WO2024084264A1

WO2024084264A1 - Immunoassay for detecting host cell proteins of high five cells

Info

Publication number: WO2024084264A1
Application number: PCT/IB2022/059928
Authority: WO
Inventors: Mahdi SHABANI; Mohammad Reza KAZEMALI; Reyhaneh MIRHASSANI; Tahereh AZARI; Payam SHAHHOSSEINI
Original assignee: HAJIAN TEHRANI, Behrooz; YOUNESI, Vahid
Priority date: 2022-10-17
Filing date: 2022-10-17
Publication date: 2024-04-25

Abstract

An immunoassay method for detecting an amount of host cell proteins (HCPs) comprising forming a plurality of HCP-immobilized antibody complexes by contacting the sample with a surface comprising a plurality of immobilized polyclonal antibodies (pAbs) against the High Five HCPs, removing unbound High Five HCPs by washing the surface with a wash buffer, contacting the plurality of HCP-immobilized antibody complexes with a plurality of labeled pAbs against the High Five HCPs, and detecting the amount of the High Five HCPs in the sample by detecting the formation of a complex between the one or more HCPs and a labeled antibody of the plurality of labeled pAbs.

Description

IMMUNOASSAY FOR DETECTING HOST CELL PROTEINS OF HIGH FIVE

CELLS

TECHNICAL FIELD

[0001] The present disclosure is generally related to an exemplary immunoassay method for detecting the amount of host cell proteins (HCPs) of High Five cells (High Five HCPs) in an exemplary sample, and more particularly to an exemplary immunoassay method for detecting the amount of High Five HCPs using exemplary polyclonal antibodies against High Five HCPs.

BACKGROUND

[0002] Genetically-modified eukaryotic or prokaryotic host cells have been commonly used for producing recombinant biopharmaceutical products, such as proteins, using cell fermentation/culture technology. One of the biggest challenges for the production of biopharmaceuticals is producing products that are free of process-related impurities to meet the regulatory guidelines for clinical applications. With regard to recombinant protein production, all expressed proteins in the expression system of a host cell line beside a protein of interest are regarded as host cell proteins (HCPs) and/or impurities. The presence of HCPs in biopharmaceutical products may have an adverse impact on patient’s safety, product stability, and stability of product- stabilizing additives.

[0003] With this regard, detection and quantification of residual contaminations (i.e., HCPs) through the entire process of producing biopharmaceutical products is of great importance. Thus, there is need to develop a detection method with a high sensitivity and specificity, and wide dynamic range.

SUMMARY [0004] This summary is intended to provide an overview of the subject matter of the present disclosure, and is not intended to identify essential elements or key elements of the subject matter, nor is it intended to be used to determine the scope of the claimed implementations. Its sole purpose is to present some concepts of one or more exemplary aspects in a simplified form as a prelude to the more detailed description that is presented later. The proper scope of the present disclosure may be ascertained from the claims set forth below in view of the detailed description below and the drawings.

[0005] One or more exemplary embodiments describe an exemplary immunoassay method for detecting an exemplary amount of exemplary host cell proteins of High Five cells (High Five HCPs) in an exemplary sample. Exemplary immunoassay method may comprise forming a plurality of exemplary HCP-immobilized antibody complexes by contacting an exemplary sample with an exemplary surface comprising a plurality of exemplary immobilized polyclonal antibodies (pAbs) against exemplary High Five HCPs at a temperature level between 20 °C and 25 °C for a time duration between 80 and 100 minutes; removing exemplary unbound High Five HCPs by washing an exemplary surface with an exemplary wash buffer; contacting a plurality of exemplary HCP-immobilized antibody complexes with a plurality of exemplary labeled pAbs; and detecting the amount of exemplary High Five HCPs in an exemplary sample by detecting the formation of an exemplary complex between exemplary one or more HCPs and an exemplary labeled antibody of the plurality of exemplary labeled pAbs.

[0006] In an exemplary embodiment, the plurality of exemplary immobilized pAbs and the plurality of exemplary labeled pAbs may comprise Immunoglobulin G (IgG) isotype and may be produced in an exemplary rabbit. In an exemplary embodiment, each respective HCP- immobilized antibody of the plurality of exemplary HCP-immobilized antibody complexes may comprise exemplary one or more HCPs of exemplary High Five HCPs that may be bound to an exemplary immobilized antibody.

[0007] This Summary may introduce a number of concepts in a simplified format; the concepts are further disclosed within the “Detailed Description” section. This Summary is not intended to configure essential/key features of the claimed subject matter, nor is intended to limit the scope of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The novel features which are believed to be characteristic of the present disclosure, as to its structure, organization, use and method of operation, together with further objectives and advantages thereof, will be better understood from the following drawings in which an exemplary embodiment will now be illustrated by way of example. It is expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limits of the present disclosure. Exemplary embodiments will now be described by way of example in association with the accompanying drawings in which: [0009] FIG. 1 illustrates an exemplary flowchart of an exemplary immunoassay method for detecting exemplary High Five host cell proteins (High Five HCPs) in an exemplary sample, consistent with one or more exemplary embodiments of the present disclosure;

[00010] FIG. 2 shows Coomassie blue stained sodium dodecyl-sulfate polyacrylamide gel electrophoresis of exemplary High Five cells lysate antigens, exemplary High Five cells broth antigens, and exemplary Diethylaminoethyl (DEAE) column eluate antigens, consistent with one or more exemplary embodiments of the present disclosure;

[00011] FIG. 3 shows Western blot analysis of exemplary DEAE column eluate antigens using exemplary anti-HCP antibodies (i.e., polyclonal Immunoglobulin G (IgG) antibodies against High Five HCPs), consistent with one or more exemplary embodiments of the present disclosure;

[00012] FIG. 4 shows Western blot analysis of exemplary High Five cells broth antigens using exemplary anti-HCP antibodies (i.e., polyclonal IgG antibodies against High Five HCPs), consistent with one or more exemplary embodiments of the present disclosure;

[00013] FIG. 5 shows Western blot analysis of exemplary High Five cells lysate antigens using exemplary anti-HCP antibodies (i.e., polyclonal IgG antibodies against High Five HCPs), consistent with one or more exemplary embodiments of the present disclosure; and

[00014] FIG. 6 illustrates an exemplary standard curve created for an exemplary High Five HCPs ELISA (enzyme-linked immunosorbent assay), consistent with one or more exemplary embodiments of the present disclosure.

