FIELD OF THE INVENTION

The present description relates to a method for determining the total carbonylation level on a polypeptide.

BACKGROUND

Metal-catalyzed oxidation products are a highly relevant quality attribute of monoclonal antibodies (mAbs) due to the contact of the mAbs with metal or metal ions during the manufacturing or storage process. During purification or storage, stainless steel is a common surface contact material. The iron ions present in the cell culture media or leached from the stainless steel can cause methionine and histidine oxidation in the Fc region of the mAb. Other than methionine and histidine oxidation, protein carbonylation, referring to the formation of aldehyde/ketone groups in proteins, is a major degradation pathway from the metal-catalyzed oxidation reaction and has not been studied extensively on mAbs. As the result of the oxidative carbonylation, the positive charge on lysine and arginine side chains and the steric constrain from proline residue are lost. Those changes could lead to increased protein aggregation propensity and thus potentially affects mAb product quality and stability profiles. In addition, the mAb aggregates induced by metal-catalyzed oxidation can be more immunogenic in transgenic mice than those induced by other stress conditions. Therefore, it is of significant importance to identify the extent of carbonylation of mAbs from metal-catalyzed oxidation during manufacturing and storage.

For quantitative measurement of total protein carbonylation levels, different hydrazides have been adopted as the derivatization reagents to react with the carbonyl groups on proteins (Yan et al., 2011). Quantitation was usually based on the unique characteristic of the hydrazides and the corresponding hydrazones after the derivatization. For example, a widely used spectrophotometric carbonylation assay measures the absorbance of a derived sample at 375 nm, where the derivatization reagent, 2,4-dinitrophenylhydrazine (DNPH), and the resulting hydrazone, strongly absorb (Levine et al., 1990). A fluorometric carbonylation assay measures the fluorescence with excitation at 485 nm and emission at 535 nm of a derived sample after the derivatization by fluorescein thiosemicarbazide (Mohanty et al., 2010). An ELISA carbonylation assay uses an anti-DNPH antibody for quantification after the derivatization of a sample by DNPH (Buss et al., 1997). While different hydrazide reagents have been employed for the derivatization and quantitation purposes, a common procedure in the protein carbonylation assays is the removal of the unbound hydrazide after the derivatization step to prevent over-quantification of the carbonylation levels (Rogowska-Wrzesinska et al., 2014). Typically, the unbound hydrazide is removed through protein precipitation and washing, which is a labor-intensive process and constitutes a major source of experimental variability. In fact, it was demonstrated that two additional washing steps reduced the carbonylation result by 15% when using the spectrophotometric carbonylation assay with DNPH (Matthijssens et al., 2007). The large assay variability and the labor-intensive nature associated with the washing step can make it difficult to broadly apply these carbonylation assays to measure mAb carbonylation during product development. Modified ELISA (Uehara et al., 2015) and DNPH (Mesquita et al., 2014) assays have been reported recently using simplified methodologies. However, these modified methods are still relatively labor-intensive and their robustness was not assessed yet. Lucifer Yellow carbohydrazide (LY-CH) has previously been used as a derivatization reagent to quantify aldehyde groups formed by periodic acid oxidation on the N-Glycan oligosaccharide residues of antibodies (Morehead et al., 1991). An assay was described to examine the quantitative labeling of oxidized antibody carbohydrate residues with LY-CH (Keener et al., 1994). In this assay, the LY-CH-to-polypeptide molar ratio in the derivatization reaction is limited due to the use of conventional buffer systems and results in a more difficult removal of unbound LY-CH. Furthermore, the assay does not correct the protein contribution to the respective absorbance of the hydrazide reagent for quantifying the carbonyl content which leads to an overestimation of the carbonylation content.

US 2006/0216756 A1 discloses a method for two-dimensional evaluation of the nature of oxidized protein, the method comprises the steps of specific fluorescent labeling of the carbonyl groups of the oxidized protein in a horny layer specimen taken from skin. US 2007/0110670 A1 discloses methods comprising the labeling of carbonyl groups of an oxidized protein with a fluorescence dye for evaluating hair damage. WO2012175519 A1 describes a method for detecting and/or quantifying protein carbonyl groups in at least one protein-containing sample comprising the steps of labeling protein carbonyl groups with hydrazide followed by an analysis of the sample using one-dimensional or two-dimensional gel electrophoresis.

In the present description, a simplified, more robust and more accurate proteinCarbonylationAssay usingLuciferYellow carbohydrazide (CALY) is described which overcomes the problems of the prior art as described above.

SUMMARY

The description relates to a method and kits for determining the total carbonylation level on a polypeptide.

In one aspect, the description relates to a method for determining the total carbonylation level on a polypeptide wherein the method comprises the steps of a) contacting the polypeptide in a solution with a hydrazide dye under conditions such that the hydrazide dye can react with the carbonyl groups present on the amino acid residues of the polypeptide to form a polypeptide-dye-complex, b) removing unbound hydrazide dye from the resultant solution from step a), c) determining in the resultant solution from step b) the concentration of the hydrazide dye bound in the polypeptide-dye-complex and the concentration of the polypeptide bound in the polypeptide-dye-complex, and d) determining the total carbonylation level of the polypeptide based on the ratio of the concentration of the hydrazide dye bound in the polypeptide-dye-complex to the concentration of the polypeptide bound in the polypeptide-dye-complex.

In one embodiment, the hydrazide dye is LY-CH. In one embodiment, step a) is performed in the presence of alkali metal ions. In one embodiment, the amino acid residues of the polypeptide are selected from the group consisting of arginine, lysine, proline and threonine. In one embodiment, the polypeptide is an antibody. In one embodiment, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multi-specific antibody, an antibody fragment, an antibody drug conjugate, a THIOMAB™ or a THIOMAB™ drug conjugate. In one embodiment, the polypeptide and the hydrazide dye is contacted in step a) in a final molar ratio of 6,000-10,000. In one embodiment, step a) is performed in the presence of a non-ionic surfactant. In one embodiment, the non-ionic surfactant is reduced Triton X-100. In one embodiment, step a) is performed at a temperature of 35° C.-39° C. In one embodiment, step a) is performed for 10-20 h. In one embodiment, step b) is performed by a method selected from the group consisting of filtration, gel filtration and dialysis. In one embodiment, step b) is performed by filtration. In one embodiment, filtration is carried out in the presence of 100-300 mM potassium phosphate and in the presence of 200-300 mM potassium chloride.

In another aspect, the description relates to a kit for carrying out the method as described herein, wherein the kit comprises hydrazide dye and a buffer. In one embodiment, the hydrazide dye is LY-CH. In one embodiment, the buffer is lithium MES buffer. In one embodiment, the kit further comprises a non-ionic surfactant, a standard for the polypeptide and a standard for hydrazide dye. In one embodiment, the non-ionic surfactant is reduced Triton X-100.

