US-20260126375-A1 - RAPID DEAMIDATION SCREENING

Abstract

Rapid comparability assessment of asparagine or glutamine deamidation of therapeutic proteins, peptides other modalities against a reference standard using two trace two-dimensional correlation spectroscopy. Asynchronous plots are generated providing information of the abundance of the components under evaluation. The method is valid for an array of protein samples providing an estimate of the extent of deamidation. The results of which are orthogonal to HPLC cation exchange, thus validating the existence of the deamidated species and if aggregate is present. This rapid method run time is only 10 minutes for up to 21 samples in their formulation condition.

Inventors

• Belinda Pastrana-Rios

Assignees

• PROTEIN DYNAMIC SOLUTIONS, INC.

Dates

Publication Date

20260507

Application Date

20251219

Claims (5)

1. 1 . A method for deamidation screening of a sample comprising: a) providing the sample in a slide containing at least one sample well and at least one reference well; b) acquiring at least one spectral image of the sample and at least one spectral image of the reference using a quantum cascade laser microscope under a controlled temperature condition; c) identifying and selecting, in at least one of the acquired spectral images of the sample and the reference, a region of interest; d) obtaining spectral data for the sample for the sample region of interest and obtaining spectral data of the reference for the reference region of interest; e) applying a baseline correction to the spectral data for the sample and the reference for the region of interest; f) applying a two-trace two-dimensional correlation to the baseline corrected sample and reference spectral data to generate a synchronous spectrum Φ(v 1 , v 2 ) and Asynchronous Spectrum Ψ(v 1 , v 2 ); generating a synchronous plot and an asynchronous plot from the synchronous and asynchronous spectra; and analyzing signature peaks to determine an estimation of deamidation of the sample.
2. 2 . The method of claim 1 , further comprising generating weighted difference spectra for the sample and reference, and comparing the weighted difference spectra to determine an estimate of deamidation.
3. 3 . The method of claim 2 , further comprising evaluating the difference spectra to determine if the deamidated sample is also aggregated.
4. 4 . The method of claim 1 , wherein acquiring at least one spectral image of the sample using a quantum cascade laser microscope includes acquiring at least one first hyperspectral image of the sample.
5. 5 . The method of claim 1 , further comprising applying a 2T2D correlation coefficient ρ(v 1 , v 2 ) to the synchronous spectrum, and applying a disrelation coefficient ξ(v 1 , v 2 ) to the asynchronous spectrum, as given by: ρ ⁡ ( v 1 , v 2 ) = Φ ⁡ ( v 1 , v 2 ) / Φ ⁡ ( v 1 , v 1 ) · Φ ⁡ ( v 2 , v 2 ) ξ ⁡ ( v 1 , v 2 ) = Ψ ⁡ ( v 1 , v 2 ) / Φ ⁡ ( v 1 , v 1 ) · Φ ⁡ ( v 2 , v 2 ) where: ρ ⁡ ( v 1 , v 2 ) 2 + ξ ⁡ ( v 1 , v 2 ) 2 = 1 .

Description

RELATED APPLICATION This application is a continuation of International Application No. PCT/US2024/034653, filed on Jun. 20, 2024, which claims the benefit of U.S. Provisional Patent Application No. 63/509,340, filed on Jun. 21, 2023, the entire contents of which are incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Award No. 1632420 awarded by the National Science Foundation. The Government has certain rights in this invention. FIELD OF THE INVENTION The present invention relates to a system and method for rapid screening of deamidated proteins in a sample by comparing to a reference standard. The system and method can screen for deamidated proteins independent of the modality. Moreover, the system and method can be used to screen both drug substances (DS) and drug products (DP) for deamidated proteins, and the testing for protein deamidation using the system and method can be assessed for samples varying in formulation and samples that are under or were subjected to forced degraded conditions providing a highly flexible method for developability, comparability and forensic evaluations. BACKGROUND The number of biologics that have been approved by regulatory authority has exceeded small molecule therapeutics for the treatment of diseases.1,2 The Biopharma industry will always require analytical tools that provide rapid, robust and reproducible results to accelerate speed to market, while ensuring safety and efficacy of the therapeutic protein to benefit the patient. Deamidation is a critical quality attribute for both drug substances (DS), as well as drug products (DP). Deamidation is a non-enzymatic degradation pathway for proteins comprised of aqueous solvent exposed asparagine and glutamine residues.3,4 This process occurs in proteins at high pH, low pH, under thermal stress or combination of these conditions. This is a critical event that introduces a negative charge in the sequence of the protein and if it occurs within the binding interface of the target can lead to loss of specificity, affinity and even efficacy. Also, if the negative charge is localized within the Fc region of an antibody, then it may limit the effector function involving the antibody dependent cellular cytotoxicity (ADCC). Furthermore importantly, asparagine and glutamine deamidation may lead to loss of stability, aggregation and immunogenicity. However, current methods of evaluating an array of therapeutic proteins with respect to deamidation quality attributes are time consuming, and may further depend on factors such as size of the sample and modality. Moreover, these methods typically require time-consuming sample preparation before any evaluation can be performed. What is needed is a system and method for reliably evaluating an array of therapeutic proteins in a short time period to provide an estimate of deamidation. SUMMARY OF THE INVENTION The system and method described herein provide a rapid method of screening an array of therapeutic proteins against one or more reference standards under controlled temperature conditions in a short time period, such as 10 minutes, to determine an estimate of the presence of deamidated proteins. This short timeframe is for the evaluation of a plurality of samples against a predetermined number of references, independent of size and modality. For example, the system and method may evaluate 21 samples against 2 references independent of size or modality in a 10 minute time period. Vibrational spectroscopy, such as infrared spectroscopy which is highly selective and sensitive, may be used to evaluate the samples. In particular, after spectral images are obtained suing vibrational spectroscopy, two-trace two-dimensional correlation spectroscopy can be applied to provide a comparative analysis between a sample protein against one or more reference standards at a controlled temperature. The controlled temperature may be in the range of range of 20-65° C., although the deamidation screening method will be routinely implemented at 25° C. Based on the comparisons, the system and method provides an estimate of asparagine and glutamine deamidation for therapeutic proteins and peptides. The system and method may also be used for screening related to Adeno-associated virus and cell and gene therapy. In addition, the system and method can be used to screen for exosomes, which may be carriers for therapeutics. To evaluate samples, the system and method use a vibrational spectroscopy apparatus, such as a quantum cascade laser (QCL) transmission microscope, Raman spectroscopy and microscopy, FT-IR, or light detection and ranging (LIDAR) system, with a slide cell array and dedicated software that provide rapid deamidation screening. The slide cell array may be comprised of a polymer, such as polyethylene, salt such as calcium fluoride or barium fluoride, or other polymer material that is transparent within most of the s