WO2022035197A1 - Stable pharmaceutical formulation - Google Patents

Stable pharmaceutical formulation Download PDF

Info

Publication number
WO2022035197A1
WO2022035197A1 PCT/KR2021/010606 KR2021010606W WO2022035197A1 WO 2022035197 A1 WO2022035197 A1 WO 2022035197A1 KR 2021010606 W KR2021010606 W KR 2021010606W WO 2022035197 A1 WO2022035197 A1 WO 2022035197A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
binding molecule
pharmaceutical formulation
present
stable pharmaceutical
Prior art date
Application number
PCT/KR2021/010606
Other languages
French (fr)
Korean (ko)
Inventor
김광우
김수정
노지원
오준석
이재빈
한원용
Original Assignee
(주)셀트리온
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by (주)셀트리온 filed Critical (주)셀트리온
Publication of WO2022035197A1 publication Critical patent/WO2022035197A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a stable pharmaceutical preparation, and more particularly, to a pharmaceutical preparation capable of stably preserving a molecule binding to a spike protein (S protein) on the surface of SARS-coronavirus-2 .
  • S protein spike protein
  • SARS-coronavirus-2 severe acute respiratory syndrome coronavirus 2, SARS-CoV-2
  • SARS-CoV-2 is a single-stranded RNA coronavirus with a positive sense in genetic sequencing (DNA sequencing).
  • SARS-CoV-2 is contagious to humans and is the cause of coronavirus disease 2019 (COVID-19). The first outbreak of COVID-19 was in Wuhan, Hubei Province, China.
  • SARS-CoV-2 may have mild to severe symptoms such as fever, cough, shortness of breath, and diarrhea. People with complications or diseases and the elderly are more likely to die.
  • coronavirus disease 2019 2019 (COVID-19)
  • the existing treatment is administered to the patient to expect a therapeutic effect.
  • Antiviral agents favipiravir, remdesivir, and galidesivir, which are Ebola treatment or treatment candidates, and hepatitis C treatment ribavirin are being used as COVID-19 treatments.
  • the antimalarial drug Chloroquine has been shown to have a therapeutic effect on COVID-19 and is undergoing public clinical trials.
  • hepatitis C treatment ribavirin may have severe side effects such as anemia, and the antiviral drug interferon is also recommended to be used with caution due to concerns about various side effects.
  • the Korea Centers for Disease Control and Prevention announced that i) that COVID-19 could also spread like influenza for a long time and that it would be included in the surveillance system like influenza, and ii) a coronavirus that spreads among people (4 species) is also prevalent in winter and spring, leaving open the possibility that COVID-19 may also become indigenous (2020.2.17).
  • a formulation capable of more stably preserving a binding molecule targeting the SARS-CoV-2 virus spike protein (S protein) has not yet been developed. Accordingly, as a result of continuous research to develop a formulation having excellent stability, the present inventors have finally completed the present invention.
  • the present inventors have developed a pharmaceutical preparation capable of stably preserving a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2.
  • S protein spike protein
  • the problem to be solved by the present invention is a stable pharmaceutical preparation, preferably comprising a neutralizing binding molecule that binds to the spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2)
  • S protein spike protein
  • SARS-CoV-2 SARS-CoV-2
  • the present invention provides (A) a neutralizing binding molecule that binds to the spike protein (S protein) on the surface of SARS-coronavirus-2 (SARS-CoV-2), (B) a buffer, (C ) a stabilizer and (D) a surfactant.
  • S protein spike protein
  • SARS-CoV-2 SARS-coronavirus-2
  • Neutralizing binding molecules such as antibodies have larger and more complex structures than conventional organic and inorganic drugs. Therefore, a pharmaceutical preparation containing a neutralizing-binding molecule such as an antibody has a special problem in that the stability of the neutralizing-binding molecule, such as an antibody, must be maintained.
  • the stability of a neutralizing binding molecule may be affected by factors such as ionic strength, pH, temperature, repeated freeze/thaw cycles, and concentration of a neutralizing binding molecule, such as an antibody.
  • Neutralizing binding molecules such as active antibodies, exhibit physical instability including denaturation, aggregation (formation of soluble and insoluble aggregates), precipitation and adsorption, or physical instability, including racemization, beta-elimination, disulfide exchange, hydrolysis, deamidation and oxidation. Loss may occur due to chemical instability.
  • Such physical and chemical instability can potentially result in the formation of neutralizing binding molecule byproducts or derivatives, such as antibodies, with reduced biological activity, increased toxicity, and/or increased immunogenicity of the neutralizing binding molecule, such as an antibody.
  • each neutralizing-binding molecule such as an antibody has its own physicochemical properties, so neutralizing-binding molecules such as other antibodies have been proposed. It is practically impossible to predict the type and amount of excipients to overcome the problem of instability of a neutralizing binding molecule such as a specific antibody in consideration of the conventional formulation used. In addition, it is difficult and time-consuming to derive optimal conditions such as the concentration of neutralizing-binding molecules such as antibodies, pH, and concentration of excipients for maintaining chemically and biologically stable neutralizing-binding molecules such as specific antibodies in pharmaceutical formulations and effort are required.
  • the present invention relates to a high molecular weight of the neutralizing binding molecule in a pharmaceutical formulation comprising a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-coronavirus-2 and a buffer, a stabilizer and a surfactant together. It was confirmed for the first time that there was an effect of stabilizing its activity by reducing the formation of the number of components and insoluble foreign substances.
  • the present invention relates to a spike protein on the surface of a coronavirus, preferably SARS-coronavirus-2 (SARS-CoV-2), more preferably SARS-coronavirus-2 (SARS-CoV-2). , S protein) to a stable pharmaceutical formulation comprising a neutralizing binding molecule that binds.
  • SARS-CoV-2 SARS-coronavirus-2
  • SARS-CoV-2 SARS-coronavirus-2
  • SARS-CoV-2 SARS-coronavirus-2
  • SARS-CoV-2 SARS-coronavirus-2
  • SARS-CoV-2 SARS-coronavirus-2
  • the binding molecule may bind to the receptor binding domain (RBD) region of the spike protein on the SARS-coronavirus-2 surface.
  • RBD receptor binding domain
  • the SARS-coronavirus-2 (SARS-CoV-2) surface spike protein (S protein, S protein) of the present invention consists of or includes the sequence of SEQ ID NO: 3841. and derivatives and/or variants thereof, but are not limited thereto.
  • the SARS-CoV-2 (SARS-CoV-2) surface spike protein region of the present invention may consist of or include the sequence of SEQ ID NO: 3842. , derivatives and/or variants thereof.
  • the pharmaceutical formulation according to the present invention may be in a liquid form, but is not limited thereto.
  • the neutralizing binding molecule of the present invention exhibits excellent binding ability and/or excellent neutralizing ability to the spike protein of SARS-CoV-2 (SARS-CoV-2). In another embodiment of the present invention, the neutralizing binding molecule of the present invention exhibits excellent binding and/or neutralizing ability to a mutant virus in which the spike protein of SARS-CoV-2 is mutated.
  • the neutralizing binding molecule of the present invention is a binding molecule that binds to the receptor binding domain (RBD) region of the spike protein of SARS-CoV-2 and is screened as a binding molecule with neutralizing ability.
  • the neutralizing binding molecule of the present invention may exhibit excellent neutralizing ability against mutant viruses of other regions of the S protein other than the RBD region, but is not limited thereto.
  • the World Health Organization classifies SARS-Coronavirus-2 into six types based on amino acid changes due to differences in gene sequence. First, it was classified into S and L types, then again into L, V, and G types, and as G was divided into GH and GR, it is classified into a total of six types: S, L, V, G, GH, and GR. At the beginning of the COVID-19 outbreak, types S and V were prevalent in Asia including Wuhan, China, and after that, different types were discovered for each continent. Among them, it has been reported that the GH type has the potential to appear high in transmission power.
  • the neutralizing binding molecule of the present invention is S-type (amino acid at position 614 of the S protein is D), G-type (the amino acid at position 614 of the S protein is G) based on the SARS-CoV-2 virus amino acid mutation. ), may exhibit neutralizing ability in strains such as V type, but is not limited to this strain.
  • An example of the SARS-CoV-2 virus type S is the BetaCoV/Korea/KCDC03/2020 strain, but is not limited thereto.
  • An example of the SARS-CoV-2 virus type G includes, but is not limited to, the hCoV-19/South Korea/KUMC17/2020 strain.
  • the neutralizing binding molecule of the present invention exhibits excellent neutralizing ability even in a mutant virus in which D614G mutation occurs at amino acid position 614 of the spike protein of SARS-CoV-2 (SARS-CoV-2).
  • the neutralizing binding molecule of the present invention is SARS-coronavirus-2 (SARS-CoV-2) surface protein (RBD) mutant proteins A435S, F342L, G476S, K458R, N354D, V367F, V483A, and W436R It exhibits excellent bonding strength.
  • the neutralizing binding molecule of the present invention is a SARS-coronavirus-2 strain isolated to date, for example, UNKNOWN-LR757996 strain (Strain), SARS-CoV-2/Hu of unknown date and place of isolation.
  • the neutralizing binding molecule of the present invention may be a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2.
  • S protein spike protein
  • the neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 1, preferably binding molecule No. 89 to No. 194, and No. 248 may be any one binding molecule selected from the group consisting of, more preferably No. 139 binding molecule. In Table 1 below, No. means the number of each binding molecule.
  • the CDRs of the variable region according to the present invention were determined by a conventional method according to the system devised by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda) , MD. (1991)).
  • the CDR numbering used in the present invention uses the Kabat method, but may be determined according to other methods such as the IMGT method, the Chothia method, and the AbM method.
  • a binding molecule comprising a CDR determined by any of the above methods is also included in the present invention.
  • the neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 2, preferably binding molecule No. 89 to No. 194, and No. 248 may be any one binding molecule selected from the group consisting of, more preferably No. 139 binding molecule. In Table 2 below, No. means the number of each binding molecule.
  • the neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 3, preferably No. 322 or No. 343 binding molecule, more preferably No. 322 binding molecule.
  • No. means the number of each binding molecule.
  • the neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 4, preferably No. 322 or No. 343 binding molecule, more preferably No. 322 binding molecule.
  • No. means the number of each binding molecule.
  • the neutralizing binding molecule of the present invention may be an scFv fragment, an scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody, but is not limited thereto.
  • the neutralizing binding molecule of the present invention may be an scFv-Fc that binds to the SARS-CoV-2 S protein.
  • a neutralizing binding molecule of the present invention comprises a light chain variable region comprising LC CDR1, LC CDR2 and LC CDR3, and a heavy chain variable region comprising HC CDR1, HC CDR2 and HC CDR3,
  • said LC CDR1 comprises the sequence SGX 1 X 2 SNIGX 3 NX 4 X 5 S, wherein X 1 is S, G or R, X 2 is S, N or T, and X 3 is N, D or K , X 4 is Y or F, X 5 is V or I,
  • said LC CDR2 comprises the sequence DNX 6 KRPS, wherein X 6 is N or D;
  • said LC CDR3 comprises the sequence GTWDX 7 X 8 LSX 9 X 10 X 11 , wherein X 7 is S or N, X 8 is S or N, X 9 is A or G, and X 10 is G or V and X 11 is V or R,
  • said HC CDR1 comprises the sequence TSGX 12 GVX 13 , wherein X 12 is M or V, X 13 is G or S,
  • said HC CDR2 comprises the sequence LIDWDDNKYX 14 TTSLKT, wherein X 14 is Y or H;
  • Said HC CDR3 may be a binding molecule comprising the sequence IPGFLRYRNRYYYYGX 15 DV, wherein X 15 is M or V.
  • the neutralizing binding molecule of the present invention comprises a light chain variable region comprising LC CDR1, LC CDR2 and LC CDR3, and a heavy chain variable region comprising HC CDR1, HC CDR2 and HC CDR3, ,
  • LC CDR1 comprises the sequence RASQSISSYLN
  • LC CDR2 comprises the sequence AASSLQS
  • said LC CDR3 comprises the sequence QQSYSTPLT
  • said HC CDR1 comprises the sequence SNYMX 16 , wherein X 16 is T or S;
  • said HC CDR2 comprises the sequence X 17 IYPGGSTX 18 X 19 ADSVX 20 G, wherein X 17 is I or V, X 18 is Y or F, X 19 is Y or F, and X 20 is K or Q ,
  • the HC CDR3 may be a binding molecule comprising the sequence DLPLTGTTLDY or SYDFLTDYTDAFDI.
  • the neutralizing binding molecule of the present invention comprises a light chain variable region comprising a CDR1 region of SEQ ID NO: 829, a CDR2 region of SEQ ID NO: 830, and a CDR3 region of SEQ ID NO: 831; and a heavy chain variable region comprising a CDR1 region of SEQ ID NO: 832, a CDR2 region of SEQ ID NO: 833, and a CDR3 region of SEQ ID NO: 834.
  • the neutralizing binding molecule of the present invention comprises a light chain variable region comprising a CDR1 region of SEQ ID NO: 2507, a CDR2 region of SEQ ID NO: 2508, and a CDR3 region of SEQ ID NO: 2509; and a heavy chain variable region comprising a CDR1 region of SEQ ID NO: 2510, a CDR2 region of SEQ ID NO: 2511, and a CDR3 region of SEQ ID NO: 2512.
  • the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 2017; and 80% to 99%, preferably 85 to 99%, more preferably 90 to 99% identical to the binding molecule comprising the heavy chain variable region of the polypeptide sequence of SEQ ID NO: 2018. It may be a binding molecule comprising a sequence .
  • the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 3523; and 80% to 99%, preferably 85 to 99%, more preferably 90 to 99% identical to the binding molecule comprising the heavy chain variable region of the polypeptide sequence of SEQ ID NO: 3524. .
  • the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 2017; and a heavy chain variable region of the polypeptide sequence of SEQ ID NO: 2018.
  • the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 3523; and a heavy chain variable region of the polypeptide sequence of SEQ ID NO: 3524.
  • the term 'antibody' is used in the broadest sense, specifically, an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more intact antibodies (eg, a bispecific antibody), and antibody fragments exhibiting the desired biological activity.
  • Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. In terms of their structure, antibodies usually have a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody mainly has two regions: a variable region and a constant region. The variable region located in the distal portion of the arm of Y binds and interacts with the target antigen.
  • variable region comprises a complementarity determining region (CDR) that recognizes and binds a specific binding site on a specific antigen.
  • CDR complementarity determining region
  • the constant region located at the tail of Y is recognized and interacted with by the immune system.
  • Target antigens have multiple binding sites, called epitopes, which are generally recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.
  • the binding molecule according to the present invention may include a functional variant of the binding molecule.
  • a variant of the invention may compete with a binding molecule of the invention for specific binding to SARS-CoV-2 or its S protein.
  • it is regarded as a functional variant of the binding molecule of the present invention if it has the ability to neutralize SARS-CoV-2.
  • the functional variant includes, but is not limited to, a derivative having a substantially similar primary structural sequence.
  • the functional variant includes in vitro or in vivo modification, modification by chemical and/or biochemical agents.
  • the functional variant is not found in the parental monoclonal antibody of the present invention.
  • Such modifications include, for example, acetylation, acylation, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis. and phosphorylation.
  • the functional variant may optionally comprise an amino acid sequence containing one or more amino acid substitutions, insertions, deletions or combinations thereof compared to the amino acid sequence of the parent antibody.
  • the functional variant may comprise a truncated form of the amino acid sequence at one or both of the amino terminus or the carboxy terminus.
  • a variable region including, but not limited to, a framework structure, a hypervariable region, in particular, a complementarity-determining region (CDR) of a light or heavy chain
  • CDR complementarity-determining region
  • a light or heavy chain region comprises three hypervariable regions, comprising three CDR regions, and a more conserved region, namely a framework region (FR).
  • FR framework region
  • a hypervariable region comprises amino acid residues from a CDR and amino acid residues from a hypervariable loop.
  • Functional variants within the scope of the present invention include about 50%-99%, about 60%-99%, about 80%-99%, about 90%-99%, about 95%-99%, or about 97%-99% amino acid sequence identity.
  • Gap or Bestfit known to those skilled in the art among computer algorithms may be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues.
  • the functional variant may be obtained by changing the parent antibody or a part thereof by a known general molecular biological method including PCR method, mutagenesis using oligomeric nucleotides and partial mutagenesis, or by organic synthesis method.
  • the present invention is not limited thereto.
  • the binding molecule may be an immunoconjugate to which one or more tags are additionally bound, for example, an immunoconjugate to which a drug is further attached to the binding molecule.
  • the binding molecule according to the present invention may be used in the form of an antibody-drug conjugate (ADC) to which a drug is bound.
  • ADC antibody-drug conjugate
  • ADCs i.e. immunoconjugates
  • the ADC form can improve the maximum efficacy and minimum toxicity of the drug by increasing the drug-connectivity and drug-releasing properties as well as the selectivity of polyclonal and monoclonal antibodies (mAbs).
  • the concentration of (A) neutralizing binding molecule of the present invention can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the stable pharmaceutical preparation according to the present invention.
  • the concentration of the neutralizing binding molecule may be 1 to 240 mg/ml.
  • the concentration of the neutralizing binding molecule may be 1 to 230 mg/ml.
  • the concentration of the neutralizing binding molecule may be 1 to 220 mg/ml.
  • the concentration of the neutralizing binding molecule may be 1 to 210 mg/ml.
  • the concentration of the neutralizing binding molecule may be between 1 and 200 mg/ml.
  • the concentration of the neutralizing binding molecule may be 5 to 240 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 5 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 220 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 5 and 210 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be 5 to 200 mg/ml.
  • the concentration of the neutralizing binding molecule may be 10-240 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 10 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 10-220 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 10 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 200 mg/ml.
  • the concentration of the neutralizing binding molecule may be 20 to 240 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 20 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 220 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 20 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 200 mg/ml.
  • the concentration of the neutralizing binding molecule may be 30 to 240 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 220 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 200 mg/ml.
  • the concentration of the neutralizing binding molecule may be 40 to 240 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 220 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 200 mg/ml.
  • the concentration of the neutralizing binding molecule may be from 1 to 100 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 1 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 1 to 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 1 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 1 and 60 mg/ml.
  • the concentration of the neutralizing binding molecule may be between 5 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 5 and 60 mg/ml.
  • the concentration of the neutralizing binding molecule may be between 10 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 10 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 60 mg/ml.
  • the concentration of the neutralizing binding molecule may be between 20 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 20 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 60 mg/ml.
  • the concentration of the neutralizing binding molecule may be between 30 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 30 to 90 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 30 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 60 mg/ml.
  • the concentration of the neutralizing binding molecule may be between 40 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 40 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 40 to 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 40 to 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 60 mg/ml.
  • the concentration of the neutralizing binding molecule may be between 50 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 50 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 50 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 50 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 50 and 60 mg/ml.
  • the buffer according to the present invention is a neutralizing material that minimizes changes in pH due to acid or alkali
  • the buffer is i) histidine, histidine salt or a mixture thereof, ii) acetate, iii) citrate, iv) succinate, v) phosphate, or vi) gluconate, but is not limited thereto.
  • the histidine salt may include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate, and the like.
  • it may be preferable in terms of pH control and stability to include histidine as a buffer.
  • the content of the buffer can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical formulation according to the present invention.
  • the content of the buffer may be 1 to 100 mM.
  • the content of the buffer may be 1 to 90 mM.
  • the content of the buffer may be 1 to 80 mM.
  • the content of the buffer may be 1 to 70 mM.
  • the content of the buffer may be 1 to 60 mM.
  • the content of the buffer may be 1 to 50 mM.
  • the content of the buffer may be 2 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 2-50 mM.
  • the content of the buffer may be 3 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 50 mM.
  • the content of the buffer may be 4 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 4-50 mM.
  • the content of the buffer may be 5 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 50 mM.
  • the content of the buffer may be 1 to 20 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 15 mM. In another embodiment of the present invention, the content of the buffer may be 7 to 13 mM. In another embodiment of the present invention, the content of the buffer may be 10 mM.
  • the stabilizer according to the present invention consists of i) a metal salt, ii) a sugar or a derivative thereof, and iii) an amino acid (provided that it is different from the amino acid contained in the (B) buffer) or a salt thereof. It may be any one or more selected from the group, preferably an amino acid.
  • the stabilizer may be a metal salt, and an anion thereof may be included together.
  • the metal salt may include sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl), or a mixture of two or more thereof.
  • the stabilizer may be a sugar or a derivative of sugar.
  • the sugar may be a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, or a mixture of two or more thereof.
  • monosaccharides include, but are not limited to, glucose, fructose, galactose, and the like.
  • disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, and the like.
  • oligosaccharides include, but are not limited to, fructooligosaccharides, galactooligosaccharides, mannan oligosaccharides, and the like.
  • polysaccharides include, but are not limited to, starch, glycogen, cellulose, chitin, pectin, and the like.
  • the sugar derivative may be a sugar alcohol, a sugar acid, or a mixture thereof.
  • sugar alcohols include glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, bolemitol, isomalt, maltitol, lactitol, maltotriitol , maltotetraitol, polyglycitol, and the like, but is not limited thereto.
  • sugar acids include, but are not limited to, aldonic acid (such as glyceric acid), ulosonic acid (such as neuramic acid), uronic acid (such as glucuronic acid), and aldaric acid (such as tartaric acid).