DETAILED DESCRIPTION

[00015] In the following detailed description, numerous specific details are set forth by way of examples to provide a thorough understanding of the relevant teachings related to the exemplary embodiments. However, it should be apparent that the present teachings may be practiced without such details. In other instances, well known methods, procedures, components, and/or circuitry have been described at a relatively high-level, without detail, in order to avoid unnecessarily obscuring aspects of the present teachings.

[00016] The following detailed description is presented to enable a person skilled in the art to make and use the methods and devices disclosed in one or more exemplary embodiments of the present disclosure. For purposes of explanation, specific nomenclature is set forth to provide a thorough understanding of the present disclosure. However, it will be apparent to one skilled in the art that these specific details are not required to practice the disclosed exemplary embodiments. Descriptions of specific exemplary embodiments are provided only as representative examples. Various modifications to the exemplary implementations will be plain to one skilled in the art, and the general principles defined herein may be applied to other implementations and applications without departing from the scope of the present disclosure. The present disclosure is not intended to be limited to the implementations shown, but is to be accorded the widest possible scope consistent with the principles and features disclosed herein. [00017] Disclosed herein is an exemplary immunoassay method and an exemplary immunoassay kit for detecting exemplary host cell proteins (HCPs) of High Five cells (High Five HCPs) in an exemplary sample, e.g., an exemplary biopharmaceutical product. HCPs may refer to proteins produced by exemplary host organisms used in biopharmaceutical industry to produce recombinant therapeutic proteins. HCPs are known as a predominant class of process- related impurities and their presence in a purified biopharmaceutical product may be undesirable due to their potential impacts on patient’s safety. In one or more exemplary embodiments, an exemplary immunoassay kit may comprise exemplary polyclonal antibodies (pAbs) against exemplary High Five HCPs. Exemplary pAbs against exemplary High Five HCPs may include Immunoglobulin G (IgG) isotype and may be produced in rabbit or goat or sheep. “Antibody” may refer to molecules capable of specific binding to a target, such as a polynucleotide, carbohydrate, polypeptide, or lipid through at least one epitope recognition site disposed in the variable region of an antibody. An “antibody” may include not only intact monoclonal or polyclonal antibodies, but also fragments of an antibody including, but not limited to, one or more of Fab, Fab', F(ab')2, Fc, Fv, scFv fragments, heavy chain, hinge region, light chain, antigen binding site, and single chain antibodies. “Polyclonal antibodies (pAbs)” may refer to a mixture of antibodies secreted by different B cell lineages of different species. pAbs may include a collection of immunoglobulin molecules that may react with a specific antigen, wherein each antibody of pAbs may identify a different epitope on an antigen. [00018] In one or more exemplary embodiments, an exemplary immunoassay method/kit may be used to detect the presence and amount of High Five HCPs in an exemplary biopharmaceutical product and/or during an exemplary bioprocessing for the production of an exemplary biopharmaceutical product. “High Five cells,” also known as BTI-TN-5B 1-4, may refer to an insect cell line originated from the ovarian cells of a cabbage looper, Trichoplusia ni. High Five cell line may be used for the production of recombinant proteins based on a Baculovirus Expression Vector System (BEVS).

[00019] FIG. 1 illustrates an exemplary flowchart of exemplary immunoassay method 100 for detecting exemplary High Five HCPs in an exemplary sample, consistent with one or more exemplary embodiments of the present disclosure. In one or more exemplary embodiments, exemplary immunoassay method 100 may include forming a plurality of exemplary HCP- immobilized antibody complexes by contacting an exemplary sample with an exemplary surface comprising a plurality of exemplary immobilized pAbs against exemplary High Five HCPs (step 102), removing unbound exemplary High Five HCPs by washing an exemplary surface with an exemplary wash buffer (step 104), contacting a plurality of exemplary HCP- immobilized antibody complexes with a plurality of exemplary labeled pAbs against exemplary High Five HCPs (step 106), and detecting the amount of exemplary High Five HCPs in an exemplary sample by detecting the formation of an exemplary complex between exemplary one or more HCPs and an exemplary labeled antibody of a plurality of exemplary labeled pAbs (step 108).

[00020] In further details, step 102 may include forming a plurality of exemplary HCP- immobilized antibody complexes by contacting an exemplary sample with an exemplary surface comprising a plurality of exemplary immobilized pAbs against exemplary High Five HCPs. In one or more exemplary embodiments, exemplary immobilized pAbs may include IgG isotype and may be produced in an exemplary rabbit or goat or sheep. Meanwhile, each HCP- immobilized antibody complex of exemplary HCP-immobilized antibody complexes (set forth in step 102) may comprise one or more exemplary HCPs of exemplary High Five HCPs bound to an exemplary immobilized antibody. In an exemplary embodiment, each respective immobilized antibody of exemplary immobilized pAbs may be attached to an exemplary surface through a Fc region of an exemplary immobilized antibody. “HCP-immobilized antibody complex” set forth in step 102 may refer to an exemplary complex formed by the binding of one or more exemplary HCPs to an exemplary immobilized antibody.

