US-20260126451-A1 - MOLECULAR RECOGNTION ASSAYS OF CRITICAL STRUCTURE ATTRIBUTES IN PROTEOFORMS

US20260126451A1US 20260126451 A1US20260126451 A1US 20260126451A1US-20260126451-A1

Abstract

A method is provided to simultaneously identify and quantify critical structure attributes (CSAs) in proteoforms or proteoform families that includes the steps of: 1. injecting a sample into a reagent stream of one or more detection enhanced molecular recognition reagents; 2. incubating the combined sample:reagent(s) during migration through an analytical platform the causes molecular recognition reagent(s) to be sequestered by a specific critical structure attribute (CSA) in the analyte to form a luminon (A n :*S as ) complex; 3. using a sequestron selector in the reagent stream to overtake the luminon complex in a down-stream size exclusion chromatography (SEC) column to achieve mixing and formation of an N c ˜P as :A n :*S as , P as :A n :*S as , or *P as :A n :*S as sequestome complex; 4. resolving this sequestome complex from any unbound affinity selector(s) such as *S as or *P as , and non-analytes in the SEC column; 5. transporting the resolved sequestome complex into a flow-through detection means that detects and quantifies CSAs in fluorescent labeled *S as or *P as bearing luminon or sequestome complex to generate data for the construction of critical structure attribute ratio plots as a function of elapsed fermentation time.

Inventors

Fred Regnier
Jinhee Kim
Meena Narsimhan
Nathan Morris
Mary Bower

Assignees

Novilytic, LLC

Dates

Publication Date: 20260507
Application Date: 20251230

Claims (10)

1 . A method for analyzing critical structure attributes (CSAs) in a proteoform or proteoform family through sequestration of one or more affinity selectors, comprising the steps of: a) providing a cell and particulate-clarified sample from a fermenter, a cell culture, or organism, b) continuously adding: (i) mobile phase bearing a fluorescent low molecular weight labeled molecular recognition agents (M ra ) to an incubation or size exclusion chromatography (SEC) column, the M ra being either a primary (P as ) or secondary (*S as ) affinity selector, or (ii) affinity selector reagents to a sample stream to form a mixture, wherein (A) at least a first reagent is labeled with a donor fluorophore (**P as ) that binds specifically to a protein analyte, and (B) at least a second reagent is labeled with an acceptor fluorophore (*S as ) that binds to a critical structure attribute in the protein analyte; c) introducing the sample to the incubation or SEC column under conditions sufficient to allow formation of fluorescence labelled complex(es), d) transporting the complex(es) thus resolved into detection means; and e) using the detection means, to detect and quantify critical structure attributes (CSAs) in analytes.
2 . The method of claim 1 , wherein the method is for use as a luminon assay.
3 . The method of claim 1 , wherein step a) further comprises chronologically selecting a cell and particulate-clarified sample from a process development or production fermenter.
4 . The method of claim 1 , wherein step c) further comprises injecting the sample and wherein; (i) the linear velocity of the analyte protein (A p ) is more than twice the size of the M ra , (ii) the greater linear velocity of the A p relative to the M ra causes the two to mix, thereby forming a luminon complex (A p :M ra ), and (iii) the high linear velocity of the A p :M ra complex causes the complex to move into a saturating concentration of M ra and be washed repeatedly as it migrates to a flow-through detector; and wherein the detection means in step e) is a flow-through detection means.
5 . The method of claim 1 , wherein the method is for in-line monitoring CSAs.
6 . The method of claim 5 , wherein step a) further comprises continuously providing a cell and particulate clarified sample stream.
7 . The method of claim 5 , wherein step c) further comprises, introducing the mixture of reagents and sample continuously through a stationary micro-mixer into an incubation or SEC column, and wherein; (i) reagents concentration exceeds anticipated levels of the analyte and CSAs, and (ii) the combined volume of reagents is less than that of the sample, and adapting the flow rate through the reactor or SEC column to allow formation of one or more fluorescence labeled (**P as ) m :A n :(*S as ) n complexes.
8 . The method of claim 5 , wherein the detection means includes Förster resonance energy transfer (FRET) spectroscopy, or detection is achieved by a change in fluorescence after association of the affinity selectors with the analyte.
9 . The method of claim 8 , wherein the identification and quantification of specific critical structure attributes (SCAs) of the proteoform or proteoform family is based on selector fluorophore spectral properties for use in process validation.
10 . The method in claim 5 , wherein: a) a first fluorescent labeled reagent (*P as ) is continuously added to the mobile phase, and b) fluorescence of the (*P as ) m :A n complex continuously monitored after transport to a detection means.