  • aldonic acid such as glyceric acid
  • ulosonic acid such as neuramic acid
  • uronic acid such as glucuronic acid
  • aldaric acid such as tartaric acid
  • the sugar or its derivative may be sorbitol, mannitol, trehalose, sucrose, or a mixture of two or more thereof, preferably trehalose.
  • the stabilizing agent may be an amino acid or a salt of an amino acid.
  • the amino acid may be glycine, arginine, threonine, methionine, or a mixture of two or more thereof.
  • the salt of the amino acid may be L- arginine monohydrochloride (monohydrochloride), but is not limited thereto.
  • the stabilizer may be preferably L-arginine monohydrochloride.
  • the content of the stabilizer can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical formulation according to the present invention.
  • the content of the stabilizer may be 50 to 300 mM or 1 to 20% (w/v), but is not limited thereto.
  • the content of the stabilizer is 50 to 300 mM, 50 to 290 mM, 50 to 280 mM, 50 to 270 mM, 50 to 260 mM or 50 to 250 mM.
  • the content of the stabilizer is 60 to 300 mM, 60 to 290 mM, 60 to 280 mM, 60 to 270 mM, 60 to 260 mM or from 60 to 250 mM.
  • the content of the stabilizer is 70 to 300 mM, 70 to 290 mM, 70 to 280 mM, 70 to 270 mM, 70 to 260 mM or 70 to 250 mM.
  • the content of the stabilizer is 80 to 300 mM, 80 to 290 mM, 80 to 280 mM, 80 to 270 mM, 80 to 260 mM or 80 to 250 mM.
  • the content of the stabilizer is 90 to 300 mM, 90 to 290 mM, 90 to 280 mM, 90 to 270 mM, 90 to 260 mM or from 90 to 250 mM.
  • the content of the stabilizer is 100 to 300 mM, 100 to 290 mM, 100 to 280 mM, 100 to 270 mM, 100 to 260 mM or from 100 to 250 mM.
  • the content of the stabilizer is 1 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 1 to 18% (w/v), 1 to 16% (w/v), 1 to 14% (w/v), 1 to 12% (w/v) or 1 to 10% (w/v) days can be 1 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 1 to 18% (w/v), 1 to 16% (w/v), 1 to 14% (w/v), 1 to 12% (w/v) or 1 to 10% (w/v) days can
  • the content of the stabilizer is 2 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 2 to 18% (w/v), 2 to 16% (w/v), 2 to 14% (w/v), 2 to 12% (w/v) or 2 to 10% (w/v) days can
  • the content of the stabilizer is 3 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 3 to 18% (w/v), 3 to 16% (w/v), 3 to 14% (w/v), 3 to 12% (w/v) or 3 to 10% (w/v) days can
  • the content of the stabilizer is 4 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 4 to 18% (w/v), 4 to 16% (w/v), 4 to 14% (w/v), 4 to 12% (w/v) or 4 to 10% (w/v) days can
  • the content of the stabilizer is 5 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 5 to 18% (w/v), 5 to 16% (w/v), 5 to 14% (w/v), 5 to 12% (w/v) or 5 to 10% (w/v) days can
  • the stabilizer Within the content range of the stabilizer, high molecular weight or low molecular weight components are maintained low for a long period of time, the content of intact immunoglobulin G component or intact heavy and light chains is maintained high, and long-term stability is excellent and low viscosity. .
  • the surfactant according to the present invention is polyoxyethylene sorbitan fatty acid ester (eg, polysorbate), polyoxyethylene alkyl ether (eg, Brij), alkylphenylpolyoxyethylene ethers (eg Triton-X), polyoxyethylene-polyoxypropylene copolymers (eg Poloxamer, Pluronic), sodium dodecyl sulfate (SDS), poloxamers and mixtures thereof, and the like, but are limited thereto. it is not going to be
  • the surfactant may be polysorbate, poloxamer, or a mixture thereof, preferably polyoxyethylene sorbitan fatty acid ester (polysorbate), poloxamer, or a mixture thereof.
  • the polysorbate may be polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture of two or more thereof, but is not limited thereto.
  • the poloxamer may be poloxamer 188, but is not limited thereto.
  • the surfactant may most preferably be polysorbate 80.
  • the concentration of the surfactant can be freely adjusted within a range that does not adversely affect the stability and viscosity of the stable pharmaceutical formulation according to the present invention.
  • the concentration of the surfactant according to the present invention is 0.01 to 0.1% (w/v), preferably 0.01 to 0.08% (w/v), more preferably 0.01 to 0.07% (w/v) days can
  • the concentration of the surfactant may be most preferably 0.05% (w/v).
  • the pH of the composition according to the present invention may be 5.0 to 7.0, 5.5 to 7.0, 5.7 to 7.0, 6.0 to 7.0, 6.3 to 7.0 or 6.5 to 7.0.
  • the pH of the composition according to the present invention may be 5.5 to 6.5, 5.7 to 6.5, 6.0 to 6.5, or 6.3 to 6.5.
  • the pH of the composition according to the present invention may be 5.5 to 6.3, 5.7 to 6.3, or 6.0 to 6.3.
  • the pH of the composition according to the present invention may be 5.5 to 6.0, and may be 5.7 to 6.0.
  • the pH of the composition according to the present invention may preferably be 6.0. Within the above pH range, it can exhibit excellent stability and low viscosity for a long period of time.
  • the pH can be adjusted using a buffer, and when the buffer is included in a predetermined amount, the pH of the above range can be exhibited without a separate pH adjusting agent.
  • stable means that the neutralizing binding molecule according to the present invention substantially retains physical stability, chemical stability and/or biological activity during the manufacturing process and/or upon storage/storage.
  • various analytical techniques for measuring the stability of an antibody that are readily available in the art may be used.
  • the physical stability can be evaluated by methods known in the art, which include measuring the sample apparent attenuation of light (absorption or optical density). This light attenuation measurement is related to the turbidity of the formulation.
  • the physical stability can be measured by high molecular weight component content, low molecular weight component content, intact protein amount, the number of insoluble foreign particles, and the like.
  • the chemical stability can be evaluated, for example, by detecting and quantifying a chemically changed form of a neutralizing binding molecule.
  • Such chemical stability includes charge changes (eg, occurring as a result of deamidation or oxidation) that can be assessed, for example, by ion exchange chromatography.
  • Chemical stability can be measured with charge variants (acidic or basic peaks) or the like.
  • the biological activity may be evaluated by a method known in the art, for example, antigen binding affinity may be measured through ELISA.
  • stable pharmaceutical preparation refers to a pharmaceutical preparation satisfying one or more of the following.
  • a pharmaceutical formulation having an absorbance A600 of 0 to 0.0700 or 0 to 0.0400 measured with a spectrophotometer after storage for 4 weeks at a temperature of 5 ⁇ 3° C.;
  • a pharmaceutical formulation having an absorbance A600 of 0 to 0.0700 or 0 to 0.0400 measured with a spectrophotometer after storage for 4 weeks at a temperature of 40 ⁇ 2° C., a relative humidity of 75 ⁇ 5%, and a closed condition;
  • the number of insoluble foreign particles (10.00 ⁇ m ⁇ , ⁇ 400.00 ⁇ m) measured by HIAC after storage for 4 weeks at a temperature of 40 ⁇ 2°C, a relative humidity of 75 ⁇ 5%, and a closed condition is 0 to 200 pharmaceutical formulations ;
  • the number of insoluble foreign particles (25.00 ⁇ m ⁇ , ⁇ 400.00 ⁇ m) measured by HIAC after storage for 4 weeks at a temperature of 40 ⁇ 2°C, a relative humidity of 75 ⁇ 5%, and a closed condition is 0 to 50 pharmaceutical formulations ;
  • the number of insoluble foreign particles (1.00 ⁇ m ⁇ , ⁇ 400.00 ⁇ m) measured by MFI after storage for 4 weeks at a temperature of 40 ⁇ 2°C, a relative humidity of 75 ⁇ 5%, and a closed condition is 0 to 20,000 pharmaceutical formulations ;
  • the number of insoluble foreign particles (10.00 ⁇ m ⁇ , ⁇ 400.00 ⁇ m) measured by MFI after storage for 4 weeks at a temperature of 40 ⁇ 2°C, a relative humidity of 75 ⁇ 5%, and a closed condition is 0 to 300 pharmaceutical formulations ;
  • the number of insoluble foreign particles (25.00 ⁇ m ⁇ , ⁇ 400.00 ⁇ m) measured by MFI after storage for 4 weeks at a temperature of 40 ⁇ 2°C, a relative humidity of 75 ⁇ 5%, and a closed condition is 0 to 30 pharmaceutical formulations ;
  • the stable pharmaceutical formulation of the present invention can be prepared using a known method, and is not limited to a specific manufacturing method. For example, after adjusting the pH by adding a buffer to a solution containing a stabilizer and a surfactant, a neutralizing binding molecule may be added to the mixed solution to prepare a pharmaceutical formulation. In addition, after preparing a solution containing some excipients in the final step of the purification process, the remaining ingredients may be added to prepare a pharmaceutical formulation.
  • the preparation may not include a freeze-drying process during manufacture. If the freeze-drying process is not included, for example, the pharmaceutical formulation of the present invention may be prepared and placed in an airtight container such as a glass vial or pre-filled syringe, which is a primary packaging material, immediately after treatment such as sterilization.
  • an airtight container such as a glass vial or pre-filled syringe, which is a primary packaging material
  • the stable pharmaceutical formulation of the present invention is (A) a neutralizing binding molecule 5 that binds to the spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2) to 240 mg/ml; (B) 1-50 mM buffer; (C) 50-200 mM stabilizer; and (D) 0.01 to 0.1% (w/v) of a surfactant.
  • S protein spike protein
  • SARS-CoV-2 SARS-CoV-2
  • D 0.01 to 0.1% (w/v) of a surfactant.
  • the present invention provides the above stable pharmaceutical formulation; And it may provide a product comprising a container for accommodating the stable pharmaceutical formulation in a closed state.
  • the stable pharmaceutical formulation is as described above.
  • the container may be formed of a material such as glass, polymer (plastic), or metal, but is not limited thereto.
  • the container may be a bottle, a vial, a cartridge, a syringe (pre-filled syringe), or a tube, but is not limited thereto.
  • the container may be a glass or polymer vial, or a glass or polymer pre-filled syringe.
  • the product may further include instructions for providing a method of use, a method of storage, or both of the stable pharmaceutical formulation.
  • the product may include other tools necessary from a commercial and user point of view, for example, needles, syringes, and the like.
  • binding molecule refers to an intact immunoglobulin, including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, or antigen-binding, which is an immunoglobulin that binds to an antigen. Includes fragments. For example, in binding to the spike protein of SARS-CoV-2, it refers to a variable domain, enzyme, receptor, or protein comprising an immunoglobulin fragment that competes with an intact immunoglobulin. Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact immunoglobulin.
  • the antigen-binding fragment comprises at least two contiguous groups of the amino acid sequence of the antibody, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues , at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, a peptide or polypeptide comprising an amino acid sequence of at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.
  • the term "antigen-binding fragment” particularly refers to Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv). , bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind a particular antigen to the polypeptide. etc.
  • the fragments may be produced synthetically or by enzymatic or chemical digestion of complete immunoglobulins, or may be genetically engineered by recombinant DNA techniques. Methods of production are well known in the art.
  • the stable pharmaceutical formulation according to the present invention has excellent long-term storage stability under temperature conditions such as accelerated conditions and severe conditions, and can maintain excellent stability even under physical stress conditions such as light, freezing/thawing, and shaking.
  • FIG. 10 is a measurement result of the content of the main component at a temperature of 5 ⁇ 3° C. in Examples 1 and 7 to 16.
  • FIG. 10 is a measurement result of the content of the main component at a temperature of 5 ⁇ 3° C. in Examples 1 and 7 to 16.
  • FIG. 11 is a measurement result of the content of the main component at a temperature of 25 ⁇ 2° C. in Examples 1 and 7 to 16.
  • FIG. 11 is a measurement result of the content of the main component at a temperature of 25 ⁇ 2° C. in Examples 1 and 7 to 16.
  • FIG. 12 is a measurement result of the content of the main component at a temperature of 40 ⁇ 2° C. in Examples 1 and 7 to 16.
  • FIG. 12 is a measurement result of the content of the main component at a temperature of 40 ⁇ 2° C. in Examples 1 and 7 to 16.
  • FIG. 23 is a measurement result of intact immunoglobulin G content of Examples 1, 20 to 31, and Comparative Example 1.
  • a buffer solution containing a stabilizer was prepared at a pH that showed the optimal buffering ability, and then SARS-CoV-2 neutralizing binding molecules were added to the solution, and a surfactant was added. was added to reach the target concentration to prepare the corresponding formulation component.
  • the absorbance at 600 nm was measured using a UV-Vis spectrophotometer.
  • the main component content (%) was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC).
  • the content (pre-peak; %) of the high molecular weight component was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC).
  • the content (post-peak; %) of low molecular weight components was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC).
  • the content of intact immunoglobulin G was measured using Labchip GXII, a non-reducing chip-based CE-SDS analysis equipment.
  • the contents of light and heavy chains of the antibody were measured using Labchip GXII, a reduced chip-based CE-SDS analysis equipment.
  • MFI Micro Flow Imaging
  • HIAC 9703 Light-shielding particle counter
  • the oxidation rate (%) of heavy chain Met 263 was measured through peptide mapping by liquid chromatography (LC-MS) through mass spectrometry.
  • SARS-CoV-2 RBD binding affinity (%) was measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
  • each buffer was prepared to suit each pH, and arginine monohydrochloride, sorbitol and trehalose were added thereto, an antibody was added thereto, and a surfactant was added to the sample of Table 5 were manufactured.
  • the pharmaceutical formulations prepared according to Examples 1 to 3 were stored at a temperature of 5 ⁇ 3° C. and a temperature of 50 ⁇ 2° C., stability after 5 days at a temperature of 5 ⁇ 3° C., and after 3 days and 5 at a temperature of 50 ⁇ 2° C. Stability after one day was measured. For physical stress, freezing and thawing at -40°C were repeated 5 times. The results of the experiment are shown in Tables 6 to 18 and FIGS. 1 to 8 .
  • the antibody used in Experimental Example 1 was No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
  • the formulation prepared with the following ingredients had excellent stability under temperature conditions such as accelerated conditions and severe conditions, and maintained excellent stability even under physical stress conditions such as freezing/thawing.
  • the main component content and the intact immunoglobulin G content in the formulation containing L-arginine monohydrochloride were more stable under high temperature conditions than the formulation containing sorbitol and trehalose.
  • Example 1 histidine 10 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL
  • Example 2 histidine 10 mM 6.0 Sorbitol 5% (w/v) Polysorbate 800.05% (w/v) 60mg/mL
  • Example 3 histidine 10 mM 6.0 Trehalose 10% (w/v) Polysorbate 800.05% (w/v) 60mg/mL
  • Example 1 0.0370 0.0055 0.0096 0.0108 0.0068
  • Example 2 0.0121 0.0162 0.0157 0.0412 0.0070
  • Example 3 0.0082 0.0037 0.0090 0.0059 0.0059
  • Example 1 the turbidity of Example 1 was 0.0400 or less under conditions of 5 ⁇ 3° C., 50 ⁇ 2° C. and freeze/thaw stress. In the case of Example 2, the turbidity of Example 1 was higher than that of 50 ⁇ 2° C. conditions.
  • Table 7 shows the measurement results of the main component content (Main peak %) by size exclusion chromatography.
  • Example 1 contained the highest content of main components under the conditions of 5 ⁇ 3 °C and 50 ⁇ 2 °C. It can be seen that the main component content of Examples 1 to 3 is stable to 98.0% or more after 5 days at 50 ⁇ 2° C. condition. The main component content by freezing/thawing stress was similar to Examples 1 to 3, and it was found that it was stable at 98.0% or more (FIG. 1).
  • Table 8 shows the results of size exclusion chromatography high molecular weight component content (pre-peak %) measurement.
  • Example 1 Referring to Table 8, the conditions of 5 ⁇ 3° C. after 5 days and 50 ⁇ 2° C. after 3 days and 5 days of Example 1 showed the lowest content of high molecular weight components. It was found that the high molecular weight component content of Examples 1 to 3 was stable at 50 ⁇ 2° C. after 5 days at 1.00% or less. The high molecular weight content due to freezing/thawing stress was similar to Examples 1 to 3, and it was found that it was stable at 1.00% or less ( FIG. 2 ).
  • Table 9 shows the measurement results of the low molecular weight component content (post-peak %) by size exclusion chromatography.
  • Example 1 0.04 0.04 0.18 0.31 0.05
  • Example 2 0.04 0.04 0.16 0.27 0.04
  • Example 3 0.04 0.05 0.16 0.30 0.03
  • Table 10 below shows the measurement results of intact immunoglobulin G content (non-reducing chip-based CE-SDS).
  • Example 1 the highest intact immunoglobulin G content was shown in Example 1 at 5 ⁇ 3° C. after 5 days and at 50 ⁇ 2° C. after 3 and 5 days. It was found that the content of intact immunoglobulin G of Examples 1 to 3 was stable to 90.0% or more after 5 days at 50 ⁇ 2°C.
  • the high molecular weight content due to freezing/thawing stress was similar to Examples 1 to 3, and it was found that it was stable at 90.0% or more (FIG. 4).
  • Table 11 below shows the measurement results of the antibody light chain and heavy chain content (reduced chip-based CE-SDS).
  • Table 12 below shows the measurement results of the charge variant (main peak %).
  • Example 1 65 65.48 64.67 63.35 65.56
  • Example 2 65.12 65.39 64.21 62.67 65.39
  • Example 3 65.14 64.92 64.03 62.92 65.25
  • Table 13 shows the measurement results of the charge variant (acid peak %).
  • Table 14 shows the measurement results of charge variants (basic peak %).
  • Table 15 shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Table 16 shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Table 17 below shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ (um)) measured by HIAC.
  • Example 1 is a formulation finally selected according to Experimental Example 1.
  • the prepared pharmaceutical formulation was stored at 5 ⁇ 3°C temperature, 40 ⁇ 2°C temperature and 75 ⁇ 5% relative humidity, and stability after 2 and 4 weeks at 5 ⁇ 3°C temperature and 40 ⁇ 2°C temperature and 75 ⁇ Stability after 2 weeks and 4 weeks at 5% relative humidity was measured. For physical stress, shaking stress was applied at 3000 rpm at room temperature for 4 hours, and the results are shown in Tables 20 to 27 and FIG. 9 .
  • Antibodies used in Experimental Example 2 were No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
  • Example 4 histidine 10 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 100mg/mL
  • Example 5 histidine 10 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 150mg/mL
  • Example 6 histidine 10 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 200mg/mL
  • Table 21 below shows the measurement results of SARS-CoV-2 RBD binding affinity (ELISA).
  • Table 22 below shows the measurement results of the main component content (Main peak %) by size exclusion chromatography.
  • Table 23 shows the measurement results of high molecular weight components (pre-peak %) by size exclusion chromatography.
  • Example 5 0.50 0.56 1.02 0.63 1.53 0.51
  • Example 6 0.55 0.67 1.19 0.88 1.31 0.65
  • Example 1 0.37 0.40 0.53 0.36 0.65 0.38
  • Table 24 shows the results of measurement of low molecular weight components (post-peak %) by size exclusion chromatography.
  • Table 25 shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ (um)) measured by HIAC.
  • Table 26 shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ (um)) measured by HIAC.
  • Example 26 the number of insoluble foreign particles (25.00 ⁇ (um)) of Examples 4 to 6 was stable to 10 or less under all experimental conditions, and it was found to be at a level similar to the result of Example 1.
  • Table 27 shows the oxidation rate Oxidation (Met 263) measurement results.
  • Example 1 is a formulation finally selected according to Experimental Example 1.
  • the prepared pharmaceutical formulations were stored at 5 ⁇ 3°C temperature, 25 ⁇ 2°C temperature, 60 ⁇ 5% relative humidity, 40 ⁇ 2°C temperature and 75 ⁇ 5% relative humidity, and each temperature condition for 3 weeks and 6 weeks and stability after 9 weeks.
  • shaking stress was applied at 3000 rpm at room temperature for 4 hours, freezing/thawing stress was repeated 5 times at -40 ° C.
  • the results are shown in Tables 29 to 43 and FIGS. 10 to 17 .
  • the antibody used in Experimental Example 3 was No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
  • Example 7 histidine 5 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL
  • Example 8 Histidine 15 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL
  • Example 9 histidine 10 mM 5.7 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL
  • Example 10 histidine 10 mM 6.3 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL
  • Example 11 histidine 10 mM 6.0 L-Arginine monohydrochloride 100 mM Polysorbate 800.05% (w/v) 60mg/mL
  • Example 12 histidine 10 mM 6.0 L-Arginine monohydrochloride 200 mM Polysorbate 800.05%
  • Example 7 0.0114 0.0019 0.0059 0.0039 0.0100 0.0066 0.0095 0.0096 0.0063 0.0104 0.0099 0.0114 0.0121
  • Example 8 0.0121 0.0103 0.0086 0.0076 0.0096 0.0144 0.0075 0.0080 0.0089 0.0148 0.0034 0.0103 0.0231
  • Example 9 0.0071 0.0064 0.0050 0.0082 0.0103 0.0089 0.0064 0.0100 0.0126 0.0090 0.0070 0.0109 0.0171
  • Example 10 0.0097 0.0095 0.0070 0.0072 0.0100 0.0108 0.0085 0.0102 0.0072 0.0111 0.00
  • Table 30 below shows the measurement results of the main component content (Main peak %) by size exclusion chromatography.