[00021] With further regards to step 102, in an exemplary embodiment, contacting an exemplary sample (e.g., an exemplary biopharmaceutical product, such as an exemplary recombinant protein produced in exemplary High Five cells) with an exemplary surface comprising a plurality of exemplary immobilized pAbs against exemplary High Five HCPs may include contacting an exemplary sample with an exemplary surface comprising a plurality of exemplary immobilized pAbs against exemplary High Five HCPs at a temperature level between about 20 °C and 25 °C (room temperature) for a time duration between about 80 and 100 minutes while shaking/spinning using a shaker/spinner. In an exemplary embodiment, exemplary immunoassay method 100 may be enzyme-linked immunosorbent assay (ELISA); thus, exemplary immunoassay method 100 may be conducted in an exemplary microtiter plate. For example, in an exemplary embodiment, step 102 may be accomplished by adding a certain amount of an exemplary sample (e.g., between about 50 pL and 200 pL) to one or more wells of an exemplary microtiter plate, followed by incubating an exemplary microtiter plate (e.g., in an exemplary incubator) at a temperature level between about 20 °C and 25 °C (room temperature) for a time duration between about 80 and 100 minutes while being shaked/spined

(e.g., with a spinning rate of about 200 rpm) using a shaker/spinner. [00022] In one or more exemplary embodiments, step 104 may include removing unbound exemplary High Five HCPs by washing an exemplary surface with an exemplary wash buffer. For example, in exemplary immunoassay method 100 conducted as an exemplary ELISA, each well of an exemplary microtiter plate may be washed, several times (e.g., 5 times), with a IX phosphate buffered saline (PBS) buffer containing between about 0.05% and 0.1% (v/v) Polysorbate 20 (Tween 20). In an exemplary implementation, an exemplary PBS buffer (used as an exemplary wash buffer) may comprise sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), and potassium phosphate monobasic (0.0018 M).

[00023] Referring again to exemplary immunoassay method 100, step 106 may include contacting a plurality of exemplary HCP-immobilized antibody complexes with a plurality of exemplary labeled pAbs against exemplary High Five HCPs. In an exemplary embodiment, each respective labeled antibody of exemplary labeled pAbs may be coupled to an exemplary labeling agent. In one or more exemplary embodiments, an exemplary labeling agent may comprise, but is not limited to, luminescent particle, a quantum dot, a chromogen, a catalyst, and a radioactive compound. In an exemplary embodiment, exemplary labeled pAbs may include an exemplary IgG isotype and may be produced in an exemplary rabbit or goat or sheep. In an exemplary embodiment, an exemplary labeling agent may include horseradish peroxidase (HRP).

[00024] In an exemplary implementation, in exemplary immunoassay method 100 conducted as an exemplary ELISA, step 106 may be accomplished by adding about 100 pL of an exemplary solution of HRP-conjugated pAbs, with an exemplary titer of about 1:16500, to each well of an exemplary microtiter plate. In an exemplary implementation, contacting a plurality of exemplary HCP-immobilized antibody complexes with a plurality of exemplary labeled pAbs against exemplary High Five HCPs may include incubating an exemplary microtiter plate (in which an exemplary solution of HRP-conjugated pAbs is added to each well) at about 20-25 °C (i.e., room temperature) for about 20-40 minutes (e.g., 30 minutes) while shaking/spinning (with a spinning rate of about 200 rpm) using a shaker/spinner. In an exemplary embodiment, step 106 may result in the formation of an exemplary complex between an exemplary labeled antibody and an exemplary HCP-immobilized antibody complex. Formation of an exemplary complex between an exemplary labeled antibody and an exemplary HCP-immobilized antibody complex may refer to the formation of an exemplary complex by binding of an exemplary labeled antibody to one or more exemplary HCPs that are bound to an exemplary immobilized antibody.

[00025] In one or more exemplary implementations, step 106 of exemplary immunoassay method 100 may further include removing unbound labeled pAbs from around the formed complexes between an exemplary labeled antibody and an exemplary HCP-immobilized antibody complex. In an exemplary implementation, removing unbound labeled pAbs may include removing unbound labeled pAbs using an exemplary wash buffer. In an exemplary embodiment, an exemplary wash buffer may include IX PBS buffer containing sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), and potassium phosphate monobasic (0.0018 M), and 0.05-0.1% (v/v) Polysorbate 20 (Tween 20). For example, in exemplary immunoassay method 100 conducted as an exemplary ELISA, each well of an exemplary microtiter plate may be washed, several times (e.g., 5 times), with an exemplary IX PBS buffer containing about 0.05-0.1% (v/v) Polysorbate 20 (Tween 20).

[00026] With further reference to FIG. 1, step 108 may include detecting the amount of exemplary High Five HCPs in an exemplary sample by detecting the formation of an exemplary complex between exemplary one or more HCPs and an exemplary labeled antibody of a plurality of exemplary labeled pAbs. In an exemplary implementation, detecting the amount of exemplary High Five HCPs in an exemplary sample by detecting the formation of an exemplary complex between exemplary one or more HCPs and an exemplary labeled antibody may include detecting the amount of exemplary High Five HCPs in an exemplary sample by detecting the formation of an exemplary complex between exemplary one or more HCPs and an exemplary HRP-conjugated antibody by detecting the reaction between an exemplary HRP-conjugated antibody and an exemplary HRP substrate, such as 3,3',5,5'-Tetramethylbenzidine (TMB), ABTS, AEC, DAB, and ECL. For example, in exemplary immunoassay method 100 conducted as an exemplary ELISA, step 108 may be accomplished by adding a certain volume (e.g., 50- 200 pL) of an exemplary HRP substrate (e.g., TMB) to each well of an exemplary microtiter plate and incubating an exemplary microtiter plate at about 20-25 °C (i.e., room temperature), in dark, for about 5-30 minutes (e.g., 15 minutes). For example, in exemplary immunoassay method 100 conducted as an exemplary ELISA, an exemplary reaction between HRP- conjugated antibody and an exemplary HRP substrate may be stopped by adding a certain amount (e.g., 100 pL) of an exemplary stop solution, such as 1 N hydrochloric acid solution, to each well of an exemplary microtiter plate. In one or more exemplary embodiments, detecting the amount of exemplary High Five HCPs in an exemplary sample by detecting the formation of an exemplary complex between exemplary one or more HCPs and an exemplary labeled antibody may include detecting a color change due to the reaction between an exemplary HRP- conjugated antibody and an exemplary HRP substrate, such as TMB . For example, in exemplary immunoassay method 100 conducted as an exemplary ELISA, the reaction between an exemplary HRP-conjugated antibody and an exemplary HRP substrate, such as TMB, may be detected by measuring the light absorbance (i.e., optical density (OD)) of each well of an exemplary microtiter plate at an exemplary wavelength of about 450 nm (e.g., with a 630 nm reference filter).

