inhibitors and DPP-4 inhibitors or other IR treatment to be administered to the patient. The system and methods comprise a feedback loop to maintain a therapeutic level of insulin, metformin or other IR treatment and decrease the risk of hypoglycemia and other complications. The current invention has the advantage of being a self-referencing IR assessment by utilizing the measurements of normal insulin compared to abnormal insulin and not relying only on glucose measurements, Body Mass Index or other references included in current IR measurement methods. This leads to a much more responsive and faster treatment for IR compared to current approaches, which employ methods such as, Oral Glucose Tolerance Test (OGTT), which takes hours to perform.

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.