  • Example 7 99.47 99.50 99.36 98.96 99.44 99.28 98.53 99.42 99.19 98.24 99.46 99.39 99.12
  • Example 8 99.45 99.53 99.41 98.99 99.44 99.29 98.54 99.45 99.14 98.20 99.48 99.43 99.09
  • Example 9 99.53 99.54 99.46 98.76 99.45 99.36 98.45 99.47 99.15 97.82 99.29 99.46 99.19
  • Example 10 99.26 99.46 99.29 98.87 99.37 99.14 98.43 99.
  • Example 30 As shown in Table 30, it was found that the main component content of Examples 7 to 16 was stable to 99% or more at 5 ⁇ 3° C. temperature conditions, and was stable to 97% or more at 40 ⁇ 2° C. temperature and 75 ⁇ 5% relative humidity conditions. . It was also found that the level was similar to that of Example 1 ( FIGS. 10 , 11 and 12 ).
  • Table 31 shows the measurement results of high molecular weight components (pre-peak %) by size exclusion chromatography.
  • Example 7 0.48 0.45 0.53 0.68 0.51 0.60 0.85 0.52 0.61 0.81 0.47 0.57 0.79
  • Example 8 0.48 0.41 0.49 0.63 0.51 0.59 0.82 0.46 0.65 0.80 0.44 0.53 0.82
  • Example 9 0.41 0.40 0.45 0.66 0.50 0.52 0.78 0.46 0.63 0.89 0.64 0.50 0.68
  • Example 10 0.66 0.49 0.60 0.77 0.59 0.73 0.97 0.55 0.74 0.93 0.52 0.66 0.75
  • Example 11 0.57 0.47 0.55 0.71 0.52 0.63 0.88 0.49 0.64 0.87 0.47 0.59 0.74
  • Example 12 0.49 0.45 0.49 0.67 0.49 0.58 0.85 0.
  • the high molecular weight component content of Examples 7 to 16 is stable to 1.0% or less at 5 ⁇ 3° C. temperature conditions, and is stable to 2.0% or less at 40 ⁇ 2° C. temperature and 75 ⁇ 5% relative humidity conditions. could In addition, it was found that the level was similar to the result of Example 1.
  • Table 32 below shows the measurement results of low molecular weight components (post-peak %) by size exclusion chromatography.
  • Example 7 0.05 0.05 0.12 0.37 0.04 0.12 0.62 0.05 0.20 0.96 0.07 0.04 0.09
  • Example 8 0.07 0.06 0.10 0.37 0.05 0.12 0.64 0.10 0.21 0.99 0.08 0.05 0.09
  • Example 9 0.06 0.06 0.09 0.58 0.05 0.12 0.77 0.06 0.22 1.29 0.07 0.04 0.13
  • Example 10 0.07 0.05 0.11 0.36 0.05 0.13 0.60 0.08 0.21 0.99 0.08 0.04 0.10
  • Example 11 0.06 0.06 0.11 0.39 0.04 0.12 0.62 0.07 0.21 0.95 0.08 0.06 0.11
  • Example 12 0.07 0.05 0.09 0.38 0.04 0.12 0.63 0.
  • Example 32 As shown in Table 32, it can be seen that the low molecular weight component content of Examples 7 to 16 is stable to 0.5% or less at 5 ⁇ 3° C. temperature conditions, and is stable to 1.5% or less at 40 ⁇ 2° C. temperature and 75 ⁇ 5% relative humidity conditions. could In addition, it was found that the level was similar to the result of Example 1.
  • Table 33 below shows the measurement results of the intact immunoglobulin G content (non-reducing chip-based CE-SDS).
  • Example 7 98.15 97.86 98.09 97.80 98.41 98.69 98.67 97.52 97.50 96.59 97.86 98.77 98.62
  • Example 8 98.17 97.90 97.90 97.68 98.43 98.74 98.75 97.61 97.32 96.58 97.74 98.95 98.80
  • Example 9 98.24 97.99 97.46 97.46 98.43 98.77 98.68 97.62 97.52 96.25 97.74 98.91
  • Table 34 below shows the measurement results of the antibody light chain and heavy chain content (reduced chip-based CE-SDS).
  • Table 35 shows the measurement results of the charge variant (main peak %).
  • Example 7 62.29 62.36 62.29 57.88 63.32 62.39 54.34 62.61 62.06 50.73 61.53 63.01 61.81
  • Example 8 62.34 62.61 62.52 58.48 63.37 63.00 54.59 62.91 63.12 49.88 61.51 63.07 61.81
  • Example 9 62.33 62.62 62.03 57.44 62.97 62.12 52.83 62.53 62.34 49.59 61.50 63.07 61.91
  • Example 10 Example 10
  • Table 36 shows the measurement results of the charge variant (acid peak %).
  • Table 37 shows the measurement results of charge variants (basic peak %).
  • Example 7 23.66 23.37 23.01 21.41 23.10 22.23 17.36 23.26 22.08 16.42 24.11 22.86 23.13
  • Example 8 23.53 23.27 22.53 19.99 22.95 21.34 16.30 22.93 21.06 15.83 24.02 22.71 23.09
  • Example 9 23.58 23.41 23.22 21.18 23.12 22.39 17.94 23.36 22.02 16.19 24.01 23.07 23.59
  • Example 10 24.20 23.21 22.41 19.07 22.86 21.06 15.61 23.07 20.69 15.18 23.78 22.59 22.62
  • Example 11 24.20 23.21 22.41 19.07 22.86 21.06 15.61 23.07 20.69 15.18 23.78 22.59 22.62
  • Example 11 24.20 23.21 22.41 19.07 22.86 21.06 15.61 23.07 20.69 15.18 23.78 22.59 22.62
  • Example 11 24.20 23.21 22.41 19.
  • Table 38 shows the oxidation rate oxidation (Met 263) measurement results.
  • Example 7 2.4 2.7 3.4 6.8 4.4
  • Example 8 2.7 2.8 3.4 7.0 4.9
  • Example 9 2.4 2.5 2.7 4.4 4.4
  • Example 10 2.4 2.8 3.9 8.4 4.8
  • Example 11 2.3 3.5 3.3 7.5 5.3
  • Example 12 2.3 2.7 5.4 8.3 5.0
  • Example 13 2.4 2.7 4.7 8.0 5.1
  • Example 14 2.4 2.8 3.8 7.7 4.5
  • Example 15 2.4 2.8 3.7 7.9 4.3
  • Example 16 2.4 2.8 4.0 8.4 5.2
  • Example 1 2.4 2.7 3.5 6.8 4.9
  • Table 39 below shows the measurement results of SARS-CoV-2 RBD binding affinity (ELISA).
  • Example 7 101 99 98 97 106 94 96 108 102 105 107
  • Example 8 100 97 106 95 104 96 101 100 105 102 107
  • Example 9 104 107 103 98 106 105 94 107 112 114
  • Example 10 101 102 98 101 96 96 99 98 107 104 106
  • Example 11 104 100 106 101 101 99 99 103 99 98 102
  • Example 12 97 100 102 101 99 105 99 108 107 113 97
  • Example 14 108 106 106 102 104 86 101 100 98 98 98
  • Table 40 shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Table 41 shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Table 42 below shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ (um)) measured by HIAC.
  • Table 43 below shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ (um)) measured by HIAC.
  • Example 1 16 mL of the stable liquid pharmaceutical formulation of Example 1 prepared by the method of Experimental Example 1 was stored in an airtight container at 5 ⁇ 3° C./ambient relative humidity. Stability was measured after 1 month, 2 months and 3 months at the above temperature and humidity.
  • Example 1 was found to be stable because the appearance was maintained without any change in appearance after 3 months at 5 ⁇ 3 °C.
  • Example 1 was found to be stable because it was kept constant without a change in the antibody concentration after 3 months at 5 ⁇ 3 °C.
  • Table 46 below shows the measurement results of the antibody light chain and heavy chain content (reduced CE-SDS).
  • Example 1 was found to be stable because it was kept constant without change in the antibody light chain and heavy chain content after 3 months at 5 ⁇ 3 °C.
  • Table 47 below shows the measurement results of intact immunoglobulin G content (non-reduced CE-SDS).
  • Example 1 was found to be stable since it was maintained at 5 ⁇ 3° C. for 3 months without change in the intact immunoglobulin G content.
  • Table 48 below shows the measurement results of SEC-HPLC (main component, high molecular weight component, low molecular weight component content).
  • Example 1 was found to be stable because it was kept constant without changes in the main component, high molecular weight component, and low molecular weight component content measured by SEC-HPLC after 3 months at 5 ⁇ 3 °C.
  • IEC-HPLC (main peak, acidic peak, and basic peak) measurement results are shown in Table 49 below.
  • Example 1 was found to be stable because it was kept constant without changes in the main peak, acidic peak, and basic peak contents measured by IEC-HPLC after 3 months at 5 ⁇ 3°C.
  • Table 50 below shows the results of SARS-CoV-2 RBD binding affinity measurement.
  • Example 1 was found to be stable because the SARS-CoV-2 RBD binding affinity was kept constant without change after 3 months at 5 ⁇ 3°C.
  • each buffer was prepared to suit each pH, and arginine monohydrochloride, sorbitol and trehalose were added thereto, an antibody was added thereto, and a surfactant was added to the sample of Table 51 were manufactured.
  • the prepared pharmaceutical formulation was stored at a temperature of 5 ⁇ 3° C. and 50 ⁇ 2° C., and stability after 5 days at a temperature of 5 ⁇ 3° C. and stability after 3 days and 5 days at a temperature of 50 ⁇ 2° C. were measured.
  • freezing and thawing were repeated 5 times at -40°C, and shaking stress was applied at room temperature at 3000 rpm for 4 hours.
  • the results of the experiment are shown in Tables 52 to 64 and FIGS. 18 to 22 .
  • Antibodies used in Experimental Example 5 were No. 1 described in Tables 3 to 4 above. It is the 322 bond molecule.
  • Table 53 below shows the measurement results of the antibody light chain and heavy chain content (reduced chip-based CE-SDS).
  • Table 54 below shows the measurement results of intact immunoglobulin G content (non-reducing chip-based CE-SDS).
  • Example 17 showed a lower content of intact immunoglobulin G compared to Examples 18 and 19 at 50 ⁇ 2° C. ( FIG. 19 ).
  • Table 55 shows the measurement results of charge variants (main peak %).
  • Table 57 shows the measurement results of charge variants (basic peak %).
  • Table 58 shows the measurement results of the main component content (Main peak %).
  • Example 17 showed a lower main component content than Examples 18 and 19 under the conditions of 50 ⁇ 2 °C.
  • the main component content due to freezing/thawing stress and shaking stress was similar to Examples 17 to 19, and it was found that it was stable at 99.0% or more ( FIG. 20 ).
  • Table 59 shows the measurement results of the high molecular weight component content (pre-peak %).
  • Example 17 was higher than that of Examples 18 and 19 at 50 ⁇ 2° C. ( FIG. 21 ).
  • Table 60 shows the measurement results of the low molecular weight component content (post-peak %).
  • Example 17 the low molecular weight component of Example 17 was higher than that of Examples 18 and 19 under the conditions of 50 ⁇ 2° C. ( FIG. 22 ).
  • Table 61 shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Table 62 shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Table 63 shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ (um)) measured by HIAC.
  • Table 64 shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ (um)) measured by HIAC.
  • each buffer solution (acetate, citrate, succinate, phosphate) was prepared according to each pH, and arginine monohydrochloride, sodium chloride, sucrose and glycine were added thereto. After addition of the antibody, it was concentrated. Thereafter, surfactants (polysorbates 20 and 80, poloxamer 188) were added (however, Comparative Example 1 was not added) to prepare the samples in Table 65.
  • Example 1 is a formulation finally selected according to Experimental Example 1. The prepared pharmaceutical formulation was stored at 5 ⁇ 3° C. temperature, 40 ⁇ 2° C. temperature, and 75 ⁇ 5% relative humidity, and stability after 2 weeks and 4 weeks at each temperature condition was measured. For physical stress, shaking stress was applied at 3000 rpm for 4 hours at room temperature. The results are shown in Tables 66 to 75 and FIGS. 23 to 25 .
  • the antibody used in Experimental Example 6 was No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
  • Example 20 Acetate 10 mM 5.5 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL Example 21 Citrate 10 mM 5.5 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL Example 22 Citrate 10 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL Example 23 Succinate 10 mM 6.5 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL Example 24 Phosphate 10 mM 6.0 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL Example 25 Phosphate 10 mM 6.5 L-Arginine monohydrochloride 150 mM Polysorbate 800.05% (w/v) 60mg/mL Example
  • Example 20 0.0098 0.0124 0.0072 0.0098 0.0112 0.0096
  • Example 21 0.0118 0.0084 0.0091 0.0099 0.0137 0.0135
  • Example 22 0.0134 0.0091 0.0134 0.0110 0.0090 0.0167
  • Example 23 0.0121 0.0086 0.0093 0.0076 0.0086 0.0430
  • Example 24 0.0122 0.0074 0.0083 0.0091 0.0087 0.0113
  • Example 25 0.0059 0.0071 0.0089 0.0088 0.0101 0.0145
  • Example 26 0.0131 0.0104 0.0114 0.0083 0.0146 0.0294
  • Example 27 0.0051 0.0049 0.0027 0.0013 0.0019 0.0023
  • Example 28 0.0020 0.0033 0.0025 0.0088 0.0028 0.0041
  • Example 29 0.0083 0.0090 0.0089 0.0099 0.0085 0.0105
  • Example 30 0.0091
  • Table 67 shows the measurement results of intact immunoglobulin G content (non-reduced CE-SDS).
  • Example 20 98.66 98.23 97.61 97.92 97.22 98.59
  • Example 21 98.65 98.24 97.62 98.00 97.26 98.59
  • Example 22 98.61 98.26 97.49 97.92 97.73 98.63
  • Example 23 98.49 98.22 97.73 98.03 97.69 98.60
  • Example 24 98.64 98.20 97.70 97.92 97.53 98.55
  • Example 25 98.64 98.22 97.62 97.97 96.99 98.50
  • Example 26 98.61 98.21 97.69 98.04 96.92 98.58
  • Example 27 98.61 98.22 97.59 98.00 97.28 98.44
  • Example 21 98.65 98.24 97.62 98.00 97.26 98.59
  • Example 22 98.61
  • Examples 20 to 31 did not differ from Example 1 in the intact immunoglobulin G content after 4 weeks at 5 ⁇ 3° C. and 40 ⁇ 2° C. and 75 ⁇ 5% relative humidity conditions. In addition, it was confirmed that there was no decrease in the effect of shaking stress (FIG. 23).
  • Table 68 shows the measurement results of the antibody light chain and heavy chain content (reduced CE-SDS).
  • Examples 20 to 31 were stable because they were kept constant without change in the light chain and heavy chain contents of the antibody after 4 weeks at 5 ⁇ 3 ° C, 40 ⁇ 2 ° C temperature, and 75 ⁇ 5% relative humidity conditions. knew that it was In addition, it was confirmed that it is stable against shaking stress (FIG. 24).
  • Examples 20 to 31 were stable with a main component content of 99.0% or more after 4 weeks at 5 ⁇ 3°C. Although a decrease in the main component content was seen at 40 ⁇ 2 ° C. temperature and 75 ⁇ 5% relative humidity conditions, it was confirmed that the main component content was stable at 98.0% or more in all examples after 4 weeks, and there was no significant difference from Example 1. could (Fig. 25).
  • Table 70 shows the measurement results of the high molecular weight component content (pre-peak %).
  • Example 20 0.29 0.36 0.60 0.35 0.66 0.31
  • Example 21 0.29 0.37 0.64 0.36 0.67 0.31
  • Example 22 0.31 0.39 0.67 0.38 0.75 0.32
  • Example 23 0.31 0.39 0.71 0.39 0.78 0.36
  • Example 24 0.34 0.43 0.71 0.44 0.82 0.36
  • Example 25 0.39 0.50 0.86 0.51 0.96 0.42
  • Example 26 0.39 0.49 0.91 0.50 1.00 0.40
  • Example 27 0.36 0.40 0.67 0.40 0.94 0.37
  • Example 28 0.34 0.42 0.61 0.43 0.66 0.34
  • Example 29 0.36 0.40 0.66 0.42 0.76 0.35
  • Example 30 0.35 0.39 0.65 0.40 0.71 0.33
  • Example 31 0.31 0.43 0.61 0.40 0.66 0.32
  • Example 1 0.35 0.40 0.66 0.42 0.80 0.34 Comparative Example 1 0.33 0.38 0.66 0.40 0.72 0.34
  • Example 20 to 31 were stable at 1.0% or less of high molecular weight components after 4 weeks and after shaking stress under all temperature conditions. In addition, it was found that there was no significant difference from the results of Example 1.
  • Table 71 shows the measurement results of the content of low molecular weight components (post-peak %).
  • Example 20 0.07 0.06 0.61 0.08 0.98 0.08
  • Example 21 0.06 0.06 0.58 0.08 0.93 0.08
  • Example 22 0.07 0.06 0.35 0.08 0.58 0.06
  • Example 23 0.06 0.06 0.34 0.08 0.54 0.06
  • Example 24 0.06 0.06 0.32 0.07 0.56 0.06
  • Example 25 0.05 0.06 0.31 0.08 0.55 0.04
  • Example 26 0.07 0.06 0.36 0.08 0.61 0.07
  • Example 27 0.05 0.06 0.29 0.07 0.48 0.07
  • Example 28 0.08 0.06 0.26 0.07 0.47 0.04
  • Example 29 0.07 0.06 0.34 0.08 0.58 0.06
  • Example 30 0.08 0.06 0.33 0.08 0.58 0.04
  • Example 31 0.04 0.06 0.32 0.07 0.56 0.04
  • Example 1 0.07 0.06 0.35 0.08 0.61 0.05 Comparative Example 1 0.06 0.06 0.33 0.57 0.06
  • Example 20 to 31 were stable at 1.0% or less of low molecular weight components after 4 weeks and after shaking stress in all temperature conditions. In addition, it was found that there was no significant difference from the results of Example 1.
  • Table 72 shows the measurement results of the number of insoluble foreign particles (10.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Table 73 shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ m ⁇ , ⁇ 100.00 ⁇ m) measured by MFI.
  • Example 20 2 2 4 2 4 25
  • Example 21 0 0 0 2 2 15
  • Example 22 2 2 6 0 0 28
  • Example 23 0 0 2 4 0 9
  • Example 24 0 0 2 0 4 100
  • Example 25 0 2 0 5 2 0
  • Example 26 2 0 0 0 2 78
  • Example 27 0 0 0 4 0 2
  • Example 28 0 3 2 0 4 21
  • Example 30 0 2 0 4 20 9
  • Example 31 0 0 0 2 0 68
  • Example 20 3 0 23 65 2 2
  • Example 21 3 2 2 2 5 0
  • Example 22 2 0 5 2 3 2
  • Example 23 0 0 28 2 0 2
  • Example 24 0 0 0 2 0 50
  • Example 25 75 2 12 8 2 0
  • Example 26 0 8 12 2 3 18
  • Example 27 7 15 10 20 18 15
  • Example 29 0 0 2 0 5 7
  • Example 30 3 3 3 2 17 365
  • Example 31 0 2 12 2 0 153
  • Example 1 2 0 5 0 8 95 Comparative Example 1 5 0 0 13 2 24603
  • Table 75 shows the measurement results of the number of insoluble foreign particles (25.00 ⁇ (um)) measured by HIAC.
  • Example 20 2 0 3 5 0 2
  • Example 21 0 0 0 0 0 0 0
  • Example 22 2 0 0 0 0 0
  • Example 23 0 0 10 0 0 0
  • Example 24 0 0 0 0 0
  • 32 Example 25
  • Example 26 0 0 10 0 0 0
  • Example 27 0 2 0 2 0 0
  • Example 28 2 0 2 7 0 8
  • Example 29 0 0 0 0 0 0
  • Example 30 0 2 0 0 3 3
  • Example 31 0 0 8 0 0 18
  • Example 1 0 0 2 0 0 0 16195

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Inorganic Chemistry (AREA)
  • Pulmonology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to a stable pharmaceutical formulation and, more particularly, to a pharmaceutical formulation comprising a binding molecule with respect to the spike protein (S protein) of the SARS-coronavirus-2 surface. The stable pharmaceutical formulation according to the present invention has excellent long-term storage stability under temperature conditions of accelerated conditions and harsh conditions, and can maintain excellent stability even under physical stress conditions such as light, freezing/thawing, and shaking.

Description

안정한 약제학적 제제stable pharmaceutical formulations
본 발명은 안정한 약제학적 제제에 관한 것으로, 보다 구체적으로는, 사스-코로나바이러스-2 표면의 스파이크 단백질(Spike protein, S protein)에 대한 결합 분자를 안정하게 보존할 수 있는 약제학적 제제에 관한 것이다.The present invention relates to a stable pharmaceutical preparation, and more particularly, to a pharmaceutical preparation capable of stably preserving a molecule binding to a spike protein (S protein) on the surface of SARS-coronavirus-2 .