EXAMPLES

[00027] Hereinafter, one or more exemplary embodiments will be described in further detail with reference to examples. It will be obvious to a person having ordinary skill in the art that these examples may be for illustrative purposes only and are not to be interpreted to limit the scope of the present disclosure.

Example 1: Producing Polyclonal Antibodies Against Host Cell Proteins of High Five

Cells

[00028] In this example, polyclonal antibodies (pAbs) against host cell proteins (HCPs) of high five cells were produced. High Five insect cells (Catalog Number: B85502) were cultured in FBS (fetal bovine serum)-free medium in a bioreactor system. Once the High Five insect cells reached a predetermined density, they were harvested. To acquire a comprehensive coverage of the High Five cells’ antigens, three groups of antigens including: i) cell lysate antigens, ii) broth antigens, and iii) Diethylaminoethyl (DEAE) column eluate antigens (i.e., antigens purified, by a DEAE column, from exemplary harvested High Five cells) were prepared. The accuracy and specificity of the three groups of antigens was confirmed by western blotting. Since High Five and Sf9 insect cell lines are morphologically similar, the production of pAbs against HCPs of High Five cells was performed using a commercially available Sf9 HCPs ELISA (enzyme-linked immunosorbent assay) kit.

[00029] Six animals (e.g., rabbit, sheep, and/or goat) were immunized with the three groups of antigens through five immunogen injections. In one or more exemplary embodiment, pAbs against High Five HCPs may be produced by immunizing an exemplary goat or sheep or rabbit. To perform exemplary initial injections, 0.2-1 mg of each antigen group was dissolved in 700 pL phosphate-buffered saline (PBS containing sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), and potassium phosphate monobasic (0.0018 M)), and then diluted with 700 pL of an exemplary complete Freund's adjuvant. All four subsequent injections (every two or three weeks, 0.2-1 mg each) were accomplished using an exemplary incomplete Freund's adjuvant. All injections were done intramuscularly by 5 mL syringes.

[0001] Before each injection, exemplary blood samples were collected from each animal. The serum of each sample was separated and stored at -20 °C. The quality of immunization to exemplary antigens was assessed by ELISA, after sample collection. For this purpose, each well of an ELISA microtiter plate was coated with 1 pg/mL of each antigen group (i.e., cell lysate antigens, broth antigens, and elution antigens). Goat anti-rabbit antibody or rabbit antigoat antibody or rabbit anti-sheep antibody were labeled with horseradish peroxidase (HRP) using an exemplary commercial HRP conjugation kit. An exemplary assay was designed based on an exemplary indirect antibody ELISA (in an exemplary 96-well microtiter plate) to test the produced polyclonal antibodies. Using the developed ELISA test, exemplary sera samples collected from the immunized rabbits were tested at 1:100-1:128000 dilution ranges. For this purpose, 100 pL of each dilution of exemplary serum samples (1:100-1:128000) was added to each well. The microtiter plate was incubated for 60 minutes while shaking at 37 °C. After incubation, the microtiter plate was washed, 5 times, with an exemplary wash buffer containing sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), potassium phosphate monobasic (0.0018 M), and 0.05-0.1% (v/v) Tween 20. Then, 100 pL of an exemplary HRP conjugate of goat anti-rabbit antibody or rabbit anti-goat antibody or rabbit anti-sheep antibody was added to each well. After 30 minutes incubation at 37 °C, the microtiter plate was washed, 5 times, with an exemplary wash buffer containing sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), potassium phosphate monobasic (0.0018 M), and 0.05-0.1% (v/v) Tween 20. After being washed with an exemplary wash buffer, the microtiter plate was incubated with 100 pL of an exemplary chromogen substrate reagent for 10 minutes while shaking. After 10 minutes, 100 pL of an exemplary stop solution (I N HC1) was added to quench color development. The light absorbance of each well was read at an exemplary wavelength of about 450 nm.

[00030] Furthermore, a bicinchoninic acid assay (BCA protein assay) was performed on the collected sera samples to measure the total protein concentration of sera samples during the immunization phase. When the level of total Immunoglobulin G (IgG) against the three groups of antigens was found to be unaltered (the constant IgG levels may be confirmed by achieving constant optical absorption in blood samples taken in separate times), serum was collected from each animal. Finally, an exemplary crude anti-HCP polyclonal antibody, obtained from the collected sera, were purified by an exemplary affinity chromatography utilizing an exemplary Cyanogen Bromide (CNBr) activated Sepharose 4B resin column. First, ImM HCL solution was loaded to the column (100 mL/1 mg gel) to swell the gel. The column was incubated for about 60 minutes at about 20-25 °C; about 3.5 mL media may be made per 1 gram of gel.

[0002] An exemplary cell lysate antigen was dialyzed using an exemplary 12-14 kDa MW cut off dialysis membrane against an exemplary coupling buffer containing NaHCOs (0.1 M) and NaCl (0.5 M) with an exemplary pH level of 8.3 — 4 repeats; each repeat for about 1 h. An exemplary coupling buffer was added to an exemplary dialyzed protein (i.e., an exemplary cell lysate antigen) with a final volume of about 5 mL (per 1 g of gel) to completely remove HCL from gel matrix. Afterwards, an exemplary chromatography column was exposed to an exemplary blocking buffer (0.1 M Tris-HCL, pH 8) to block the binding of cell lysate antigens to Sepharose. The gel particles were transferred to a previously prepared column that may consist of a washer at the top of the column. To improve gel trapping, an exemplary column may also have grooves at the bottom. An exemplary column may be washed with an exemplary blocking buffer using a peristaltic pump until the UV absorbance of the collected fractions (with a volume of about 1 mL), at an exemplary wavelength of 280 nm, may reach zero.