Description

REFERENCE TO RELATED APPLICATION This application is a continuation of and claims priority to U.S. application Ser. No. 18/060,200, which was filed on Nov. 30, 2022, and which issued as U.S. Pat. No. 12,510,546 on Dec. 30, 2025, the entire disclosure of which is incorporated herein by reference. BACKGROUND Therapeutic recombinant proteins are complex products produced in fermenters operated in either a batch or a continuous harvesting mode. In both cases it is desirable to ensure continuous batch-to-batch consistency and quality through real-time testing. The need for continuous process validation (CPV) during therapeutic protein production was first proposed by the FDA in a 1987 document entitled “Guidelines on General Principles of Process Validation”. This was followed in 2004 and 2011 by additional guidelines enumerating the need for “a planned set of controls, derived from product and process understanding that confirms process performance and product quality” within and between lots. The FDA guidelines suggest this be done by continuously monitoring critical quality attributes (CQAs). A CQA has been defined as the impact a biopharmaceutical protein, product-related impurity, process-related impurity, or contaminant might have on the biological activity, pharmacokinetics, pharmacodynamics, immunogenicity, toxicity or overall safety and efficacy of a therapeutic product. The present disclosure relates to analytical platforms and methods that identify and quantify critical structural attributes (CSAs) of proteoforms on a recurring basis. CSAs are a subset of critical quality attributes (CQAs), being defined here as structural features of a substance required for its subsequent use. Enabling features of the systems and methods in the present disclosure are: i) CSA analyses of a product proteoform or proteoform family during synthesis; ii) automation of the requisite sample preparation, identification, and quantification steps in CSA assays; iii) achieving CSA assays within the time-window required for remediation of process deviations before product quality is compromised; and iv) execution of CQA analyses at-line (or on-line) during a manufacturing campaign. The genetic component of therapeutic protein expression in a host-cell culture is of major importance in explaining the origin and deviation in critical structure attributes (CSAs) of a product protein within a fermenter. An exon from a single gene supplies sequence code for multiple forms of the protein, producing a genetically related family of structural isoforms referred to as a proteoform family or “proteoforms”. Proteoforms vary in biological activity and half-life even though they are of similar structure. Current understanding of the origin of proteoform families is that their synthesis starts with DNA transcription via the formation of pre- and primary-mRNA species. This process involves a combination of intron excisions, exon rearrangements and/or shuffling, exon fusion, RNA copy number regulation, and epigenetic imprinting; all of which are enabled by a series of enhancers and silencers. Post-transcriptional processing subsequently leads to the production of mature mRNA species, this process being accompanied by variations in splicing, enzymatic editing, and reading frame shifts. The net outcome is that multiple mRNA species arise from a single protein-coding gene during these processing steps, each of which produce different proteoforms. Alternative splicing of mRNA, single amino acid polymorphism, and post-translational modifications (PTMs) play a further role in proteoform complexity. This leads to the expression of a genetically related family of many members referred to as a proteoform family (or proteoforms). Clearly, the potential for variation in regulatory control during proteoform expression in a fermenter can lead to alterations in the structure of family members and concomitantly therapeutic protein quality. Although liquid chromatography-mass spectral (LC-MS) analysis of a purified proteoform family is the current gold standard in protein quality control, it is not the best choice for continuous process validation (CPV). Primary structure data obtained by LC-MS does not easily correlate with individual critical structure attributes (CSAs) and sample preparation can take 6-12 hours. This is too long to allow process remediation before product quality is compromised. Additionally, LC-MS instrumentation is expensive, of high maintenance, and must be operated by one of skill in the art. Circumventing these limitations is the focus of the present disclosure. Like LC-MS, chromatographic and electrophoretic techniques provide well known avenues for sample analysis. The primary shortcoming of these techniques in continuous process validation is that they are overwhelmed by the sample complexity and by the time allowed for the analyses of individual critical structure attributes. Separation peak capacity in these systems seldom exceeds