사스-코로나바이러스-2(severe acute respiratory syndrome coronavirus 2, SARS-CoV-2)는 유전적 배열(DNA sequencing)상 전도 기능(Positive sense)이 있는 단일 가닥 RNA(single-stranded RNA) 코로나바이러스이다. SARS-CoV-2는 인간에게 전염성이 있고 코로나바이러스감염증-19(coronavirus disease 2019, COVID-19)의 원인이다. COVID-19의 최초 발생지는 중국 후베이성의 우한시이다. SARS-coronavirus-2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is a single-stranded RNA coronavirus with a positive sense in genetic sequencing (DNA sequencing). SARS-CoV-2 is contagious to humans and is the cause of coronavirus disease 2019 (COVID-19). The first outbreak of COVID-19 was in Wuhan, Hubei Province, China.
SARS-CoV-2에 감염된 사람들은 열, 기침, 호흡 곤란, 설사와 같이 경증 내지 중증의 증상을 보일 수 있다. 합병증이나 병을 가진 사람들, 노인은 사망할 가능성이 크다.People infected with SARS-CoV-2 may have mild to severe symptoms such as fever, cough, shortness of breath, and diarrhea. People with complications or diseases and the elderly are more likely to die.
특히 심장질환 및 당뇨병 등의 기저질환 보유자가 감염에 더 취약하며, 합병증이나 장기 손상 등을 겪기 때문에 조기 발견과 치료가 매우 중요하다. 2019년 12월 8일부터 2020년 3월 20일 현재까지 245,550명의 환자가 발생하였고, 그 중 10,049명이 사망하여 치사율은 4.09%에 달한다(WHO). 2020년 현재까지 한국을 포함한 177개국에서 발생하였다. In particular, people with underlying diseases such as heart disease and diabetes are more susceptible to infection and suffer complications or organ damage, so early detection and treatment are very important. From December 8, 2019 to March 20, 2020, there were 245,550 cases, of which 10,049 died, resulting in a fatality rate of 4.09% (WHO). As of 2020, it has occurred in 177 countries including Korea.
현재 코로나바이러스감염증-19(coronavirus disease 2019, COVID-19)의 치료제는 없고, 기존 치료제를 환자에게 투여하여 치료 효과를 기대하고 있는 실정이다. 에볼라 치료제 혹은 치료 후보 물질인 항바이러스제 파비피라비르 (favipiravir), 렘데시비르 (remdesivir), 갈리데시비어 (galidesivir)와 C형 간염 치료제인 리바비린 (ribavirin) 등을 COVID-19 치료제로 사용하고 있다. 또한, 말라리아치료제 클로로퀸 (Chloroquine)도 COVID-19에 치료 효과를 보이는 것으로 나타나 공개 임상시험 진행 중에 있다. 그러나 C형 간염 치료제인 리바비린은 빈혈과 같은 부작용이 심할 수 있고, 항바이러스제인 인터페론 (interferon)도 여러 가지 부작용을 우려하여 주의해서 사용할 것을 권고하고 있다. Currently, there is no treatment for coronavirus disease 2019 (COVID-19), and the existing treatment is administered to the patient to expect a therapeutic effect. Antiviral agents favipiravir, remdesivir, and galidesivir, which are Ebola treatment or treatment candidates, and hepatitis C treatment ribavirin are being used as COVID-19 treatments. In addition, the antimalarial drug Chloroquine has been shown to have a therapeutic effect on COVID-19 and is undergoing public clinical trials. However, hepatitis C treatment ribavirin may have severe side effects such as anemia, and the antiviral drug interferon is also recommended to be used with caution due to concerns about various side effects.
비록 이러한 약물들이 COVID-19 환자 치료에 활용되어 치료 효과를 보고 있지만, 아직 어떠한 근거로 치료 효과를 내는지는 아직 명확히 입증되지 않았다. 중국에서 COVID-19 회복 환자의 현장을 주입하는 혈장 요법을 시행하여 중증 환자의 치료에 효과를 보였다고 발표하였으나, 치료 효과가 불분명하고 불확실성이 크다.Although these drugs have been used to treat COVID-19 patients and are seeing therapeutic effects, it has not yet been clearly demonstrated on what basis they have therapeutic effects. In China, it was announced that the on-site injection of plasma therapy for patients recovering from COVID-19 was effective in the treatment of severely ill patients, but the therapeutic effect is unclear and there is great uncertainty.
한국의 경우, COVID-19 중앙임상 TF(테스크포스)가 2020년 2월 13일 COVID-19의 치료 원칙을 마련하여, 1차 치료제로 에이즈 치료제인 칼레트라 (Kaletra), 말라리아치료제인 클로로퀸과 하이드록시클로로퀸(Hydroxychloroquine)을 권하며, 리바비린과 인터페론은 부작용을 우려해 1차 치료제로 권하지 않기로 발표했다. 경증이거나 젊은 환자, 발병 10일이 지난 경우에는 항바이러스제를 투여하지 않아도 증상이 호전된다고 판단하고, 고령자, 기저질환자, 중증 환자에게는 항바이러스 치료제를 투여하기로 합의했다. In Korea, the COVID-19 Central Clinical Task Force (Task Force) prepared the treatment principle for COVID-19 on February 13, 2020, and as the first-line treatment, AIDS treatment Kaletra, malaria treatment chloroquine and hydroxy Chloroquine (Hydroxychloroquine) is recommended, and ribavirin and interferon are not recommended as first-line treatment due to concerns about side effects. In mild or young patients, if 10 days have passed since the onset of the disease, it was judged that the symptoms improved even if antiviral drugs were not administered.
미국 CDC는 i) COVID-19가 계절성 유행 바이러스가 아닌 메르스처럼 토착화되어 감염을 일으킬 수 있다고 발표하였고, ii) 바이러스가 올해 또는 내년 어느 시점에 커뮤니티로 전파될 수 있으며, 비록 코로나 바이러스가 실제 커뮤니티에 잠복되어 있다는 증거는 없으나, 데이터 기반으로 결론을 내릴 수 있도록 감시강화 필요성을 언급하였다(2020.2.13).The U.S. CDC has announced that i) COVID-19 can become indigenous and cause infections like MERS rather than a seasonal virus, and ii) the virus may spread to communities at some point this year or next year, even though the coronavirus is Although there is no evidence that it is hidden in the
한국 질병관리본부(현, 질병관리청)는 i) COVID-19도 인플루엔자처럼 장기적으로 유행할 수 있다고 판단하여, 인플루엔자와 같이 감시 체계에 포함하겠다고 발표하였고, ii) 사람 사이에 유행하는 코로나바이러스(4종)도 겨울~봄에 유행하고 있어서 COVID-19도 토착화될 수 있다는 가능성을 열어두고 있다(2020.2.17).The Korea Centers for Disease Control and Prevention (currently, the Korea Centers for Disease Control and Prevention) announced that i) that COVID-19 could also spread like influenza for a long time and that it would be included in the surveillance system like influenza, and ii) a coronavirus that spreads among people (4 species) is also prevalent in winter and spring, leaving open the possibility that COVID-19 may also become indigenous (2020.2.17).
사스나 메르스와는 다르게 COVID-19의 세계적 유행 (pandemic) 현실화에 대한 우려가 있지만 봄 이후 (4월) 소강 상태가 될 가능성도 있어, 추이를 보며 신중하게 접근하는 전문가들이 많다. 아직 코로나19에 대한 정보 부족으로 전문가들도 추후 전개 양상에 대해서는 의견이 분분하나, 단시일 내에 해결되리라 전망하는 전문가는 거의 없다. COVID-19의 유행 양상과 특징이 정확히 분석되고 이번 COVID-19로 인한 위기 상황이 얼마나 지속되는지에 영향을 받겠지만, 무증상 감염자가 전세계에 퍼지게 될 경우 풍토병화 될 가능성에 대한 우려가 있다. 세계적으로 COVID-19 재발병 가능성에 대한 대응책 마련이 시급하다. Unlike SARS and MERS, there are concerns about the realization of a global pandemic of COVID-19, but there is a possibility that there may be a lull after spring (April). Due to the lack of information on COVID-19, experts also have differing opinions about the future development, but few experts predict that it will be resolved in a short time. Although the pattern and characteristics of the COVID-19 epidemic will be accurately analyzed and will be affected by how long the crisis caused by this COVID-19 will last, there are concerns about the possibility that asymptomatic infections may become endemic if they spread around the world. It is urgent to prepare countermeasures against the possibility of a recurrence of COVID-19 worldwide.
SARS-CoV-2 바이러스의 스파이크 단백질(Spike protein, S protein)을 표적으로 하는 결합 분자를 보다 안정하게 보존할 수 있는 제형은 아직까지 개발되지 않았다. 이에 따라, 본 발명자들은 안정성이 우수한 제형을 개발하고자 지속적인 연구를 거듭한 결과, 본 발명을 최종 완성하였다. A formulation capable of more stably preserving a binding molecule targeting the SARS-CoV-2 virus spike protein (S protein) has not yet been developed. Accordingly, as a result of continuous research to develop a formulation having excellent stability, the present inventors have finally completed the present invention.
본 발명자들은 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자를 안정하게 보존할 수 있는 약제학적 제제를 개발하였다. The present inventors have developed a pharmaceutical preparation capable of stably preserving a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2.
이에, 본 발명이 해결하고자 하는 과제는 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자를 포함하는, 안정한 약제학적 제제, 바람직하게는 장기 보관시 안정한 약제학적 제제를 제공하는 것이다.Accordingly, the problem to be solved by the present invention is a stable pharmaceutical preparation, preferably comprising a neutralizing binding molecule that binds to the spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2) The goal is to provide a pharmaceutical formulation that is stable for long-term storage.
상기 과제를 해결하고자, 본 발명은 (A) 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자, (B) 완충제, (C) 안정화제 및 (D) 계면활성제를 포함하는 안정한 약제학적 제제를 제공한다. In order to solve the above problems, the present invention provides (A) a neutralizing binding molecule that binds to the spike protein (S protein) on the surface of SARS-coronavirus-2 (SARS-CoV-2), (B) a buffer, (C ) a stabilizer and (D) a surfactant.
항체 등의 중화 결합 분자는 통상적인 유기 및 무기 약물에 비해 크고 복잡한 구조를 가진다. 그러므로, 항체 등의 중화 결합 분자를 포함하는 약제학적 제제는 항체 등의 중화 결합 분자의 안정성을 유지해야 하는 특별한 문제를 지니고 있다. Neutralizing binding molecules such as antibodies have larger and more complex structures than conventional organic and inorganic drugs. Therefore, a pharmaceutical preparation containing a neutralizing-binding molecule such as an antibody has a special problem in that the stability of the neutralizing-binding molecule, such as an antibody, must be maintained.
항체 등의 중화 결합 분자의 안정성은 이온 강도, pH, 온도, 동결/해동의 반복 주기, 항체 등의 중화 결합 분자 농도와 같은 인자들에 의해 영향을 받을 수 있다. 활성 항체 등의 중화 결합 분자는 변성, 응집(용해성 및 불용성 응집물의 형성), 침전 및 흡착을 포함하는 물리적 불안정성 또는 라세미화, 베타-제거, 이황화 교환, 가수분해, 탈아미드화 및 산화를 포함하는 화학적 불안정성에 의하여 손실이 발생할 수 있다. 이러한 물리적 및 화학적 불안정성은 잠재적으로 항체 등의 중화 결합 분자의 저하된 생물학적 활성, 증가된 독성 및/또는 증가된 면역원성을 갖는 항체 등의 중화 결합 분자 부산물 또는 유도체의 형성을 초래할 수 있다.The stability of a neutralizing binding molecule, such as an antibody, may be affected by factors such as ionic strength, pH, temperature, repeated freeze/thaw cycles, and concentration of a neutralizing binding molecule, such as an antibody. Neutralizing binding molecules, such as active antibodies, exhibit physical instability including denaturation, aggregation (formation of soluble and insoluble aggregates), precipitation and adsorption, or physical instability, including racemization, beta-elimination, disulfide exchange, hydrolysis, deamidation and oxidation. Loss may occur due to chemical instability. Such physical and chemical instability can potentially result in the formation of neutralizing binding molecule byproducts or derivatives, such as antibodies, with reduced biological activity, increased toxicity, and/or increased immunogenicity of the neutralizing binding molecule, such as an antibody.
항체 등의 중화 결합 분자의 안정한 액체 약제학적 제제의 개발에 사용될 수 있는 다양한 부형제들이 제시되고 있지만, 항체 등의 중화 결합 분자마다 고유의 물리화학적인 성질을 가지기 때문에, 다른 항체 등의 중화 결합 분자를 사용한 종래의 제제를 고려해 특정 항체 등의 중화 결합 분자가 가지는 불안정성 문제를 극복하기 위한 부형제의 종류 및 양을 예측하는 것은 실질적으로 불가능하다. 또한, 특정 항체 등의 중화 결합 분자를 약제학적 제제 내에서 화학적으로 그리고 생물학적으로 안정하게 유지시키기 위한 항체 등의 중화 결합 분자의 농도, pH, 부형제의 농도 등의 최적 조건을 도출하는 것은 어려우며 많은 시간과 노력이 필요하다.Although various excipients that can be used for the development of stable liquid pharmaceutical formulations of neutralizing-binding molecules such as antibodies have been proposed, each neutralizing-binding molecule such as an antibody has its own physicochemical properties, so neutralizing-binding molecules such as other antibodies have been proposed. It is practically impossible to predict the type and amount of excipients to overcome the problem of instability of a neutralizing binding molecule such as a specific antibody in consideration of the conventional formulation used. In addition, it is difficult and time-consuming to derive optimal conditions such as the concentration of neutralizing-binding molecules such as antibodies, pH, and concentration of excipients for maintaining chemically and biologically stable neutralizing-binding molecules such as specific antibodies in pharmaceutical formulations and effort are required.
현재, 사스-코로나바이러스-2 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자를 포함하는 안정한 약제학적 제제에 관한 선행 기술은 존재하지 않는다. 본 발명은 사스-코로나바이러스-2 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자와 완충제, 안정화제 및 계면활성제를 함께 포함하는 약제학적 제제 내에서 상기 중화 결합 분자의 고분자량 성분 및 불용성 이물의 숫자 형성을 감소시켜 이의 활성을 안정화 시키는 효과가 있음을 최초로 확인하였다.Currently, there is no prior art relating to a stable pharmaceutical preparation comprising a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-coronavirus-2. The present invention relates to a high molecular weight of the neutralizing binding molecule in a pharmaceutical formulation comprising a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-coronavirus-2 and a buffer, a stabilizer and a surfactant together. It was confirmed for the first time that there was an effect of stabilizing its activity by reducing the formation of the number of components and insoluble foreign substances.
이하 본 발명을 더욱 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명은 코로나바이러스(Coronavirus), 바람직하게는 사스-코로나바이러스-2(SARS-CoV-2), 더욱 바람직하게는 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자를 포함하는 안정한 약제학적 제제에 관한 것이다.The present invention relates to a spike protein on the surface of a coronavirus, preferably SARS-coronavirus-2 (SARS-CoV-2), more preferably SARS-coronavirus-2 (SARS-CoV-2). , S protein) to a stable pharmaceutical formulation comprising a neutralizing binding molecule that binds.
본 발명에 있어, 상기 결합 분자는 사스-코로나바이러스-2 표면의 스파이크 단백질의 RBD(Receptor binding domain) 영역에 결합할 수 있다.In the present invention, the binding molecule may bind to the receptor binding domain (RBD) region of the spike protein on the SARS-coronavirus-2 surface.
본 발명에 있어, 본 발명의 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein, S 단백질)은 서열번호: 3841의 서열로 구성되거나, 이를 포함하는 것일 수 있으며, 이들의 유도체 및/또는 변이체를 포함하나, 이에 한정되지 않는다.In the present invention, the SARS-coronavirus-2 (SARS-CoV-2) surface spike protein (S protein, S protein) of the present invention consists of or includes the sequence of SEQ ID NO: 3841. and derivatives and/or variants thereof, but are not limited thereto.
본 발명에 있어, 본 발명의 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질의 RBD(Receptor binding domain) 영역은 서열번호: 3842의 서열로 구성되거나, 이를 포함하는 것일 수 있으며, 이들의 유도체 및/또는 변이체를 포함하나, 이에 한정되지 않는다.In the present invention, the SARS-CoV-2 (SARS-CoV-2) surface spike protein region of the present invention may consist of or include the sequence of SEQ ID NO: 3842. , derivatives and/or variants thereof.
본 발명에 있어, 본 발명에 따른 약제학적 제제는 액상일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pharmaceutical formulation according to the present invention may be in a liquid form, but is not limited thereto.
본 발명의 일 구체예에서, 본 발명의 중화 결합 분자는 사스-코로나바이러스-2(SARS-CoV-2)의 스파이크 단백질에 우수한 결합능 및/또는 우수한 중화능을 나타낸다. 본 발명의 다른 구체예에서, 본 발명의 중화 결합 분자는 사스-코로나바이러스-2(SARS-CoV-2)의 스파이크 단백질에 돌연변이가 생긴 변이 바이러스에 우수한 결합능 및/또는 중화능을 나타낸다. 본 발명에 있어, 본 발명의 중화 결합 분자는 사스-코로나바이러스-2(SARS-CoV-2)의 스파이크 단백질의 RBD(Receptor binding domain) 영역에 결합하며 중화능 있는 결합 분자로 스크리닝된 결합 분자일 수 있다. 본 발명에 있어, 본 발명의 중화 결합 분자는 상기 RBD 영역 이외의 S 단백질의 다른 부위의 변이 바이러스에도 우수한 중화능을 나타낼 수 있으나, 이에 한정되는 것은 아니다. In one embodiment of the present invention, the neutralizing binding molecule of the present invention exhibits excellent binding ability and/or excellent neutralizing ability to the spike protein of SARS-CoV-2 (SARS-CoV-2). In another embodiment of the present invention, the neutralizing binding molecule of the present invention exhibits excellent binding and/or neutralizing ability to a mutant virus in which the spike protein of SARS-CoV-2 is mutated. In the present invention, the neutralizing binding molecule of the present invention is a binding molecule that binds to the receptor binding domain (RBD) region of the spike protein of SARS-CoV-2 and is screened as a binding molecule with neutralizing ability. can In the present invention, the neutralizing binding molecule of the present invention may exhibit excellent neutralizing ability against mutant viruses of other regions of the S protein other than the RBD region, but is not limited thereto.
세계보건기구(WHO)는 사스-코로나 바이러스-2를 유전자 염기서열 차이로 인한 아미노산 변화를 기준으로 6개 유형으로 분류하고 있다. 먼저 S, L 유형으로 분류되었다가 다시 L, V, G 유형으로 나뉘고 G가 GH와 GR로 나뉘면서 S, L, V, G, GH, GR 의 총 6개 유형으로 분류하고 있다. COVID-19 발생 초기에 중국 우한을 비롯한 아시아 지역에는 S와 V 유형이 유행하였고, 이후 대륙별로 서로 다른 유형이 발견되었다. 이 중 GH 유형이 전파력이 높게 나타날 가능성이 있다고 보고된바 있다. 국내의 경우 코로나바이러스 감염증 환자에서 채취한 유전자를 분류한 결과, 대부분은 유럽과 미국에서 유행한 G형의 변종인 GH형인 것으로 나타났고, 이유형은 바이러스 전파력이 높은 것으로 알려져 있다. 이 중 바이러스의 세포 내 침입 시 중요한 역할을 하는 스파이크 단백질의 614번 아미노산이 아스파트산 (D)에서 글리신 (G)으로 바뀐 G형의 바이러스는 3월 이후 유럽과 미국에서 급격히 증가해 현재는 거의 대부분 지역에서 나타나고 있다. 최근 보고된 바에 따르면 70여개 넘는 코로나바이러스 변이가 발생한 것으로 확인되었고, 전파력이 증가된 변이가 8개 (D614G 등), 중화항체를 회피하는 변이가 10개 (A841V 등), 혈장치료 효과가 낮은 변이 17개 (I472V 등)가 확인되었다.The World Health Organization (WHO) classifies SARS-Coronavirus-2 into six types based on amino acid changes due to differences in gene sequence. First, it was classified into S and L types, then again into L, V, and G types, and as G was divided into GH and GR, it is classified into a total of six types: S, L, V, G, GH, and GR. At the beginning of the COVID-19 outbreak, types S and V were prevalent in Asia including Wuhan, China, and after that, different types were discovered for each continent. Among them, it has been reported that the GH type has the potential to appear high in transmission power. In Korea, as a result of classifying the genes collected from patients with coronavirus infection, most of them were found to be the GH type, a variant of the G type prevalent in Europe and the United States. Among these, type G virus, in which amino acid 614 of the spike protein, which plays an important role in virus invasion, has been changed from aspartic acid (D) to glycine (G), has increased rapidly in Europe and the United States since March, and is now almost It appears in most areas. According to a recent report, more than 70 coronavirus mutations have been identified, 8 mutations with increased transmission power (D614G, etc.), 10 mutations that avoid neutralizing antibodies (A841V, etc.), mutations with low plasma treatment effect 17 (I472V, etc.) were identified.