[00031] The column was periodically washed by an exemplary 0.1 M sodium acetate acetic acid washing buffer with a pH level of 4.6 containing 0.5 M NaCl, and then at pH level of 8 by an exemplary 0.1 M Tris-HCl base washing buffer containing 0.5 M NaCl, at least 3 times (about 5-10 times for each wash). Finally, the column was washed using an exemplary PBS buffer containing 0.137 M sodium chloride, 0.002 M potassium chloride, 0.01 M sodium phosphate dibasic, and 0.0018 M potassium phosphate monobasic. To purify the produced antibodies, about 5 mL of the collected serum from immunized rabbits was diluted with about 20 mL of PBS. Finally, about 25 mL of the diluted serum was loaded in an exemplary chromatography column at a flow rate of about 75 cm/hour. Afterwards, an exemplary column was washed with about 50 mL PBS. The optical density (OD) of all fractions, obtained from an exemplary column, were measured at a wavelength of about 280 nm. The loaded column was eluted with an exemplary elution buffer (0.1 M Glycine-HCL buffer, pH 2.5). The flowthrough fractions were collected in 1 mL volumes.

[00032] After measuring the light absorbance of each fraction at 280 nm, the flowthrough fractions were evaluated for antibody titers against High Five antigens. The column was again washed with PBS containing 1% NaNs and then incubated at 2-8 °C. The fractions with pH 7.5 were neutralized with 2 M Tris. To concentrate the obtained antibody solutions, an exemplary saturated ammonium persulfate solution (4.1 M) was added to the antibody solutions with a volume ratio of about 1:1 and incubated for about 1 hour at about 4 °C, and finally centrifuged at 8000 g for 30 min at 4 °C. After removing the supernatant, about 500 pL of ice-cold PBS was slowly added to the obtained sediment. The concentration process was continued until the final concentration of antibody reached 5 mg/mL. The concentrated antibody solutions were transferred to a dialysis tube and dialyzed within 12-18 hours against PBS (1:100 of sample to PBS buffer). After dialysis, the antibody solutions were collected and stored at -20 °C. The purified anti-High Five antibodies were conjugated to HRP using an exemplary commercial HRP conjugation kit.

Example 2: Developing an Enzyme-Linked Immunosorbent Assay (ELISA)

[00033] In this example, an exemplary High Five HCPs ELISA was developed based on one or more exemplary embodiments described in the present disclosure to detect HCPs of High Five cells. In one or more exemplary embodiments, an exemplary High Five HCPs ELISA may comprise a 96-well microtiter plate in which each well may be coated with anti-HCP antibodies (i.e., antibodies against HCPs of High Five cells). Exemplary components of an exemplary High Five HCPs ELISA may further comprise exemplary HRP-conjugated anti-HCP antibodies (i.e., antibodies against HCPs of High Five cells), an exemplary chromogenic substrate (i.e., TMB), exemplary reference standards, and an exemplary stop solution.

[00034] To develop an exemplary High Five HCPs ELISA, a microtiter plate was coated with anti-HCP antibodies by adding 100 pL of bicarbonate coating buffer (15 mM Na2COs, 35 mM NaHCCE; pH 9.6-9.7) comprising 1 pg/mL of anti-HCP antibodies to each well and incubating the microtiter plate at 2-8 °C for 12-18 hours. Following 14-17 hours of incubation, the microtiter plate was washed with an exemplary wash buffer containing PBS buffer and 0.05- 0.1% (v/v) Tween 20. 110 pL of an exemplary blocking buffer containing 1% bovine serum albumin (BSA) in PBS was added to each well and the microtiter plate was incubated for 1 hour at 25 °C. After 1 hour, the blocking buffer was discarded and the microtiter plate was again incubated at 25 °C until the wells were dried (blocking the wells may reduce the background noise). High Five cells lysate with a concentration of about 780 |ag/mL was used to prepare an exemplary sample source. The concentration of cell lysate antigens was determined by Bradford method. Using Bradford method, 10 pL of High Five cells lysate plus 200 pL of Bradford reagent was added to each well and incubated in dark at 25 °C for 5 minutes. Furthermore, BSA with a concentration between 0-100 pg/mL in PBS was prepared as an exemplary standard solution. Finally, the OD of exemplary BSA solutions was measured at 595 nm and the measured absorbance values were used to create an exemplary Bradford standard curve. The concentration of High Five cells lysate antigens was determined based on the obtained standard curve. The concentration of cell lysate antigens was further confirmed by Lowry assay. The standard solutions of an exemplary High Five HCPs ELISA were prepared with concentrations including 0, 3, 6, 12, 25, 50 and 100 ng/mL.

[00035] The HRP-conjugated anti-HCP antibody was prepared with a titer of about 1:16500 and was diluted with an exemplary diluent solution consisting of distilled water (filtered through a 0.45 pm syringe filter) with a volume ratio of about 2:3 and Isothiazolin with a concentration of about 0.05%. An exemplary wash solution/buffer, developed for an exemplary High Five HCPs ELISA, may comprise PBS buffer containing sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), and potassium phosphate monobasic (0.0018 M), and 0.05-0.1% (v/v) Tween 20. An exemplary High Five HCPs ELISA may further comprise TMB as an exemplary HRP substrate and 1 N hydrochloric acid solution as an exemplary stop solution.