본 발명에 있어, 본 발명의 중화 결합 분자는 SARS-CoV-2 바이러스 아미노산 변이 기준으로 S형(S 단백질의 614번 위치의 아미노산이 D), G형(S 단백질의 614번 위치의 아미노산이 G), V형 등의 균주(strain)에 중화능을 나타낼 수 있으나, 이 균주(strain)에 한정되는 것은 아니다. 상기 SARS-CoV-2 바이러스 S형의 일 예로는 BetaCoV/Korea/KCDC03/2020 균주가 있으나, 이 균주에 한정되는 것은 아니다. 상기 SARS-CoV-2 바이러스 G형의 일 예로는 hCoV-19/South Korea/KUMC17/2020 균주가 있으나, 이 균주에 한정되는 것은 아니다. 본 발명의 일 구체예에서, 본 발명의 중화 결합 분자는 사스-코로나바이러스-2(SARS-CoV-2)의 스파이크 단백질의 614번 아미노산 위치에서 D614G 변이가 일어난 변이 바이러스에도 우수한 중화능을 나타낸다. 일 실시예로, 본 발명의 중화 결합 분자는 사스-코로나바이러스-2(SARS-CoV-2)의 표면 단백질 (RBD)의 변이 단백질 A435S, F342L, G476S, K458R, N354D, V367F, V483A, 및 W436R에 대해 우수한 결합력을 나타낸다.In the present invention, the neutralizing binding molecule of the present invention is S-type (amino acid at position 614 of the S protein is D), G-type (the amino acid at position 614 of the S protein is G) based on the SARS-CoV-2 virus amino acid mutation. ), may exhibit neutralizing ability in strains such as V type, but is not limited to this strain. An example of the SARS-CoV-2 virus type S is the BetaCoV/Korea/KCDC03/2020 strain, but is not limited thereto. An example of the SARS-CoV-2 virus type G includes, but is not limited to, the hCoV-19/South Korea/KUMC17/2020 strain. In one embodiment of the present invention, the neutralizing binding molecule of the present invention exhibits excellent neutralizing ability even in a mutant virus in which D614G mutation occurs at amino acid position 614 of the spike protein of SARS-CoV-2 (SARS-CoV-2). In one embodiment, the neutralizing binding molecule of the present invention is SARS-coronavirus-2 (SARS-CoV-2) surface protein (RBD) mutant proteins A435S, F342L, G476S, K458R, N354D, V367F, V483A, and W436R It exhibits excellent bonding strength.
본 발명에 있어, 본 발명의 중화 결합 분자는 현재까지 단리된 사스-코로나바이러스-2 균주, 예를 들어 단리 일시와 장소를 알 수 없는 UNKNOWN-LR757996 균주(Strain), SARS-CoV-2/Hu/DP/Kng/19-027 균주; 2019년 12월 중국에서 단리된 Wuhan-Hu-1 균주; 2019년 12월 23일 최초로 중국에서 단리된 BetaCoV/Wuhan/IPBCAMS-WH-01/2019 균주; 2019년 12월 30일 중국에서 단리된 BetaCoV/Wuhan/IPBCAMS-WH-02/2019 균주, BetaCoV/Wuhan/IPBCAMS-WH-03/2019 균주, BetaCoV/Wuhan/IPBCAMS-WH-04/2019 균주, WIV02 균주, WIV04 균주, WIV05 균주, WIV06 균주, WIV07 균주; 2020년 1월 일본에서 단리된 2019-nCoV/Japan/TY/WK-521/2020 균주, 2019-nCoV/Japan/TY/WK-501/2020 균주, 2019-nCoV/Japan/TY/WK-012/2020 균주, 2019-nCoV/Japan/KY/V-029/2020 균주; 2020년 1월 대한민국에서 단리된 SNU01 균주; 대한민국에서 단리된 BetaCoV/Korea/KCDC03/2020 균주; 2020년 1월 1일 중국에서 단리된 BetaCoV/Wuhan/IPBCAMS-WH-05/2020 균주; 2020년 1월 2일 중국에서 단리된 2019-nCoV WHU02 균주, 2019-nCoV WHU01 균주; 2020년 1월 8일 중국에서 단리된 SARS-CoV-2/WH-09/human/2020/CHN 균주; 2020년 1월 10일 중국에서 단리된 2019-nCoV_HKU-SZ-002a_2020 균주; 2020년 1월 11일 중국에서 단리된 2019-nCoV_HKU-SZ-005b_2020 균주; 2020년 1월 17일 중국에서 단리된 SARS-CoV-2/Yunnan-01/human/2020/CHN 균주; 2020년 1월 19일 미국에서 단리된 2019-nCoV/USA-WA1/2020 균주; 2020년 1월 20일 중국에서 단리된 HZ-1 균주; 2020년 1월 21일 미국에서 단리된 2019-nCoV/USA-IL1/2020 균주; 2020년 1월 22일 미국에서 단리된 2019-nCoV/USA-CA2/2020 균주, 2019-nCoV/USA-AZ1/2020 균주; 2020년 1월 23일 미국에서 단리된 2019-nCoV/USA-CA1/2020 균주; 2020년 1월 25일 호주에서 단리된 Australia/VIC01/2020 균주; 2020년 1월 25일 미국에서 단리된 2019-nCoV/USA-WA1-F6/2020 균주, 2019-nCoV/USA-WA1-A12/2020 균주; 2020년 1월 27일 미국에서 단리된 2019-nCoV/USA-CA6/2020 균주; 2020년 1월 28일 미국에서 단리된 2019-nCoV/USA-IL2/2020 균주; 2020년 1월 29일 미국에서 단리된 2019-nCoV/USA-MA1/2020 균주, 2019-nCoV/USA-CA5/2020 균주, 2019-nCoV/USA-CA4/2020 균주, 2019-nCoV/USA-CA3/2020 균주; 2020년 1월 29일 핀란드에서 단리된 nCoV-FIN-29-Jan-2020 균주; 2020년 1월 29일 중국에서 단리된 SARS-CoV-2/IQTC02/human/2020/CHN 균주; 2020년 1월 31일 미국에서 단리된 2019-nCoV/USA-WI1/2020 균주; 2020년 1월 31일 타이완에서 단리된 SARS-CoV-2/NTU01/2020/TWN 균주; 2020년 2월 5일 타이완에서 단리된 SARS-CoV-2/NTU02/2020/TWN 균주; 2020년 2월 6일 미국에서 단리된 2019-nCoV/USA-CA7/2020 균주; 2020년 2월 7일 스웨덴에서 단리된 SARS-CoV-2/01/human/2020/SWE 균주; 2020년 2월 10일 미국에서 단리된 2019-nCoV/USA-CA8/2020 균주; 2020년 2월 11일 미국에서 단리된 2019-nCoV/USA-TX1/2020 균주; 2020년 2월 23일 미국에서 단리된 2019-nCoV/USA-CA9/2020 균주; 2020년 2월 28일 브라질에서 단리된 SARS-CoV-2/SP02/human/2020/BRA 균주; 단리 일시와 장소를 알 수 없는 UNKNOWN-LR757995, UNKNOWN-LR757997, UNKNOWN-LR757998, SARS-CoV-2/Hu/DP/Kng/19-020; 2020년 1월 일본에서 단리된 2019-nCoV/Japan/AI/I-004/2020; 2020년 1월 13일 네팔에서 단리된 SARS0CoV-2/61-TW/human/2020/ NPL; 2020년 2월 5일 중국에서 단리된 SARS-CoV-2/IQTC01/human/2020/CHN 균주; 대한민국에서 단리된 hCoV-19/South Korea/KUMC17/2020 균주와 향후 단리될 사스-코로나바이러스-2 균주에 대하여 중화 가능하나, 이들 균주에 한정되는 것은 아니다.In the present invention, the neutralizing binding molecule of the present invention is a SARS-coronavirus-2 strain isolated to date, for example, UNKNOWN-LR757996 strain (Strain), SARS-CoV-2/Hu of unknown date and place of isolation. /DP/Kng/19-027 strain; Wuhan-Hu-1 strain isolated from China in December 2019; BetaCoV/Wuhan/IPBCAMS-WH-01/2019 strain first isolated in China on December 23, 2019; BetaCoV/Wuhan/IPBCAMS-WH-02/2019 strain, BetaCoV/Wuhan/IPBCAMS-WH-03/2019 strain, BetaCoV/Wuhan/IPBCAMS-WH-04/2019 strain, WIV02 isolated on December 30, 2019 in China strain, WIV04 strain, WIV05 strain, WIV06 strain, WIV07 strain; 2019-nCoV/Japan/TY/WK-521/2020 strain isolated from Japan in January 2020, 2019-nCoV/Japan/TY/WK-501/2020 strain, 2019-nCoV/Japan/TY/WK-012/ 2020 strain, 2019-nCoV/Japan/KY/V-029/2020 strain; SNU01 strain isolated from Korea in January 2020; BetaCoV/Korea/KCDC03/2020 strain isolated from Korea; BetaCoV/Wuhan/IPBCAMS-WH-05/2020 strain isolated from China on January 1, 2020; 2019-nCoV WHU02 strain, 2019-nCoV WHU01 strain isolated on January 2, 2020 in China; SARS-CoV-2/WH-09/human/2020/CHN strain isolated from China on January 8, 2020; 2019-nCoV_HKU-SZ-002a_2020 strain isolated from China on January 10, 2020; 2019-nCoV_HKU-SZ-005b_2020 strain isolated from China on January 11, 2020; SARS-CoV-2/Yunnan-01/human/2020/CHN strain isolated from China on January 17, 2020; 2019-nCoV/USA-WA1/2020 strain isolated in the United States on January 19, 2020; HZ-1 strain isolated in China on January 20, 2020; 2019-nCoV/USA-IL1/2020 strain isolated in the United States on January 21, 2020; 2019-nCoV/USA-CA2/2020 strain, 2019-nCoV/USA-AZ1/2020 strain isolated in the United States on January 22, 2020; 2019-nCoV/USA-CA1/2020 strain isolated in the United States on January 23, 2020; Australia/VIC01/2020 strain isolated in Australia on 25 January 2020; 2019-nCoV/USA-WA1-F6/2020 strain, 2019-nCoV/USA-WA1-A12/2020 strain isolated in the United States on January 25, 2020; 2019-nCoV/USA-CA6/2020 strain isolated in the United States on January 27, 2020; 2019-nCoV/USA-IL2/2020 strain isolated in the United States on January 28, 2020; 2019-nCoV/USA-MA1/2020 strain, 2019-nCoV/USA-CA5/2020 strain, 2019-nCoV/USA-CA4/2020 strain, 2019-nCoV/USA-CA3 isolated in USA on January 29, 2020 /2020 strain; nCoV-FIN-29-Jan-2020 strain isolated on January 29, 2020 in Finland; SARS-CoV-2/IQTC02/human/2020/CHN strain isolated from China on January 29, 2020; 2019-nCoV/USA-WI1/2020 strain isolated in the United States on January 31, 2020; SARS-CoV-2/NTU01/2020/TWN strain isolated in Taiwan on January 31, 2020; SARS-CoV-2/NTU02/2020/TWN strain isolated on February 5, 2020 in Taiwan; 2019-nCoV/USA-CA7/2020 strain isolated in the United States on February 6, 2020; SARS-CoV-2/01/human/2020/SWE strain isolated on February 7, 2020 in Sweden; 2019-nCoV/USA-CA8/2020 strain isolated in the United States on February 10, 2020; 2019-nCoV/USA-TX1/2020 strain isolated in the United States on February 11, 2020; 2019-nCoV/USA-CA9/2020 strain isolated in the United States on February 23, 2020; SARS-CoV-2/SP02/human/2020/BRA strain isolated on February 28, 2020 in Brazil; UNKNOWN-LR757995, UNKNOWN-LR757997, UNKNOWN-LR757998, SARS-CoV-2/Hu/DP/Kng/19-020 of unknown date and place of isolation; 2019-nCoV/Japan/AI/I-004/2020 isolated from Japan in January 2020; SARS0CoV-2/61-TW/human/2020/NPL isolated from Nepal on January 13, 2020; SARS-CoV-2/IQTC01/human/2020/CHN strain isolated from China on February 5, 2020; It is possible to neutralize the hCoV-19/South Korea/KUMC17/2020 strain isolated in Korea and the SARS-coronavirus-2 strain to be isolated in the future, but is not limited to these strains.
(A) 중화 결합 분자(A) neutralizing binding molecule
본 발명에 있어, 본 발명의 중화 결합 분자는 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자일 수 있다.In the present invention, the neutralizing binding molecule of the present invention may be a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2.
본 발명의 중화 결합 분자는 하기 표 1의 결합 분자들로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있으며, 바람직하게는 하기 표 1의 결합 분자 No. 89 내지 No. 194, 및 No. 248로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있으며, 더욱 바람직하게는 하기 표 1의 No. 139 결합 분자일 수 있다. 하기 표 1에서 No.는 각 결합 분자의 번호를 의미한다.The neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 1, preferably binding molecule No. 89 to No. 194, and No. 248 may be any one binding molecule selected from the group consisting of, more preferably No. 139 binding molecule. In Table 1 below, No. means the number of each binding molecule.
Figure PCTKR2021010606-appb-img-000001
Figure PCTKR2021010606-appb-img-000001
Figure PCTKR2021010606-appb-img-000002
Figure PCTKR2021010606-appb-img-000002
Figure PCTKR2021010606-appb-img-000003
Figure PCTKR2021010606-appb-img-000003
Figure PCTKR2021010606-appb-img-000004
Figure PCTKR2021010606-appb-img-000004
Figure PCTKR2021010606-appb-img-000005
Figure PCTKR2021010606-appb-img-000005
본 발명에 있어서, 본 발명에 따른 가변영역의 CDR은 Kabat 등에 의해 고안된 시스템에 따라 통상적인 방법으로 결정되었다(문헌 Kabat et al., Sequences of Proteins of Immunological Interest(5th), National Institutes of Health, Bethesda, MD. (1991) 참조). 본 발명에 사용된 CDR 넘버링은 Kabat 방법을 사용했으나, 이외에 IMGT 방법, Chothia 방법, AbM 방법 등 다른 방법에 따라 결정될 수 있다. 본 발명의 일 구체예에서, 상기 방법 중 어느 하나로 결정된 CDR을 포함하는 결합 분자도 본 발명에 포함된다. In the present invention, the CDRs of the variable region according to the present invention were determined by a conventional method according to the system devised by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda) , MD. (1991)). The CDR numbering used in the present invention uses the Kabat method, but may be determined according to other methods such as the IMGT method, the Chothia method, and the AbM method. In one embodiment of the present invention, a binding molecule comprising a CDR determined by any of the above methods is also included in the present invention.
본 발명에 있어, 본 발명의 중화 결합 분자는 하기 표 2의 결합 분자들로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있으며, 바람직하게는 하기 표 2의 결합 분자 No. 89 내지 No. 194, 및 No. 248로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있으며, 더욱 바람직하게는 No. 139 결합 분자일 수 있다. 하기 표 2에서 No.는 각 결합 분자의 번호를 의미한다.In the present invention, the neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 2, preferably binding molecule No. 89 to No. 194, and No. 248 may be any one binding molecule selected from the group consisting of, more preferably No. 139 binding molecule. In Table 2 below, No. means the number of each binding molecule.
Figure PCTKR2021010606-appb-img-000006
Figure PCTKR2021010606-appb-img-000006
Figure PCTKR2021010606-appb-img-000007
Figure PCTKR2021010606-appb-img-000007
Figure PCTKR2021010606-appb-img-000008
Figure PCTKR2021010606-appb-img-000008
Figure PCTKR2021010606-appb-img-000009
Figure PCTKR2021010606-appb-img-000009
Figure PCTKR2021010606-appb-img-000010
Figure PCTKR2021010606-appb-img-000010
Figure PCTKR2021010606-appb-img-000011
Figure PCTKR2021010606-appb-img-000011
Figure PCTKR2021010606-appb-img-000012
Figure PCTKR2021010606-appb-img-000012
Figure PCTKR2021010606-appb-img-000013
Figure PCTKR2021010606-appb-img-000013
Figure PCTKR2021010606-appb-img-000014
Figure PCTKR2021010606-appb-img-000014
Figure PCTKR2021010606-appb-img-000015
Figure PCTKR2021010606-appb-img-000015
Figure PCTKR2021010606-appb-img-000016
Figure PCTKR2021010606-appb-img-000016
Figure PCTKR2021010606-appb-img-000017
Figure PCTKR2021010606-appb-img-000017
Figure PCTKR2021010606-appb-img-000018
Figure PCTKR2021010606-appb-img-000018
또한, 본 발명의 중화 결합 분자는 하기 표 3의 결합 분자들로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있으며, 바람직하게는 하기 표 3의 No. 322 또는 No. 343 결합 분자일 수 있으며, 더욱 바람직하게는 No. 322 결합 분자일 수 있다. 하기 표 3에서 No.는 각 결합 분자의 번호를 의미한다.In addition, the neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 3, preferably No. 322 or No. 343 binding molecule, more preferably No. 322 binding molecule. In Table 3 below, No. means the number of each binding molecule.
Figure PCTKR2021010606-appb-img-000019
Figure PCTKR2021010606-appb-img-000019
Figure PCTKR2021010606-appb-img-000020
Figure PCTKR2021010606-appb-img-000020
Figure PCTKR2021010606-appb-img-000021
Figure PCTKR2021010606-appb-img-000021
또한, 본 발명의 중화 결합 분자는 하기 표 4의 결합 분자들로 이루어진 군으로부터 선택되는 어느 하나의 결합 분자일 수 있으며, 바람직하게는 하기 표 4의 No. 322 또는 No. 343 결합 분자일 수 있으며, 더욱 바람직하게는 No. 322 결합 분자일 수 있다. 하기 표 4에서 No.는 각 결합 분자의 번호를 의미한다.In addition, the neutralizing binding molecule of the present invention may be any one binding molecule selected from the group consisting of binding molecules shown in Table 4, preferably No. 322 or No. 343 binding molecule, more preferably No. 322 binding molecule. In Table 4 below, No. means the number of each binding molecule.
Figure PCTKR2021010606-appb-img-000022
Figure PCTKR2021010606-appb-img-000022
Figure PCTKR2021010606-appb-img-000023
Figure PCTKR2021010606-appb-img-000023
Figure PCTKR2021010606-appb-img-000024
Figure PCTKR2021010606-appb-img-000024
Figure PCTKR2021010606-appb-img-000025
Figure PCTKR2021010606-appb-img-000025
Figure PCTKR2021010606-appb-img-000026
Figure PCTKR2021010606-appb-img-000026
Figure PCTKR2021010606-appb-img-000027
Figure PCTKR2021010606-appb-img-000027
Figure PCTKR2021010606-appb-img-000028
Figure PCTKR2021010606-appb-img-000028
Figure PCTKR2021010606-appb-img-000029
Figure PCTKR2021010606-appb-img-000029
Figure PCTKR2021010606-appb-img-000030
Figure PCTKR2021010606-appb-img-000030
본 발명에 있어, 본 발명의 중화 결합 분자는 scFv 절편, scFv-Fc 절편, Fab 절편, Fv 절편, 디아바디(diabody), 키메라 항체, 인간화 항체 또는 인간 항체일 수 있으나, 이에 한정되지 않는다. 본 발명의 중화 결합 분자는 SARS-CoV-2 S 단백질에 결합하는 scFv-Fc일 수 있다. 또한, 본 발명의 다른 일 실시예에 따르면, SARS-CoV-2 S 단백질에 결합하는 완전한 인간 항체(Full IgG)를 제공할 수 있다.In the present invention, the neutralizing binding molecule of the present invention may be an scFv fragment, an scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody, but is not limited thereto. The neutralizing binding molecule of the present invention may be an scFv-Fc that binds to the SARS-CoV-2 S protein. In addition, according to another embodiment of the present invention, it is possible to provide a fully human antibody (Full IgG) that binds to the SARS-CoV-2 S protein.
본 발명의 일 구체예에서, 본 발명의 중화 결합 분자는 LC CDR1, LC CDR2 및 LC CDR3을 포함하는 경쇄 가변 영역, 및 HC CDR1, HC CDR2 및 HC CDR3을 포함하는 중쇄 가변 영역을 포함하며,In one embodiment of the present invention, a neutralizing binding molecule of the present invention comprises a light chain variable region comprising LC CDR1, LC CDR2 and LC CDR3, and a heavy chain variable region comprising HC CDR1, HC CDR2 and HC CDR3,
상기 LC CDR1은 서열 SGX1X2SNIGX3NX4X5S를 포함하고, 여기서 X1은 S, G 또는 R이고, X2는 S, N 또는 T이고, X3은 N, D 또는 K이고, X4는 Y 또는 F이고, X5는 V 또는 I이고,said LC CDR1 comprises the sequence SGX 1 X 2 SNIGX 3 NX 4 X 5 S, wherein X 1 is S, G or R, X 2 is S, N or T, and X 3 is N, D or K , X 4 is Y or F, X 5 is V or I,
상기 LC CDR2는 서열 DNX6KRPS를 포함하고, 여기서 X6은 N 또는 D이고,wherein said LC CDR2 comprises the sequence DNX 6 KRPS, wherein X 6 is N or D;
상기 LC CDR3은 서열 GTWDX7X8LSX9X10X11을 포함하고, 여기서 X7은 S 또는 N이고, X8은 S 또는 N이고, X9는 A 또는 G이고, X10은 G 또는 V이고, X11은 V 또는 R이고,wherein said LC CDR3 comprises the sequence GTWDX 7 X 8 LSX 9 X 10 X 11 , wherein X 7 is S or N, X 8 is S or N, X 9 is A or G, and X 10 is G or V and X 11 is V or R,
상기 HC CDR1은 서열 TSGX12GVX13을 포함하고, 여기서 X12는 M 또는 V이고, X13은 G 또는 S이고,wherein said HC CDR1 comprises the sequence TSGX 12 GVX 13 , wherein X 12 is M or V, X 13 is G or S,
상기 HC CDR2는 서열 LIDWDDNKYX14TTSLKT를 포함하고, 여기서 X14는 Y 또는 H이고,wherein said HC CDR2 comprises the sequence LIDWDDNKYX 14 TTSLKT, wherein X 14 is Y or H;
상기 HC CDR3은 서열 IPGFLRYRNRYYYYGX15DV를 포함하고, 여기서 X15는 M 또는 V인 결합 분자일 수 있다.Said HC CDR3 may be a binding molecule comprising the sequence IPGFLRYRNRYYYYGX 15 DV, wherein X 15 is M or V.
또한, 본 발명의 일 구체예에서, 본 발명의 중화 결합 분자는 LC CDR1, LC CDR2 및 LC CDR3을 포함하는 경쇄 가변 영역, 및 HC CDR1, HC CDR2 및 HC CDR3을 포함하는 중쇄 가변 영역을 포함하며, Further, in one embodiment of the present invention, the neutralizing binding molecule of the present invention comprises a light chain variable region comprising LC CDR1, LC CDR2 and LC CDR3, and a heavy chain variable region comprising HC CDR1, HC CDR2 and HC CDR3, ,
상기 LC CDR1은 서열 RASQSISSYLN을 포함하고, wherein said LC CDR1 comprises the sequence RASQSISSYLN,
상기 LC CDR2는 서열 AASSLQS를 포함하고, wherein the LC CDR2 comprises the sequence AASSLQS,
상기 LC CDR3은 서열 QQSYSTPLT를 포함하고, wherein said LC CDR3 comprises the sequence QQSYSTPLT,
상기 HC CDR1은 서열 SNYMX16을 포함하고, 여기서 X16은 T 또는 S이고,said HC CDR1 comprises the sequence SNYMX 16 , wherein X 16 is T or S;
상기 HC CDR2는 서열 X17IYPGGSTX18X19ADSVX20G를 포함하고, 여기서 X17은 I 또는 V이고, X18은 Y 또는 F이고, X19는 Y 또는 F이고, X20은 K또는 Q이고,wherein said HC CDR2 comprises the sequence X 17 IYPGGSTX 18 X 19 ADSVX 20 G, wherein X 17 is I or V, X 18 is Y or F, X 19 is Y or F, and X 20 is K or Q ,
상기 HC CDR3은 서열 DLPLTGTTLDY 또는 SYDFLTDYTDAFDI를 포함하는 결합 분자일 수 있다.The HC CDR3 may be a binding molecule comprising the sequence DLPLTGTTLDY or SYDFLTDYTDAFDI.
본 발명의 일 구체예에서, 본 발명의 중화 결합 분자는 서열번호: 829의 CDR1 영역, 서열번호: 830의 CDR2 영역, 및 서열번호: 831의 CDR3 영역을 포함하는 경쇄 가변영역; 및 서열번호: 832의 CDR1 영역, 서열번호: 833의 CDR2 영역, 및 서열번호: 834의 CDR3영역을 포함하는 중쇄 가변영역을 포함할 수 있다.In one embodiment of the present invention, the neutralizing binding molecule of the present invention comprises a light chain variable region comprising a CDR1 region of SEQ ID NO: 829, a CDR2 region of SEQ ID NO: 830, and a CDR3 region of SEQ ID NO: 831; and a heavy chain variable region comprising a CDR1 region of SEQ ID NO: 832, a CDR2 region of SEQ ID NO: 833, and a CDR3 region of SEQ ID NO: 834.
또한, 본 발명의 일 구체예에서 본 발명의 중화 결합 분자는 서열번호: 2507의 CDR1 영역, 서열번호: 2508의 CDR2 영역, 및 서열번호: 2509의 CDR3 영역을 포함하는 경쇄 가변영역; 및 서열번호: 2510의 CDR1 영역, 서열번호: 2511의 CDR2 영역, 및 서열번호: 2512의 CDR3 영역을 포함하는 중쇄 가변영역을 포함할 수 있다.Further, in one embodiment of the present invention, the neutralizing binding molecule of the present invention comprises a light chain variable region comprising a CDR1 region of SEQ ID NO: 2507, a CDR2 region of SEQ ID NO: 2508, and a CDR3 region of SEQ ID NO: 2509; and a heavy chain variable region comprising a CDR1 region of SEQ ID NO: 2510, a CDR2 region of SEQ ID NO: 2511, and a CDR3 region of SEQ ID NO: 2512.
본 발명의 일 구체예에서, 상기 중화 결합 분자는 서열번호: 2017의 폴리펩티드 서열의 경쇄 가변영역; 및 서열번호: 2018의 폴리펩티드 서열의 중쇄 가변영역을 포함하는 결합 분자와 80% 내지 99%, 바람직하게는 85 내지 99%, 보다 바람직하게는 90 내지 99% 동일한 서열을 포함하는 결합 분자일 수 있다.In one embodiment of the present invention, the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 2017; and 80% to 99%, preferably 85 to 99%, more preferably 90 to 99% identical to the binding molecule comprising the heavy chain variable region of the polypeptide sequence of SEQ ID NO: 2018. It may be a binding molecule comprising a sequence .
또한, 본 발명의 일 구체예에서, 상기 중화 결합 분자는 서열번호: 3523의 폴리펩티드 서열의 경쇄 가변영역; 및 서열번호: 3524의 폴리펩티드 서열의 중쇄 가변영역을 포함하는 결합 분자와 80% 내지 99%, 바람직하게는 85 내지 99%, 보다 바람직하게는 90 내지 99% 동일한 서열을 포함하는 결합 분자일 수 있다.Also, in one embodiment of the present invention, the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 3523; and 80% to 99%, preferably 85 to 99%, more preferably 90 to 99% identical to the binding molecule comprising the heavy chain variable region of the polypeptide sequence of SEQ ID NO: 3524. .
본 발명의 일 구체예에서, 상기 중화 결합 분자는 서열번호: 2017의 폴리펩티드 서열의 경쇄 가변영역; 및 서열번호: 2018의 폴리펩티드 서열의 중쇄 가변영역을 포함할 수 있다.In one embodiment of the present invention, the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 2017; and a heavy chain variable region of the polypeptide sequence of SEQ ID NO: 2018.
또한, 본 발명의 일 구체예에서, 상기 중화 결합 분자는 서열번호: 3523의 폴리펩티드 서열의 경쇄 가변영역; 및 서열번호: 3524의 폴리펩티드 서열의 중쇄 가변영역을 포함할 수 있다.Also, in one embodiment of the present invention, the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 3523; and a heavy chain variable region of the polypeptide sequence of SEQ ID NO: 3524.
본 명세서에서 용어 '항체'는 최대한 넓은 의미로 사용되며, 구체적으로 온전한(intact) 단일클론 항체, 다클론 항체, 2종 이상의 온전한 항체로부터 형성된 다중특이성 항체(예를 들어, 이중특이성 항체), 및 목적하는 생물학적 활성을 나타내는 항체 단편을 포함한다. 항체는 특이적인 항원을 인식하고 결합할 수 있는 면역계에 의하여 생성되는 단백질이다. 그 구조적인 면에서, 항체는 통상적으로 4개의 아미노산 쇄(2개의 중쇄 및 2개의 경쇄)로 이루어진 Y-형상의 단백질을 가진다. 각각의 항체는 주로 가변 영역 및 불변 영역의 2개의 영역을 가진다. Y의 팔의 말단 부분에 위치한 가변 영역은 표적 항원에 결합하고 상호작용한다. 상기 가변 영역은 특정 항원 상의 특이적 결합 부위를 인식하고 결합하는 상보성 결정 영역(CDR)을 포함한다. Y의 꼬리 부분에 위치한 불변 영역은 면역계에 의하여 인식되고 상호작용한다. 표적 항원은 일반적으로 다수의 항체 상의 CDR에 의하여 인식되는, 에피토프라고 하는 다수의 결합 부위를 가지고 있다. 상이한 에피토프에 특이적으로 결합하는 각각의 항체는 상이한 구조를 가진다. 그러므로 한 항원은 하나 이상의 상응하는 항체를 가질 수 있다.As used herein, the term 'antibody' is used in the broadest sense, specifically, an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from two or more intact antibodies (eg, a bispecific antibody), and antibody fragments exhibiting the desired biological activity. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. In terms of their structure, antibodies usually have a Y-shaped protein consisting of four amino acid chains (two heavy chains and two light chains). Each antibody mainly has two regions: a variable region and a constant region. The variable region located in the distal portion of the arm of Y binds and interacts with the target antigen. The variable region comprises a complementarity determining region (CDR) that recognizes and binds a specific binding site on a specific antigen. The constant region located at the tail of Y is recognized and interacted with by the immune system. Target antigens have multiple binding sites, called epitopes, which are generally recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.
본 발명에 있어, 본 발명에 따른 결합 분자는 상기 결합 분자의 기능적 변이체를 포함할 수 있다. 본 발명의 변이체는 SARS-CoV-2 또는 이것의 S 단백질에 특이적으로 결합하기 위해 본 발명의 결합 분자와 경쟁할 수 있다. 본 발명에 있어, SARS-CoV-2에 중화 능력을 보유한다면 본 발명의 결합 분자의 기능적 변이체로 간주된다. 본 발명에 있어, 상기 기능적 변이체는 1차 구조적 서열이 실질적으로 유사한 유도체를 포함하지만, 이에 제한되는 것은 아니다. 예를 들면, 상기 기능적 변이체는 생체외(in vitro) 또는 생체내(in vivo) 변형, 화학약품 및/또는 생화학 약품에 의한 변형을 포함한다. 본 발명의 일 구체예에 있어, 상기 기능적 변이체는 본원 발명의 부모 단일클론 항체에서는 발견되지 않는다. 이와 같은 변형으로는 예를 들어 아세틸화, 아실화, 뉴클레오티드 또는 뉴클레오티드 유도체의 공유 결합, 지질 또는 지질 유도체의 공유 결합, 가교, 이황화 결합 형성, 글리코실화, 수산화, 메틸화, 산화, 페길화, 단백질 분해 및 인산화 등이 포함된다. 본 발명에 있어, 상기 기능적 변이체는 선택적으로 부모 항체의 아미노산 서열과 비교하여 하나 이상의 아미노산의 치환, 삽입, 결실 또는 그들의 조합을 함유하는 아미노산 서열을 포함할 수 있다. 더욱이, 상기 기능적 변이체는 아미노 말단 또는 카르복시 말단 중 하나 또는 모두에서 아미노산 서열의 절단체(truncated form)를 포함할 수 있다. 본 발명에 있어, 본 발명의 기능적 변이체는 본 발명의 부모 항체와 비교하여 동일하거나 다르거나, 더 높거나 낮은 결합 친화력을 가질 수 있지만, 여전히 SARS-CoV-2 또는 이것의 S 단백질에 결합할 수 있다. 일 예로, 골격구조, 초가변(Hypervariable) 영역, 특히 경쇄 또는 중쇄의 상보성 결정 영역(Complementarity-determining region, CDR)을 포함하나 이에 한정되지 않는 가변 영역의 아미노산 서열이 변형될 수 있다. 일반적으로 경쇄 또는 중쇄 영역은 3개의 CDR 영역을 포함하는, 3개의 초가변 영역 및 더욱 보존된 영역, 즉 골격 영역(FR)을 포함한다. 일반적으로 초가변 영역은 CDR로부터의 아미노산 잔기와 초가변 루프로부터의 아미노산 잔기를 포함한다. 본 발명의 범위에 속하는 기능적 변이체는 본 명세서의 부모 항체와 약 50%~99%, 약 60%~99%, 약 80%~99%, 약 90%~99%, 약 95%~99%, 또는 약 97%~99% 아미노산 서열 동질성을 가질 수 있다. 본 발명에 있어, 비교될 아미노산 서열을 최적으로 배열하고 유사하거나 또는 동일한 아미노산 잔기를 정의하기 위해 컴퓨터 알고리즘 중 당업자에게 알려진 Gap 또는 Bestfit를 사용할 수 있다. 본 발명에 있어, 상기 기능적 변이체는 부모 항체 또는 그것의 일부를 PCR 방법, 올리고머 뉴클레오티드를 이용한 돌연변이 생성 및 부분 돌연변이 생성을 포함하는 공지의 일반 분자생물학적 방법에 의해 변화시키거나 유기합성 방법으로 얻을 수 있으나 이에 제한되는 것은 아니다. In the present invention, the binding molecule according to the present invention may include a functional variant of the binding molecule. A variant of the invention may compete with a binding molecule of the invention for specific binding to SARS-CoV-2 or its S protein. In the present invention, it is regarded as a functional variant of the binding molecule of the present invention if it has the ability to neutralize SARS-CoV-2. In the present invention, the functional variant includes, but is not limited to, a derivative having a substantially similar primary structural sequence. For example, the functional variant includes in vitro or in vivo modification, modification by chemical and/or biochemical agents. In one embodiment of the present invention, the functional variant is not found in the parental monoclonal antibody of the present invention. Such modifications include, for example, acetylation, acylation, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis. and phosphorylation. In the present invention, the functional variant may optionally comprise an amino acid sequence containing one or more amino acid substitutions, insertions, deletions or combinations thereof compared to the amino acid sequence of the parent antibody. Moreover, the functional variant may comprise a truncated form of the amino acid sequence at one or both of the amino terminus or the carboxy terminus. In the present invention, functional variants of the present invention may have the same, different, higher or lower binding affinity compared to the parent antibody of the present invention, but still be capable of binding to SARS-CoV-2 or its S protein. there is. As an example, the amino acid sequence of a variable region including, but not limited to, a framework structure, a hypervariable region, in particular, a complementarity-determining region (CDR) of a light or heavy chain may be modified. Generally a light or heavy chain region comprises three hypervariable regions, comprising three CDR regions, and a more conserved region, namely a framework region (FR). In general, a hypervariable region comprises amino acid residues from a CDR and amino acid residues from a hypervariable loop. Functional variants within the scope of the present invention include about 50%-99%, about 60%-99%, about 80%-99%, about 90%-99%, about 95%-99%, or about 97%-99% amino acid sequence identity. In the present invention, Gap or Bestfit known to those skilled in the art among computer algorithms may be used to optimally align amino acid sequences to be compared and to define similar or identical amino acid residues. In the present invention, the functional variant may be obtained by changing the parent antibody or a part thereof by a known general molecular biological method including PCR method, mutagenesis using oligomeric nucleotides and partial mutagenesis, or by organic synthesis method. However, the present invention is not limited thereto.
또한, 본 발명의 일 구체예에서, 상기 결합 분자는 추가적으로 하나 이상의 태그가 결합된 이뮤노컨쥬게이트일 수 있으며, 예를 들어, 상기 결합 분자에 약물이 추가로 부착된 이뮤노컨쥬게이트일 수 있다. 즉, 본 발명에 따른 결합 분자는 약물이 결합된 항체-약물 접합체(antibody-drug conjugate; ADC)의 형태로 사용될 수 있다. 약물을 국소 전달하기 위해 항체-약물 접합체(ADC), 즉 면역접합체를 사용하게 되면 약물 모이어티를 감염된 세포에 표적화 전달할 수 있는 반면, 접합시키지 않은 채로 투여하게 되면, 정상 세포에 대해서도 허용될 수 없는 수준의 독성이 야기될 수 있다. 상기 ADC 형태는 약물-연결성 및 약물-방출성 뿐만 아니라 폴리클로날(polyclonal) 및 모노클로날(monoclonal) 항체(mAb)의 선택성을 높임으로써 약물의 최대 효능과 최소 독성을 개선할 수 있다.Also, in one embodiment of the present invention, the binding molecule may be an immunoconjugate to which one or more tags are additionally bound, for example, an immunoconjugate to which a drug is further attached to the binding molecule. . That is, the binding molecule according to the present invention may be used in the form of an antibody-drug conjugate (ADC) to which a drug is bound. The use of antibody-drug conjugates (ADCs), i.e. immunoconjugates, for local delivery of drugs allows for targeted delivery of drug moieties to infected cells, whereas unconjugated administration is unacceptable for normal cells as well. A level of toxicity may be caused. The ADC form can improve the maximum efficacy and minimum toxicity of the drug by increasing the drug-connectivity and drug-releasing properties as well as the selectivity of polyclonal and monoclonal antibodies (mAbs).
약물 모이어티를 항체에 부착시키는, 예를 들어 공유 결합을 통하여 연결시키는 통상적인 수단으로는, 일반적으로 약물 모이어티가 항체 상의 수많은 부위에 부착되는 불균질한 분자 혼합물이 유발된다. 예를 들어, 세포독성 약물을 항체의 수많은 리신 잔기를 통하여 항체와 접합시켜 불균질한 항체-약물 접합체 혼합물을 생성시킬 수 있다. 반응 조건에 따라서, 이러한 불균질한 혼합물은 전형적으로, 약물 모이어티에 부착된 항체 분포도가 0 내지 약 8 이상이다. 또한, 특별한 정수 비의 약물 모이어티 대 항체를 수반한 접합체의 각 아군은, 약물 모이어티가 항체 상의 각종 부위에 부착되는 잠재적으로 불균질한 혼합물이다. 일 예로, 링커 시약 및 약물-링커 중간체와의 반응성은 pH, 농도, 염 농도 및 조용매와 같은 요인들에 의해 좌우된다.Conventional means of attaching drug moieties to antibodies, for example linking them via covalent bonds, generally result in heterogeneous molecular mixtures in which drug moieties are attached to numerous sites on the antibody. For example, a cytotoxic drug can be conjugated with the antibody through the numerous lysine residues of the antibody to produce a heterogeneous mixture of antibody-drug conjugates. Depending on the reaction conditions, such heterogeneous mixtures typically have a distribution of 0 to about 8 or greater of antibody attached to the drug moiety. In addition, each subgroup of conjugates with a particular integer ratio of drug moieties to antibody is a potentially heterogeneous mixture in which drug moieties are attached to various sites on the antibody. In one example, the reactivity of linker reagents and drug-linker intermediates depends on factors such as pH, concentration, salt concentration and cosolvent.
본 발명의 일 구체예에서, 본 발명의 (A) 중화 결합 분자의 농도는 본 발명에 따른 안정한 약제학적 제제의 안정성 및 점도에 악영향을 실질적으로 미치지 않는 범위 내에서 자유롭게 조절할 수 있다. 본 발명의 일 구현예에서, 중화 결합 분자의 농도는 1 내지 240 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 230 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 220 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 210 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 200 mg/ml일 수 있다. In one embodiment of the present invention, the concentration of (A) neutralizing binding molecule of the present invention can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the stable pharmaceutical preparation according to the present invention. In one embodiment of the present invention, the concentration of the neutralizing binding molecule may be 1 to 240 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be 1 to 230 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be 1 to 220 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be 1 to 210 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 1 and 200 mg/ml.
본 발명의 일 구현예에서, 중화 결합 분자의 농도는 5 내지 240 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 230 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 220 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 210 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 200 mg/ml일 수 있다. In one embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 240 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 5 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 220 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 5 and 210 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be 5 to 200 mg/ml.
또한, 본 발명의 일 구현예에서, 중화 결합 분자의 농도는 10 내지 240 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 230 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 220 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 210 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 200 mg/ml일 수 있다. Also, in one embodiment of the present invention, the concentration of the neutralizing binding molecule may be 10-240 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 10 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 10-220 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 10 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 200 mg/ml.
또한, 본 발명의 일 구현예에서, 중화 결합 분자의 농도는 20 내지 240 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 230 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 220 mg/ml일 수 있다. 본 발명의 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 210 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 200 mg/ml일 수 있다. Also, in one embodiment of the present invention, the concentration of the neutralizing binding molecule may be 20 to 240 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 20 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 220 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 20 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 200 mg/ml.
또한, 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 240 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 230 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 220 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 210 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 200 mg/ml일 수 있다. Also, in another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 30 to 240 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 220 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 200 mg/ml.
또한, 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 240 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 230 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 220 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 210 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 200 mg/ml일 수 있다. Also, in another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 40 to 240 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 230 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 220 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 210 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 200 mg/ml.
또한, 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 100 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 90 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 80 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 70 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 1 내지 60 mg/ml일 수 있다. Also, in another embodiment of the present invention, the concentration of the neutralizing binding molecule may be from 1 to 100 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 1 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 1 to 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 1 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 1 and 60 mg/ml.
본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 100 mg/ml일 수 있다. 본 발명의 또 다른 구현예에 따르면, 중화 결합 분자의 농도는 5 내지 90 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 80 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 70 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 5 내지 60 mg/ml일 수 있다.In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 5 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 5 to 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 5 and 60 mg/ml.