[0003] In an exemplary implementation, an exemplary sample containing HCPs of High Five cells may be detected by an exemplary High Five HCPs ELISA through an exemplary process. In an exemplary implementation, an exemplary process may comprise: adding about 100 pL of an exemplary sample (e.g., a pharmaceutical sample) and 100 pL of different dilutions of an exemplary standard solution with a concentration range between 0 ng/mL and 100 ng/mL to each well of an exemplary High Five HCPs ELISA microtiter plate; incubating an exemplary High Five HCPs ELISA microtiter plate at about 20-25 °C (i.e., room temperature) for about 90 minutes while shaking/spinning (with a spinning rate of about 200 rpm) using a shaker/spinner; washing the wells with an exemplary wash buffer (PBS buffer containing sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), and potassium phosphate monobasic (0.0018 M), and 0.05-0.1% (v/v) Tween 20) for 5 times; adding about 100 pL of an exemplary HRP-conjugated anti-HCP antibody with a titer of about 1:16500 to each well (including sample and standard wells) and incubating the microtiter plate at about 20-25 °C (i.e., room temperature) for about 30 minutes while shaking/spinning (with a spinning rate of about 200 rpm) using a shaker/spinner; washing the wells with an exemplary wash buffer (PBS buffer containing sodium chloride (0.137 M), potassium chloride (0.002 M), sodium phosphate dibasic (0.01 M), and potassium phosphate monobasic (0.0018 M), and 0.05-0.1% Tween 20) for 5 times; adding about 100 pL TMB to each well (including sample and standard wells) and incubating the microtiter plate at about 20- 25 °C (i.e., room temperature), in dark, for about 15 minutes; adding about 100 pL of an exemplary stop solution (i.e., 1 N hydrochloric acid solution) to each well (including sample and standard wells) for about 15 minutes; measuring the light absorbance (OD) of the wells at the wavelength of about 450 nm (with a 630 nm reference filter); creating an exemplary standard curve based on the obtained light absorbance (OD) values of the standard wells; and calculating the amount of High Five HCPs based on the created standard curve.

Example 3: Characterization of the ELISA Assay and Anti-HCP Antibodies [00036] In this example, an exemplary ELISA assay developed according to one or more exemplary embodiments was qualified by assessing a plurality of indicators including precision, specificity, accuracy, interference, limit of blank (LOB), limit of detection (LOD), limit of quantification, hook effect, linearity, and range.

[00037] Precision was assessed in two ways, inter-assay and intra-assay. Inter-assay precision may provide for the assessment of assay reproducibility. In an exemplary implementation, 20 replicates of three samples containing different concentrations of HCPs were tested in 5 days by an exemplary High Five HCPs ELISA as described in one or more exemplary embodiments. Table 1 bellow shows the inter-assay test results and precision values obtained from the detection of samples containing High Five HCPs using an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments.

Table 1:

The inter-assay test results and precision values obtained from the detection of samples containing High Five HCPs using an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00038] The mean Coefficient of Variation (CV%) of inter-assay precision was less than 10% which may show that an exemplary High Five HCPs ELISA (for the detection of High Five HCPs) had a significantly high reproducibility. Intra-assay precision was assessed by testing 20 replicates of three samples, each having a specific concentration of High Five HCPs. Similar to the obtained inter-assay results, the mean CV of intra-assay precision was less than 10%. Table 2 bellow shows the intra-assay test results and precision values obtained from the detection of samples containing High Five HCPs using an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments.

Table 2:

The intra-assay test results and precision values obtained from the detection of samples containing High Five HCPs using an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00039] Specificity of exemplary anti-HCP antibodies (i.e., IgG antibodies against HCPs of High Five cells) was evaluated to make sure that exemplary anti-HCP antibodies may specifically detect High Five HCPs. For this purpose, exemplary pharmaceutical products obtained from Escherichia coli (E. coll) and Chinese Hamster Ovary (CHO) cells were assessed using an exemplary High Five HCPs ELISA described in one or more exemplary embodiments. On the other hand, an exemplary pharmaceutical product extracted from High Five cells, whether during an upstream process or downstream process, with a wide range of High Five HCPs content (from high to low) were assessed by an exemplary High Five HCPs ELISA. Table 3 below shows ELISA test results of E. coli HCPs, CHO HCPs, and High Five HCPs, consistent with one or more exemplary embodiments. The obtained results, as set forth in Table 3, demonstrates that an exemplary High Five HCPs ELISA may specifically detect the High Five HCPs and may not interfere with proteins from other host cells.

Table 3:

ELISA test results of E. coli HCPs, CHO HCPs, and High Five HCPs, consistent with one or more exemplary embodiments of the present disclosure.

[00040] The ability of polyclonal antibodies for identification of High Five HCPs antigens was evaluated by a Western blot experiment. The three antigen groups, set forth in Example 1, including High Five cells lysate antigens, High Five cells broth antigens, and DEAE column eluate antigens were analyzed by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting. FIG. 2 shows Coomassie blue stained SDS-PAGE 200 of exemplary High Five cells lysate antigens, exemplary High Five cells broth antigens, and exemplary DEAE column eluate antigens, consistent with one or more exemplary embodiments of the present disclosure. FIG. 3 shows Western blot analysis 300 of exemplary DEAE column eluate antigens using exemplary anti-HCP antibodies (i.e., polyclonal IgG antibodies against High Five HCPs), consistent with one or more exemplary embodiments of the present disclosure. FIG. 4 shows Western blot analysis 400 of exemplary High Five cells broth antigens using exemplary anti-HCP antibodies (i.e., polyclonal IgG antibodies against High Five HCPs), consistent with one or more exemplary embodiments of the present disclosure. FIG. 5 shows Western blot analysis 500 of exemplary High Five cells lysate antigens using exemplary anti-HCP antibodies (i.e., polyclonal IgG antibodies against High Five HCPs), consistent with one or more exemplary embodiments of the present disclosure. Referring to FIGs. 3-5, exemplary anti-HCP antibodies may be capable of detecting different HCPs bands with different sizes as antigen determinants of High Five cells. Thus, an exemplary High Five HCPs ELISA may be capable of detecting High Five HCPs in pharmaceutical products produced in High Five cells.