본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 100 mg/ml일 수 있다. 본 발명의 또 다른 구현예에 따르면, 중화 결합 분자의 농도는 10 내지 90 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 80 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 70 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 10 내지 60 mg/ml일 수 있다.In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 10 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 10 and 60 mg/ml.
본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 100 mg/ml일 수 있다. 본 발명의 또 다른 구현예에 따르면, 중화 결합 분자의 농도는 20 내지 90 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 80 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 70 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 20 내지 60 mg/ml일 수 있다.In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 20 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 20 and 60 mg/ml.
본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 100 mg/ml일 수 있다. 본 발명의 또 다른 구현예에 따르면, 중화 결합 분자의 농도는 30 내지 90 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 80 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 70 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 30 내지 60 mg/ml일 수 있다.In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 30 to 90 mg/ml. In another embodiment of the invention, the concentration of the neutralizing binding molecule may be between 30 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 30 and 60 mg/ml.
본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 100 mg/ml일 수 있다. 본 발명의 또 다른 구현예에 따르면, 중화 결합 분자의 농도는 40 내지 90 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 80 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 70 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 40 내지 60 mg/ml일 수 있다.In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 40 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 40 to 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 40 to 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 40 and 60 mg/ml.
본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 50 내지 100 mg/ml일 수 있다. 본 발명의 또 다른 구현예에 따르면, 중화 결합 분자의 농도는 50 내지 90 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 50 내지 80 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 50 내지 70 mg/ml일 수 있다. 본 발명의 또 다른 구현예에서, 중화 결합 분자의 농도는 50 내지 60 mg/ml일 수 있다.In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 50 and 100 mg/ml. According to another embodiment of the present invention, the concentration of the neutralizing binding molecule may be 50 to 90 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 50 and 80 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 50 and 70 mg/ml. In another embodiment of the present invention, the concentration of the neutralizing binding molecule may be between 50 and 60 mg/ml.
(B) 완충제(B) buffer
본 발명에 있어, 본 발명에 따른 완충제는 산이나 알칼리에 의한 pH의 변화를 최소화시키는 중화성 물질이며, 상기 완충제는 i) 히스티딘, 히스티딘염 또는 이들의 혼합물,ii) 아세테이트(acetate), iii) 시트레이트(citrate), iv) 숙시네이트(succinate), v) 포스페이트(phosphate), 또는 vi) 글루코네이트(gluconate)를 포함할 수 있으나, 이에 한정되는 것은 아니다. In the present invention, the buffer according to the present invention is a neutralizing material that minimizes changes in pH due to acid or alkali, and the buffer is i) histidine, histidine salt or a mixture thereof, ii) acetate, iii) citrate, iv) succinate, v) phosphate, or vi) gluconate, but is not limited thereto.
본 발명의 일 구현예에서, 상기 히스티딘염은 히스티딘 클로라이드, 히스티딘 아세테이트, 히스티딘 포스페이트, 히스티딘 설페이트 등을 포함할 수 있다. 본 발명에 있어, 완충제로 히스티딘을 포함하는 것이, pH 조절 및 안정성 측면에서 바람직할 수 있다.In one embodiment of the present invention, the histidine salt may include histidine chloride, histidine acetate, histidine phosphate, histidine sulfate, and the like. In the present invention, it may be preferable in terms of pH control and stability to include histidine as a buffer.
본 발명의 일 구현예에서, 상기 완충제의 함량은 본 발명에 따른 약제학적 제제의 안정성 및 점도에 악영향을 실질적으로 미치지 않는 범위 내에서 자유롭게 조절할 수 있다. 본 발명의 일 구현예에서, 완충제의 함량은 1 내지 100 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 1 내지 90 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 1 내지 80 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 1 내지 70 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 1 내지 60 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 1 내지 50 mM일 수 있다.In one embodiment of the present invention, the content of the buffer can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical formulation according to the present invention. In one embodiment of the present invention, the content of the buffer may be 1 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 1 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 1 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 1 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 1 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 1 to 50 mM.
본 발명의 일 구현예에서, 완충제의 함량은 2 내지 100 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 2 내지 90 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 2 내지 80 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 2 내지 70 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 2 내지 60 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 2 내지 50 mM일 수 있다.In one embodiment of the present invention, the content of the buffer may be 2 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 2 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 2-50 mM.
본 발명의 일 구현예에서, 완충제의 함량은 3 내지 100 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 3 내지 90 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 3 내지 80 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 3 내지 70 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 3 내지 60 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 3 내지 50 mM일 수 있다.In one embodiment of the present invention, the content of the buffer may be 3 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 3 to 50 mM.
본 발명의 일 구현예에서, 완충제의 함량은 4 내지 100 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 4 내지 90 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 4 내지 80 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 4 내지 70 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 4 내지 60 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 4 내지 50 mM일 수 있다.In one embodiment of the present invention, the content of the buffer may be 4 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 4 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 4-50 mM.
본 발명의 일 구현예에서, 완충제의 함량은 5 내지 100 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 5 내지 90 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 5 내지 80 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 5 내지 70 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 5 내지 60 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 5 내지 50 mM일 수 있다.In one embodiment of the present invention, the content of the buffer may be 5 to 100 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 90 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 80 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 70 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 60 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 50 mM.
본 발명의 일 구현예에서, 완충제의 함량은 1 내지 20 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 5 내지 15 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 7 내지 13 mM일 수 있다. 본 발명의 다른 구현예에서, 완충제의 함량은 10 mM일 수 있다.In one embodiment of the present invention, the content of the buffer may be 1 to 20 mM. In another embodiment of the present invention, the content of the buffer may be 5 to 15 mM. In another embodiment of the present invention, the content of the buffer may be 7 to 13 mM. In another embodiment of the present invention, the content of the buffer may be 10 mM.
(C) 안정화제(C) Stabilizer
본 발명의 일 구현예에서, 본 발명에 따른 안정화제는 i) 금속염, ii) 당 또는 이의 유도체 및 iii) 아미노산(단, 상기 (B) 완충제에 포함되는 아미노산과 상이함) 또는 이의 염으로 구성된 군으로부터 선택되는 어느 하나 이상, 바람직하게는 아미노산일 수 있다. In one embodiment of the present invention, the stabilizer according to the present invention consists of i) a metal salt, ii) a sugar or a derivative thereof, and iii) an amino acid (provided that it is different from the amino acid contained in the (B) buffer) or a salt thereof. It may be any one or more selected from the group, preferably an amino acid.
본 발명의 일 구현예에서, 상기 안정화제는 금속염일 수 있으며, 이의 음이온이 함께 포함될 수 있다. 예를 들어, 상기 금속염은 염화나트륨(NaCl), 염화칼륨(KCl), 염화칼슘(CaCl) 또는 이들 중 2 이상의 혼합물을 포함할 수 있다. In one embodiment of the present invention, the stabilizer may be a metal salt, and an anion thereof may be included together. For example, the metal salt may include sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl), or a mixture of two or more thereof.
본 발명의 일 구현예에서, 상기 안정화제는 당 또는 당의 유도체일 수 있다. 상기 당은 단당류, 이당류, 올리고당, 다당류 또는 이들 중 2 이상의 혼합물일 수 있다. 단당류의 예로는 글루코스, 프룩토스, 갈락토스 등이 있으나, 이에 제한되지 않는다. 이당류의 예로는 수크로오스, 락토스, 말토스, 트레할로스 등이 있으나, 이에 제한되지 않는다. 올리고당의 예로는 프룩토올리고당, 갈락토올릭고당, 만난올리고당 등이 있으나, 이에 제한되지 않는다. 다당류의 예로는 전분, 글리코겐, 셀룰로스, 키틴, 펙틴 등이 있으나, 이에 제한되지 않는다.In one embodiment of the present invention, the stabilizer may be a sugar or a derivative of sugar. The sugar may be a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide, or a mixture of two or more thereof. Examples of monosaccharides include, but are not limited to, glucose, fructose, galactose, and the like. Examples of disaccharides include, but are not limited to, sucrose, lactose, maltose, trehalose, and the like. Examples of oligosaccharides include, but are not limited to, fructooligosaccharides, galactooligosaccharides, mannan oligosaccharides, and the like. Examples of polysaccharides include, but are not limited to, starch, glycogen, cellulose, chitin, pectin, and the like.
상기 당의 유도체는 당 알코올, 당 산 또는 이들의 혼합물일 수 있다. 당 알코올의 예로는 글리세롤, 에리스리톨, 트레이톨, 아라비톨, 자이리톨, 리비톨, 만니톨, 소르비톨, 갈락티톨, 푸시톨, 이디톨, 이노시톨, 볼레미톨, 아이소말트, 말티톨, 락티톨, 말토트리이톨, 말토테트라이톨, 폴리글리시톨 등이 있으나, 이에 제한되지 않는다. 당 산의 예로는 알돈산(글리세르산 등), 울로손산(뉴라민산 등), 우론산(글루쿠론산 등), 알다르산(타르타르산 등) 등이 있으나, 이에 제한되지 않는다. 본 발명의 일 구현예에서, 당 또는 이의 유도체는 소르비톨, 만니톨, 트레할로스, 수크로오스 또는 이들 중 2 이상의 혼합물일 수 있고, 바람직하게는 트레할로스일 수 있다. The sugar derivative may be a sugar alcohol, a sugar acid, or a mixture thereof. Examples of sugar alcohols include glycerol, erythritol, threitol, arabitol, xylitol, ribitol, mannitol, sorbitol, galactitol, fusitol, iditol, inositol, bolemitol, isomalt, maltitol, lactitol, maltotriitol , maltotetraitol, polyglycitol, and the like, but is not limited thereto. Examples of sugar acids include, but are not limited to, aldonic acid (such as glyceric acid), ulosonic acid (such as neuramic acid), uronic acid (such as glucuronic acid), and aldaric acid (such as tartaric acid). In one embodiment of the present invention, the sugar or its derivative may be sorbitol, mannitol, trehalose, sucrose, or a mixture of two or more thereof, preferably trehalose.
본 발명의 일 구현예에서, 상기 안정화제는 아미노산 또는 아미노산의 염일 수 있다. 상기 아미노산은 글리신, 아르기닌, 트레오닌, 메티오닌, 또는 이들 중 2 이상의 혼합물일 수 있으나. 이에 한정되는 것은 아니다. 상기 아미노산의 염은 L-아르기닌 모노하이드로클로라이드(monohydrochloride)일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 일 구현예에 따르면, 상기 안정화제는 바람직하게는 L-아르기닌 모노하이드로클로라이드일 수 있다. In one embodiment of the present invention, the stabilizing agent may be an amino acid or a salt of an amino acid. The amino acid may be glycine, arginine, threonine, methionine, or a mixture of two or more thereof. However, the present invention is not limited thereto. The salt of the amino acid may be L- arginine monohydrochloride (monohydrochloride), but is not limited thereto. According to one embodiment of the present invention, the stabilizer may be preferably L-arginine monohydrochloride.
본 발명의 일 구현예에서, 상기 안정화제의 함량은 본 발명에 따른 약제학적 제제의 안정성 및 점도에 악영향을 실질적으로 미치지 않는 범위 내에서 자유롭게 조절할 수 있다. 본 발명의 일 구현예에서, 안정화제의 함량은 50 내지 300 mM 또는 1 내지 20%(w/v)일 수 있으나, 이에 한정되지 않는다.In one embodiment of the present invention, the content of the stabilizer can be freely adjusted within a range that does not substantially adversely affect the stability and viscosity of the pharmaceutical formulation according to the present invention. In one embodiment of the present invention, the content of the stabilizer may be 50 to 300 mM or 1 to 20% (w/v), but is not limited thereto.
본 발명의 일 구현예에서, 안정화제로써 아미노산 또는 금속염을 포함하는 경우, 안정화제의 함량은 50 내지 300 mM, 50 내지 290 mM, 50 내지 내지 280 mM, 50 내지 내지 270 mM, 50 내지 내지 260 mM 또는 50 내지 내지 250 mM일 수 있다.In one embodiment of the present invention, when an amino acid or a metal salt is included as a stabilizer, the content of the stabilizer is 50 to 300 mM, 50 to 290 mM, 50 to 280 mM, 50 to 270 mM, 50 to 260 mM or 50 to 250 mM.
본 발명의 다른 구현예에서, 안정화제로써 아미노산 또는 금속염을 포함하는 경우, 안정화제의 함량은 60 내지 300 mM, 60 내지 290 mM, 60 내지 내지 280 mM, 60 내지 내지 270 mM, 60 내지 내지 260 mM 또는 60 내지 내지 250 mM 일 수 있다. In another embodiment of the present invention, when an amino acid or a metal salt is included as a stabilizer, the content of the stabilizer is 60 to 300 mM, 60 to 290 mM, 60 to 280 mM, 60 to 270 mM, 60 to 260 mM or from 60 to 250 mM.
본 발명의 다른 구현예에서, 안정화제로써 아미노산 또는 금속염을 포함하는 경우, 안정화제의 함량은 70 내지 300 mM, 70 내지 290 mM, 70 내지 내지 280 mM, 70 내지 내지 270 mM, 70 내지 내지 260 mM 또는 70 내지 내지 250 mM 일 수 있다.In another embodiment of the present invention, when an amino acid or a metal salt is included as a stabilizer, the content of the stabilizer is 70 to 300 mM, 70 to 290 mM, 70 to 280 mM, 70 to 270 mM, 70 to 260 mM or 70 to 250 mM.
본 발명의 다른 구현예에서, 안정화제로써 아미노산 또는 금속염을 포함하는 경우, 안정화제의 함량은 80 내지 300 mM, 80 내지 290 mM, 80 내지 내지 280 mM, 80 내지 내지 270 mM, 80 내지 내지 260 mM 또는 80 내지 내지 250 mM 일 수 있다.In another embodiment of the present invention, when an amino acid or a metal salt is included as a stabilizer, the content of the stabilizer is 80 to 300 mM, 80 to 290 mM, 80 to 280 mM, 80 to 270 mM, 80 to 260 mM or 80 to 250 mM.
본 발명의 다른 구현예에서, 안정화제로써 아미노산 또는 금속염을 포함하는 경우, 안정화제의 함량은 90 내지 300 mM, 90 내지 290 mM, 90 내지 내지 280 mM, 90 내지 내지 270 mM, 90 내지 내지 260 mM 또는 90 내지 내지 250 mM 일 수 있다.In another embodiment of the present invention, when an amino acid or a metal salt is included as a stabilizer, the content of the stabilizer is 90 to 300 mM, 90 to 290 mM, 90 to 280 mM, 90 to 270 mM, 90 to 260 mM or from 90 to 250 mM.
본 발명의 다른 구현예에서, 안정화제로써 아미노산 또는 금속염을 포함하는 경우, 안정화제의 함량은 100 내지 300 mM, 100 내지 290 mM, 100 내지 내지 280 mM, 100 내지 내지 270 mM, 100 내지 내지 260 mM 또는 100 내지 내지 250 mM 일 수 있다.In another embodiment of the present invention, when an amino acid or a metal salt is included as a stabilizer, the content of the stabilizer is 100 to 300 mM, 100 to 290 mM, 100 to 280 mM, 100 to 270 mM, 100 to 260 mM or from 100 to 250 mM.
본 발명의 일 구현예에서, 안정화제로써 당 또는 당의 유도체를 포함하는 경우, 안정화제의 함량은 본 발명의 안정한 약제학적 제제 총 100 부피 %를 기준으로, 1 내지 20%(w/v), 1 내지 18%(w/v), 1 내지 16%(w/v), 1 내지 14%(w/v), 1 내지 12%(w/v) 또는 1 내지 10%(w/v)일 수 있다.In one embodiment of the present invention, when sugar or a derivative of sugar is included as a stabilizer, the content of the stabilizer is 1 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 1 to 18% (w/v), 1 to 16% (w/v), 1 to 14% (w/v), 1 to 12% (w/v) or 1 to 10% (w/v) days can
본 발명의 다른 구현예에서, 안정화제로써 당 또는 당의 유도체를 포함하는 경우, 안정화제의 함량은 본 발명의 안정한 약제학적 제제 총 100 부피 %를 기준으로, 2 내지 20%(w/v), 2 내지 18%(w/v), 2 내지 16%(w/v), 2 내지 14%(w/v), 2 내지 12%(w/v) 또는 2 내지 10%(w/v)일 수 있다.In another embodiment of the present invention, when a sugar or a derivative of sugar is included as a stabilizer, the content of the stabilizer is 2 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 2 to 18% (w/v), 2 to 16% (w/v), 2 to 14% (w/v), 2 to 12% (w/v) or 2 to 10% (w/v) days can
본 발명의 다른 구현예에서, 안정화제로써 당 또는 당의 유도체를 포함하는 경우, 안정화제의 함량은 본 발명의 안정한 약제학적 제제 총 100 부피 %를 기준으로, 3 내지 20%(w/v), 3 내지 18%(w/v), 3 내지 16%(w/v), 3 내지 14%(w/v), 3 내지 12%(w/v) 또는 3 내지 10%(w/v)일 수 있다.In another embodiment of the present invention, when sugar or a derivative of sugar is included as a stabilizer, the content of the stabilizer is 3 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 3 to 18% (w/v), 3 to 16% (w/v), 3 to 14% (w/v), 3 to 12% (w/v) or 3 to 10% (w/v) days can
본 발명의 다른 구현예에서, 안정화제로써 당 또는 당의 유도체를 포함하는 경우, 안정화제의 함량은 본 발명의 안정한 약제학적 제제 총 100 부피 %를 기준으로, 4 내지 20%(w/v), 4 내지 18%(w/v), 4 내지 16%(w/v), 4 내지 14%(w/v), 4 내지 12%(w/v) 또는 4 내지 10%(w/v)일 수 있다.In another embodiment of the present invention, when sugar or a derivative of sugar is included as a stabilizer, the content of the stabilizer is 4 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 4 to 18% (w/v), 4 to 16% (w/v), 4 to 14% (w/v), 4 to 12% (w/v) or 4 to 10% (w/v) days can
본 발명의 다른 구현예에서, 안정화제로써 당 또는 당의 유도체를 포함하는 경우, 안정화제의 함량은 본 발명의 안정한 약제학적 제제 총 100 부피 %를 기준으로, 5 내지 20%(w/v), 5 내지 18%(w/v), 5 내지 16%(w/v), 5 내지 14%(w/v), 5 내지 12%(w/v) 또는 5 내지 10%(w/v)일 수 있다.In another embodiment of the present invention, when sugar or a derivative of sugar is included as a stabilizer, the content of the stabilizer is 5 to 20% (w/v), based on 100% by volume of the total stable pharmaceutical formulation of the present invention; 5 to 18% (w/v), 5 to 16% (w/v), 5 to 14% (w/v), 5 to 12% (w/v) or 5 to 10% (w/v) days can
상기 안정화제의 함량 범위 내에서, 장기간동안 고분자량 또는 저분자량 성분은 낮게 유지되고, 온전한 면역글로불린 G성분 또는 온전한 중쇄 및 경쇄의 함량은 높게 유지되며, 장기간 안정성이 우수하며 저점도를 나타낼 수 있다.Within the content range of the stabilizer, high molecular weight or low molecular weight components are maintained low for a long period of time, the content of intact immunoglobulin G component or intact heavy and light chains is maintained high, and long-term stability is excellent and low viscosity. .
(D) 계면활성제(D) surfactant
본 발명의 일 구현예에서, 본 발명에 따른 계면활성제는 폴리옥시에틸렌소르비탄지방산에스테르 (예를 들면, 폴리소르베이트), 폴리옥시에틸렌알킬에테르 (예를 들면, Brij), 알킬페닐폴리옥시에틸렌에테르 (예를 들면, Triton-X), 폴리옥시에틸렌-폴리옥시프로필렌 코폴리머 (예를 들면, Poloxamer, Pluronic), 나트륨 도데실 설페이트 (SDS), 폴록사머 및 이들의 혼합물 등일 수 있으나, 이에 한정되는 것은 아니다.In one embodiment of the present invention, the surfactant according to the present invention is polyoxyethylene sorbitan fatty acid ester (eg, polysorbate), polyoxyethylene alkyl ether (eg, Brij), alkylphenylpolyoxyethylene ethers (eg Triton-X), polyoxyethylene-polyoxypropylene copolymers (eg Poloxamer, Pluronic), sodium dodecyl sulfate (SDS), poloxamers and mixtures thereof, and the like, but are limited thereto. it is not going to be
본 발명의 일 구현예에서, 상기 계면활성제는 폴리소르베이트, 폴록사머 또는 이들의 혼합물일 수 있으며, 바람직하게는 폴리옥시에틸렌소르비탄지방산에스테르 (폴리소르베이트), 폴록사머 또는 이들의 혼합물일 수 있다. 상기 폴리소르베이트는 폴리소르베이트 20, 폴리소르베이트 40, 폴리소르베이트 60, 폴리소르베이트 80 또는 이들 중 2 이상의 혼합물일 수 있으나, 이에 한정되는 것은 아니다. 상기 폴록사머는 폴록사머 188일 수 있으나, 이에 한정되는 것은 아니다. 본 발명의 다른 구현예에서, 상기 계면활성제는 가장 바람직하게 폴리소르베이트 80일 수 있다.In one embodiment of the present invention, the surfactant may be polysorbate, poloxamer, or a mixture thereof, preferably polyoxyethylene sorbitan fatty acid ester (polysorbate), poloxamer, or a mixture thereof. there is. The polysorbate may be polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, or a mixture of two or more thereof, but is not limited thereto. The poloxamer may be poloxamer 188, but is not limited thereto. In another embodiment of the present invention, the surfactant may most preferably be polysorbate 80.