[00041] Matrix interference of an exemplary High Five HCPs ELISA, described in one or more exemplary embodiments, was also assessed in this example. For this purpose, High Five HCPs antigens with a certain concentration were spiked into a pharmaceutical sample and tested with an exemplary High Five HCPs ELISA to assess the possible assay interference with other proteins of the pharmaceutical sample. Table 4 below shows the matrix interference results obtained from testing a pharmaceutical sample — spiked with different dilutions of a 32 ng/mL solution of High Five HCPs antigens — using an exemplary High Five HCPs ELISA.

Table 4:

Matrix interference results obtained from testing a pharmaceutical sample — spiked with different dilutions of a 32 ng/mL solution of High Five HCPs antigens — using an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00042] High Five HCPs antigens with a certain concentration were spiked into a solution of PBS containing 1% (v/v) Tween 20 and tested with an exemplary High Five HCPs ELISA to assess the possible assay interference with the PBS sample components. Table 5 below shows the matrix interference results obtained from testing a solution of PBS containing 1% (v/v) Tween 20 — spiked with different dilutions of a 32 ng/mL solution of High Five HCPs antigens — using an exemplary High Five HCPs ELISA.

Table 5:

Matrix interference results obtained from testing a solution of PBS containing 1% (v/v) Tween 20 — spiked with different dilutions of a 32 ng/mL solution of High Five HCPs antigens — using an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00043] Furthermore, High Five HCPs antigens (with a concentration of about 30 ng/mL and 60 ng/mL) were spiked into exemplary solutions containing different concentrations of BSA (1-8 mg) and tested with an exemplary High Five HCPs ELISA to assess the possible assay interference with BSA. Table 6 below shows the matrix interference results obtained from testing exemplary BSA solutions — spiked with a 30 ng/mL and a 60 ng/mL solution of High Five HCPs antigens — using an exemplary High Five HCPs ELISA.

Table 6:

Matrix interference results obtained from testing exemplary BSA solutions — spiked with a 30 ng/mL and a 60 ng/mL solution of High Five HCPs antigens — using an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00044] When the recovery percentage is between 80% and 120%, an ELISA test may have a significantly low matrix interference. The obtained results, as set forth above, demonstrated a recovery percentage in range of 80-120%; thus, an exemplary High Five HCPs as described in one or more exemplary embodiments may have a significantly low matrix interference.

[00045] Another ELISA characteristic evaluated in this example includes Limit of Blank (LOB), Limit of detection (LOD), and Limit of Quantification (LOQ). LOB may be defined as the lowest value of optical density (OD) measured for an exemplary blank sample. To measure LOB, an exemplary test was performed containing 20 replicates of an exemplary blank sample in 3 working runs. Table 7 below shows the assay results for measuring LOB of an exemplary High Five HCPs ELISA. The obtained LOB of blank samples tested by an exemplary High Five HCPs ELISA was measured to be about 0.5 ng/mL.

Table 7:

Assay results for measuring LOB of an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00046] LOQ may refer to the lowest concentration of an analyte at which it may be detected with a significantly high accuracy and precision. To evaluate LOQ, an exemplary High Five HCPs on samples with a concentration range (20 replicates of each concentration were tested in a single ELISA run). Table 8 below shows the assay results for measuring LOQ of an exemplary High Five HCPs ELISA. Accuracy and precision were measured by calculating Recovery (%) and CV%. Recovery rate was between 80% and 120%, and a CV% was less than 10% at all concentrations. The obtained LOQ for an exemplary High Five HCPs ELISA was measured to be about 3 ng/mL.

Table 8:

Assay results for measuring LOQ of an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00047] LOD may refer to the lowest concentration of an analyte that may be distinguished by an assay. In an exemplary implementation, an exemplary High Five HCPs was performed on 20 replicates of the obtained LOB (i.e., 0.5 ng/mL) in 3 working runs. Herein, LOD is expressed as mean±3SD. Table 9 below shows the assay results for measuring LOD of an exemplary High Five HCPs ELISA. The obtained LOD for an exemplary High Five HCPs ELISA was measured to be about 0.8 ng/mL.

Table 9:

Assay results for measuring LOD of an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00048] Hook effect may refer to a phenomenon that may result in the decrease of OD in the presence of an excessive amount of HCPs. Thus, an exemplary test was designed to evaluate the effect of an increase in High Five HCPs concentration on Hook effect occurrence. In an exemplary implementation, High Five HCPs antigen — with different concentrations — was added to exemplary HCP-free pharmaceutical samples. After conducting an exemplary ELISA, the OD of each sample was measured. An exemplary High Five HCPs may be confirmed when an increase in High Five HCPs concentration has no significant effect on the decrease of OD. Table 10 below shows the assay results for evaluating the Hook effect of an exemplary High Five HCPs ELISA. According to the obtained results, Hook effect was not observed at HCPs concentrations ranging from 0 to 200 pg/mL.

Table 10:

Assay results for evaluating the Hook effect of an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00049] The linearity of an exemplary High Five HCPs ELISA was assessed by testing 3 exemplary pharmaceutical samples prepared at dilutions ranging from 1/2 to 1/64 (OD was measured at 450/630 nm). Table 11 below shows the assay results for evaluating the linearity of an exemplary High Five HCPs ELISA. Referring to Table 11, the recovery (%) of the performed tests did not fall between 80-120% demonstrates that the measured OD for exemplary samples using an exemplary High Five HCPs ELISA may have a non-linear relationship with High Five HCPs concentration.

Table 11: Assay results for evaluating the linearity of an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00050] The rang of an assay may be described as a difference between the lowest and the highest concentrations of an analyte measured by an assay. Table 12 below shows the assay results for evaluating the range of an exemplary High Five HCPs ELISA. An assay may have an acceptable range when the CV% falls below 10% and an average recovery (%) falls between

80% and 120%. The obtained range for an exemplary High Five HCPs ELISA was from 3 to 100 ng/mL.

Table 12:

Assay results for evaluating the range of an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00051] Furthermore, an exemplary standard curve was created for an exemplary High Five HCPs ELISA based on standard solutions, prepared from the HCPs of High Five cells lysate, with HCPs concentrations including 0, 3, 6, 12, 25, 50, and 100 ng/mL. FIG. 6 illustrates exemplary standard curve 600 created for an exemplary High Five HCPs ELISA, consistent with one or more exemplary embodiments of the present disclosure.

[00052] While the foregoing has described what are considered to be the best mode and/or other examples, it is understood that various modifications may be made therein and that the subject matter disclosed herein may be implemented in various forms and examples, and that the teachings may be applied in numerous applications, only some of which have been described herein. It is intended by the following claims to claim any and all applications, modifications and variations that fall within the true scope of the present teachings.

[00053] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, including in the claims that follow, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[00054] The scope of protection is limited solely by the claims that now follow. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language that is used in the claims when interpreted in light of this specification and the prosecution history that follows and to encompass all structural and functional equivalents. Notwithstanding, none of the claims are intended to embrace subject matter that fails to satisfy the requirement of Sections 101, 102, or 103 of the Patent Act, nor should they be interpreted in such a way. Any unintended embracement of such subject matter is hereby disclaimed.

[00055] Except as stated immediately above, nothing that has been stated or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[00056] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study except where specific meanings have otherwise been set forth herein. Relational terms such as first and second and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions. An element proceeded by “a” or “an” does not, without further constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[00057] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications that are set forth in this specification, are approximate, not exact. They are intended to have a reasonable range that is consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[00058] It will be understood that the terms and expressions used herein have the ordinary meaning as is accorded to such terms and expressions with respect to their corresponding respective areas of inquiry and study, except where specific meanings have otherwise been set forth herein. Relational terms such as “first” and “second” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[00059] The Abstract of the Disclosure is provided to allow the reader to quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, in the foregoing Detailed Description, it may be seen that various features are grouped together in various implementations. This is for purposes of streamlining the disclosure, and is not to be interpreted as reflecting an intention that the claimed implementations require more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed implementation. Thus, the following claims are hereby incorporated into the Detailed Description, with each claim standing on its own as a separately claimed subject matter. While various implementations have been described, the description is intended to be exemplary, rather than limiting and it will be apparent to those of ordinary skill in the art that many more implementations and implementations are possible that are within the scope of the implementations. Although many possible combinations of features are shown in the accompanying figures and discussed in this detailed description, many other combinations of the disclosed features are possible. Any feature of any implementation may be used in combination with or substituted for any other feature or element in any other implementation unless specifically restricted. Therefore, it will be understood that any of the features shown and/or discussed in the present disclosure may be implemented together in any suitable combination. Accordingly, the implementations are not to be restricted except in light of the attached claims and their equivalents. Also, various modifications and changes may be made within the scope of the attached claims.

Claims

What is claimed is:

1. An immunoassay method for detecting an amount of host cell proteins (HCPs) of High Five cells (High Five HCPs) in a sample, the immunoassay method comprising: forming a plurality of HCP-immobilized antibody complexes by contacting the sample with a surface comprising a plurality of immobilized polyclonal antibodies (pAbs) against the High Five HCPs at a temperature level between 20 °C and 25 °C for a time duration between 80 and 100 minutes, the plurality of immobilized pAbs comprising Immunoglobulin G (IgG) isotype produced in a rabbit, each respective HCP-immobilized antibody complex of the plurality of HCP- immobilized antibody complexes comprising one or more HCPs of the High Five HCPs bound to an immobilized antibody of the plurality of immobilized pAbs against the High Five HCPs; removing unbound High Five HCPs by washing the surface with a wash buffer; contacting the plurality of HCP-immobilized antibody complexes with a plurality of labeled pAbs against the High Five HCPs, wherein each respective labeled antibody of the plurality of labeled pAbs is coupled to a labeling agent, the plurality of labeled pAbs comprising the IgG isotype produced in the rabbit; and detecting the amount of the High Five HCPs in the sample by detecting the formation of a complex between the one or more HCPs and a labeled antibody of the plurality of labeled pAbs.

2. The immunoassay method of claim 1, wherein washing the surface with the wash buffer comprises washing the surface with a IX phosphate buffered saline (PBS) buffer comprising between 0.05% and 0.1% (v/v) Polysorbate 20.

3. The immunoassay method of claim 1, wherein the labeling agent comprises at least one of a luminescent particle, a quantum dot, a chromogen, a catalyst, and a radioactive compound.

4. The immunoassay method of claim 1, wherein contacting the plurality of HCP-immobilized antibody complexes with the plurality of labeled pAbs against the High Five HCPs comprises contacting the plurality of HCP-immobilized antibody complexes with a plurality of horseradish peroxidase (HRP)-conjugated pAbs against the High Five HCPs.

5. The immunoassay method of claim 4, wherein contacting the plurality of HCP-immobilized antibody complexes with the plurality of HRP-conjugated pAbs against the High Five HCPs comprises contacting the plurality of HCP-immobilized antibody complexes with the plurality of HRP-labeled pAbs against the High Five HCPs at a temperature level between 20 °C and 25 °C for a time duration between 20 and 40 minutes.

6. The immunoassay method of claim 1, wherein detecting the amount of the High Five HCPs in the sample comprises detecting the amount of the High Five HCPs in the sample by conducting at least one of chemiluminescent assay, immunofluorescent assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, Western blot assay, enzyme immunoassay, immunoprecipitation assay, immunohistochemical assay, immunochromatographic assay, dot blot assay, and slot blot assay.

7. The immunoassay method of claim 6, wherein detecting the amount of the High Five HCPs in the sample comprises detecting the amount of the High Five HCPs in the sample by conducting ELISA.

8. The immunoassay method of claim 1, wherein detecting the amount of the High Five HCPs in the sample comprises detecting at least 0.8 ng/mL of the High Five HCPs in the sample.