본 발명의 일 구현예에서, 상기 계면활성제의 농도는 본 발명에 따른 안정한 약제학적 제제의 안정성 및 점도에 악영향을 미치지 않는 범위 내에서 자유롭게 조절할 수 있다. 예를 들어, 본 발명에 따른 계면활성제의 농도는 0.01 내지 0.1 %(w/v), 바람직하게는 0.01 내지 0.08%(w/v), 더 바람직하게는 0.01 내지 0.07%(w/v) 일 수 있다. 본 발명에 있어, 상기 계면활성제의 농도는 가장 바람직하게 0.05%(w/v)일 수 있다.In one embodiment of the present invention, the concentration of the surfactant can be freely adjusted within a range that does not adversely affect the stability and viscosity of the stable pharmaceutical formulation according to the present invention. For example, the concentration of the surfactant according to the present invention is 0.01 to 0.1% (w/v), preferably 0.01 to 0.08% (w/v), more preferably 0.01 to 0.07% (w/v) days can In the present invention, the concentration of the surfactant may be most preferably 0.05% (w/v).
(E) pH(E) pH
본 발명의 일 구현예에서, 본 발명에 따른 조성물의 pH는 5.0 내지 7.0일 수 있으며, 5.5 내지 7.0, 5.7 내지 7.0, 6.0 내지 7.0, 6.3 내지 7.0 또는 6.5 내지 7.0일수 있다. In one embodiment of the present invention, the pH of the composition according to the present invention may be 5.0 to 7.0, 5.5 to 7.0, 5.7 to 7.0, 6.0 to 7.0, 6.3 to 7.0 or 6.5 to 7.0.
본 발명의 다른 구현예에서, 본 발명에 따른 조성물의 pH는 5.5 내지 6.5일 수 있으며, 5.7 내지 6.5, 6.0 내지 6.5, 또는 6.3 내지 6.5일수 있다. In another embodiment of the present invention, the pH of the composition according to the present invention may be 5.5 to 6.5, 5.7 to 6.5, 6.0 to 6.5, or 6.3 to 6.5.
본 발명의 다른 구현예에서, 본 발명에 따른 조성물의 pH는 5.5 내지 6.3일 수 있으며, 5.7 내지 6.3, 또는 6.0 내지 6.3일수 있다. In another embodiment of the present invention, the pH of the composition according to the present invention may be 5.5 to 6.3, 5.7 to 6.3, or 6.0 to 6.3.
본 발명의 다른 구현예에서, 본 발명에 따른 조성물의 pH는 5.5 내지 6.0일 수 있으며, 5.7 내지 6.0일수 있다. In another embodiment of the present invention, the pH of the composition according to the present invention may be 5.5 to 6.0, and may be 5.7 to 6.0.
본 발명의 다른 구현예에서, 본 발명에 따른 조성물의 pH는 바람직하게는 6.0일 수 있다. 상기 pH 범위 내에서, 장기간 동안 안정성 및 저점도를 우수하게 나타낼 수 있다. pH는 완충제를 이용하여 조절할 수 있으며, 완충제를 소정의 함량으로 포함하는 경우 별도의 pH 조절제 없이도 상기 범위의 pH를 나타낼 수 있다. In another embodiment of the present invention, the pH of the composition according to the present invention may preferably be 6.0. Within the above pH range, it can exhibit excellent stability and low viscosity for a long period of time. The pH can be adjusted using a buffer, and when the buffer is included in a predetermined amount, the pH of the above range can be exhibited without a separate pH adjusting agent.
(F) "안정한" 약제학적 제제(F) “stable” pharmaceutical formulations
본 명세서에서 용어 "안정한"은 본 발명에 따른 중화 결합 분자가 제조 공정 동안 및/또는 보관/저장 시에 물리적 안정성, 화학적 안정성 및/또는 생물학적 활성을 실질적으로 보유하는 것을 의미한다. 안정성 측정시, 당해 기술분야에서 용이하게 이용할 수 있는, 항체의 안정성을 측정하는 다양한 분석학적 기술을 이용할 수 있다.As used herein, the term “stable” means that the neutralizing binding molecule according to the present invention substantially retains physical stability, chemical stability and/or biological activity during the manufacturing process and/or upon storage/storage. When measuring stability, various analytical techniques for measuring the stability of an antibody that are readily available in the art may be used.
본 발명에 있어, 상기 물리적 안정성은 당해 기술분야에 공지된 방법으로 평가할 수 있으며, 이러한 방법은 광(흡광 또는 광학 밀도)의 샘플 겉보기 감쇠 측정을 포함한다. 이러한 광 감쇠 측정은 제제의 탁도와 관련된다. 또한, 물리적 안정성을 고분자량 성분 함량, 저분자량 성분 함량, 온전한 단백질량, 불용성 이물 입자수 등으로 측정할 수 있다.In the present invention, the physical stability can be evaluated by methods known in the art, which include measuring the sample apparent attenuation of light (absorption or optical density). This light attenuation measurement is related to the turbidity of the formulation. In addition, the physical stability can be measured by high molecular weight component content, low molecular weight component content, intact protein amount, the number of insoluble foreign particles, and the like.
본 발명에 있어, 상기 화학적 안정성은, 예를 들어, 화학적으로 변화된 형태의 중화 결합 분자를 검출하고 정량함으로써 평가할 수 있다. 상기 화학적 안정성은, 예를 들어 이온 교환 크로마토그래피에 의해 평가될 수 있는 하전 변화 (예: 탈아미드화 또는 산화의 결과로서 발생)를 포함한다. 화학적 안정성을 전하 변형체(산성 또는 염기성 피크) 등으로 측정할 수 있다.In the present invention, the chemical stability can be evaluated, for example, by detecting and quantifying a chemically changed form of a neutralizing binding molecule. Such chemical stability includes charge changes (eg, occurring as a result of deamidation or oxidation) that can be assessed, for example, by ion exchange chromatography. Chemical stability can be measured with charge variants (acidic or basic peaks) or the like.
본 발명에 있어, 상기 생물학적 활성은 당해 기술분야에 공지된 방법으로 평가할 수 있으며, 예를 들어 ELISA를 통해 항원 결합 친화도를 측정할 수 있다.In the present invention, the biological activity may be evaluated by a method known in the art, for example, antigen binding affinity may be measured through ELISA.
본 발명의 일 구현예에서, 용어 "안정한 약제학적 제제"는 다음 중 하나 이상을 만족하는 약제학적 제제를 의미한다.In one embodiment of the present invention, the term "stable pharmaceutical preparation" refers to a pharmaceutical preparation satisfying one or more of the following.
(F)-1 탁도 (F)-1 Turbidity
- 온도 5±3℃ 조건에서 4주 동안 보관한 후 분광 광도계로 측정한 흡광도 A600이 0 내지 0.0700 또는 0 내지 0.0400인 약제학적 제제;- A pharmaceutical formulation having an absorbance A600 of 0 to 0.0700 or 0 to 0.0400 measured with a spectrophotometer after storage for 4 weeks at a temperature of 5±3° C.;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 분광 광도계로 측정한 흡광도 A600이 0 내지 0.0700 또는 0 내지 0.0400인 약제학적 제제;- a pharmaceutical formulation having an absorbance A600 of 0 to 0.0700 or 0 to 0.0400 measured with a spectrophotometer after storage for 4 weeks at a temperature of 40±2° C., a relative humidity of 75±5%, and a closed condition;
(F)-2 주성분 함량(메인 피크)(F)-2 main component content (main peak)
- 온도 5±3℃ 조건에서 4주 동안 보관한 후 SE-HPLC로 측정한 주성분이 98 내지 100%인 약제학적 제제;- A pharmaceutical formulation containing 98 to 100% of the main component as measured by SE-HPLC after storage for 4 weeks at a temperature of 5±3° C.;
- 온도 40±2℃, 상대습도 75±5% 및 밀폐 조건에서 4주 동안 보관한 후 SE-HPLC로 측정한 주성분이 97 내지 100%인 약제학적 제제;- A pharmaceutical formulation containing 97 to 100% of the main component as measured by SE-HPLC after storage for 4 weeks at a temperature of 40±2° C., a relative humidity of 75±5% and a closed condition;
(F)-3 고분자량 성분(메인 피크(온전한 중화 결합 분자)를 기준으로 체류 시간(retention time)이 앞쪽인 피크)(F)-3 high molecular weight component (peak with retention time ahead of the main peak (intact neutralizing binding molecule))
- 온도 5±3℃ 조건에서 4주 동안 보관한 후 SE-HPLC로 측정한 고분자량 성분이 0 내지 1.50%인 약제학적 제제;- A pharmaceutical formulation containing 0 to 1.50% of a high molecular weight component measured by SE-HPLC after storage at a temperature of 5±3° C. for 4 weeks;
- 온도 40±2℃, 상대습도 75±5% 및 밀폐 조건에서 4주 동안 보관한 후 SE-HPLC로 측정한 고분자량 성분이 0 내지 2.0%인 약제학적 제제;- A pharmaceutical formulation containing 0 to 2.0% of high molecular weight components as measured by SE-HPLC after storage for 4 weeks at a temperature of 40±2° C., a relative humidity of 75±5% and a closed condition;
(F)-4 저분자량 성분(메인 피크(온전한 중화 결합 분자)를 기준으로 체류 시간(retention time)이 뒤쪽인 피크)(F)-4 low-molecular-weight component (peak with retention time lag behind the main peak (intact neutralizing binding molecule))
- 온도 5±3℃ 조건에서 4주 동안 보관한 후 SE-HPLC로 측정한 저분자량 성분이 0.0 내지 0.5%인 약제학적 제제;- A pharmaceutical formulation having a low molecular weight component of 0.0 to 0.5% as measured by SE-HPLC after storage at a temperature of 5±3° C. for 4 weeks;
- 온도 40±2℃, 상대습도 75±5% 및 밀폐 조건에서 4주 동안 보관한 후 SE-HPLC로 측정한 저분자량 성분이 0.0 내지 1.5%인 약제학적 제제;- A pharmaceutical formulation containing 0.0 to 1.5% of low molecular weight components measured by SE-HPLC after storage for 4 weeks at a temperature of 40±2° C., a relative humidity of 75±5% and a closed condition;
(F)-5 불용성 이물 입자수(F)-5 Number of insoluble foreign matter particles
- 온도 5±3℃에서 4주 동안 보관 후 HIAC으로 측정한 불용성 이물 입자(10.00㎛≤, <400.00㎛)의 개수는 0 내지 100개인 약제학적 제제;- A pharmaceutical formulation in which the number of insoluble foreign particles (10.00 μm≤, <400.00 μm) measured by HIAC after storage for 4 weeks at a temperature of 5±3° C. is 0 to 100;
- 온도 5±3℃에서 4주 동안 보관 후 HIAC으로 측정한 불용성 이물 입자(25.00㎛≤, <400.00㎛)의 개수는 0 내지 50개인 약제학적 제제;- A pharmaceutical formulation in which the number of insoluble foreign particles (25.00 μm≤, <400.00 μm) measured by HIAC after storage for 4 weeks at a temperature of 5±3° C. is 0 to 50;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 HIAC으로 측정한 불용성 이물 입자(10.00㎛≤, <400.00㎛)의 개수는 0 내지 200개인 약제학적 제제;- The number of insoluble foreign particles (10.00㎛≤, <400.00㎛) measured by HIAC after storage for 4 weeks at a temperature of 40±2℃, a relative humidity of 75±5%, and a closed condition is 0 to 200 pharmaceutical formulations ;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 HIAC으로 측정한 불용성 이물 입자(25.00㎛≤, <400.00㎛)의 개수는 0 내지 50개인 약제학적 제제;- The number of insoluble foreign particles (25.00㎛≤, <400.00㎛) measured by HIAC after storage for 4 weeks at a temperature of 40±2℃, a relative humidity of 75±5%, and a closed condition is 0 to 50 pharmaceutical formulations ;
- 온도 5±3℃에서 4주 동안 보관 후 MFI로 측정한 불용성 이물 입자(1.00㎛≤, <400.00㎛)의 개수는 0 내지 5,000개인 약제학적 제제;- A pharmaceutical formulation in which the number of insoluble foreign particles (1.00 μm≤, <400.00 μm) measured by MFI after storage for 4 weeks at a temperature of 5±3° C. is 0 to 5,000;
- 온도 5±3℃에서 4주 동안 보관 후 MFI로 측정한 불용성 이물 입자(10.00㎛≤, <400.00㎛)의 개수는 0 내지 300개인 약제학적 제제;- A pharmaceutical formulation in which the number of insoluble foreign particles (10.00 μm≤, <400.00 μm) measured by MFI after storage for 4 weeks at a temperature of 5±3° C. is 0 to 300;
- 온도 5±3℃에서 4주 동안 보관 후 MFI로 측정한 불용성 이물 입자(25.00㎛≤, <400.00㎛)의 개수는 0 내지 30개인 약제학적 제제;- A pharmaceutical formulation in which the number of insoluble foreign particles (25.00 μm≤, <400.00 μm) measured by MFI after storage for 4 weeks at a temperature of 5±3° C. is 0 to 30;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 MFI로 측정한 불용성 이물 입자(1.00㎛≤, <400.00㎛)의 개수는 0 내지 20,000개인 약제학적 제제;- The number of insoluble foreign particles (1.00㎛≤, <400.00㎛) measured by MFI after storage for 4 weeks at a temperature of 40±2℃, a relative humidity of 75±5%, and a closed condition is 0 to 20,000 pharmaceutical formulations ;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 MFI로 측정한 불용성 이물 입자(10.00㎛≤, <400.00㎛)의 개수는 0 내지 300개인 약제학적 제제;- The number of insoluble foreign particles (10.00㎛≤, <400.00㎛) measured by MFI after storage for 4 weeks at a temperature of 40±2℃, a relative humidity of 75±5%, and a closed condition is 0 to 300 pharmaceutical formulations ;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 MFI로 측정한 불용성 이물 입자(25.00㎛≤, <400.00㎛)의 개수는 0 내지 30개인 약제학적 제제;- The number of insoluble foreign particles (25.00㎛≤, <400.00㎛) measured by MFI after storage for 4 weeks at a temperature of 40±2℃, a relative humidity of 75±5%, and a closed condition is 0 to 30 pharmaceutical formulations ;
(F)-6 산화율(F)-6 oxidation rate
- 온도 5±3℃에서 4주 동안 보관한 후 LC-MS로 측정한 중쇄 Met 263의 산화율이 0% 내지 10%인 약제학적 제제;- a pharmaceutical formulation having an oxidation rate of 0% to 10% of heavy chain Met 263 measured by LC-MS after storage at a temperature of 5±3° C. for 4 weeks;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 LC-MS로 측정한 중쇄 Met 263의 산화율이 0% 내지 10%인 약제학적 제제;- A pharmaceutical formulation having an oxidation rate of 0% to 10% of heavy chain Met 263 measured by LC-MS after storage for 4 weeks at a temperature of 40±2° C., a relative humidity of 75±5%, and a closed condition;
(F)-7 전하 변형체(F)-7 charge variant
- 온도 5±3℃에서 4주 동안 보관한 후 IEC-HPLC로 측정한 메인 피크 함량이 61% 내지 67%인 약제학적 제제;- a pharmaceutical formulation having a main peak content of 61% to 67% as measured by IEC-HPLC after storage at a temperature of 5±3° C. for 4 weeks;
- 온도 40±2℃, 상대습도 75±5%, 및 밀폐 조건에서 4주 동안 보관한 후 IEC-HPLC로 측정한 메인 피크 함량이 86% 내지 87%인 약제학적 제제.- A pharmaceutical formulation having a main peak content of 86% to 87% as measured by IEC-HPLC after storage for 4 weeks at a temperature of 40±2° C., a relative humidity of 75±5%, and sealed conditions.
[안정한 약제학적 제제의 제조방법][Method for preparing stable pharmaceutical preparations]
본 발명의 안정한 약제학적 제제는 공지된 방법을 이용하여 제조할 수 있으며, 특정 제조 방법으로 제한되지 않는다. 예를 들어, 안정화제 및 계면활성제를 포함하는 용액에 완충제를 첨가하면서 pH를 조절한 후, 이 혼합 용액에 중화 결합 분자를 넣어 약제학적 제제를 제조할 수 있다. 또한, 정제 공정의 최종 단계에서 일부 부형제를 포함하는 용액을 제조한 후 나머지 성분을 첨가하여 약제학적 제제를 제조할 수 있다. The stable pharmaceutical formulation of the present invention can be prepared using a known method, and is not limited to a specific manufacturing method. For example, after adjusting the pH by adding a buffer to a solution containing a stabilizer and a surfactant, a neutralizing binding molecule may be added to the mixed solution to prepare a pharmaceutical formulation. In addition, after preparing a solution containing some excipients in the final step of the purification process, the remaining ingredients may be added to prepare a pharmaceutical formulation.
본 발명의 일 구체예에서, 상기 제제는 제조 시 동결 건조 공정을 포함하지 않을 수 있다. 동결 건조 공정을 포함하지 않은 경우, 예를 들어, 본 발명의 약제학적 제제를 제조하고 멸균 등의 처리 후 바로 1차 포장재인 유리 바이알이나 프리-필드 시린지와 같은 밀폐 용기에 담길 수 있다.In one embodiment of the present invention, the preparation may not include a freeze-drying process during manufacture. If the freeze-drying process is not included, for example, the pharmaceutical formulation of the present invention may be prepared and placed in an airtight container such as a glass vial or pre-filled syringe, which is a primary packaging material, immediately after treatment such as sterilization.
본 발명의 다른 구체예에서, 본 발명의 안정한 약제학적 제제는 (A) 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자 5 내지 240 mg/ml; (B) 완충제 1 내지 50 mM; (C) 안정화제 50 내지 200 mM; 및 (D) 계면활성제 0.01 내지 0.1 %(w/v)를 포함하는 것일 수 있다.In another embodiment of the present invention, the stable pharmaceutical formulation of the present invention is (A) a neutralizing binding molecule 5 that binds to the spike protein (S protein) on the surface of SARS-CoV-2 (SARS-CoV-2) to 240 mg/ml; (B) 1-50 mM buffer; (C) 50-200 mM stabilizer; and (D) 0.01 to 0.1% (w/v) of a surfactant.
본 발명의 다른 구체예에서, 본 발명은 상기 안정한 약제학적 제제; 및 상기 안정한 약제학적 제제를 밀폐된 상태로 수용하는 용기를 포함하는 제품을 제공할 수 있다. In another embodiment of the present invention, the present invention provides the above stable pharmaceutical formulation; And it may provide a product comprising a container for accommodating the stable pharmaceutical formulation in a closed state.
상기 안정한 약제학적 제제는 상술한 바와 같다. The stable pharmaceutical formulation is as described above.
본 발명의 일 구현예에서, 상기 용기는 유리, 폴리머(플라스틱), 금속 등의 물질로부터 형성될 수 있으나, 이에 제한되지 않는다. 본 발명의 일 구현예에서, 상기 용기는 병, 바이알(vial), 카트리지, 주사기(프리-필드 시린지(pre-filled syringe)), 또는 튜브일 수 있으나, 이에 제한되지 않는다. 본 발명의 일 구현예에서, 상기 용기는 유리 또는 폴리머 바이알, 또는 유리 또는 폴리머 프리-필드 시린지일 수 있다. In one embodiment of the present invention, the container may be formed of a material such as glass, polymer (plastic), or metal, but is not limited thereto. In one embodiment of the present invention, the container may be a bottle, a vial, a cartridge, a syringe (pre-filled syringe), or a tube, but is not limited thereto. In one embodiment of the present invention, the container may be a glass or polymer vial, or a glass or polymer pre-filled syringe.
상기 바이알, 카트리지, 프리-필드 시린지 등의 구체적인 제품 형태와 상기 안정한 약제학적 제제를 상기 바이알, 카트리지, 프리-필드 시린지 등에 충진하는 방법은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면 용이하게 입수하거나 실시할 수 있다. 예를 들어, 미국 특허 제4,861,335호, 제6,331,174호 등은 프리-필드 시린지의 구체적인 제품 형태 및 충진 방법을 개시한다. 또한, 상기 바이알, 카트리지, 프리-필드 시린지 등으로서 상용화된 제품을 그대로 이용하거나, 상기 안정한 약제학적 제제의 물성, 투여부위, 투여량 등을 고려하여 별도로 주문 제작한 제품을 이용할 수 있다. The specific product form of the vial, cartridge, pre-filled syringe, etc. and the method of filling the stable pharmaceutical formulation into the vial, cartridge, pre-filled syringe, etc. are easy for those of ordinary skill in the art to which the present invention pertains can be obtained or practiced. For example, US Pat. Nos. 4,861,335, 6,331,174, etc. disclose specific product forms and filling methods of pre-filled syringes. In addition, commercially available products as the vial, cartridge, pre-filled syringe, etc. may be used as it is, or a separately customized product may be used in consideration of the physical properties, administration site, dosage, etc. of the stable pharmaceutical formulation.
본 발명의 일 구현예에서, 상기 제품은 상기 안정한 약제학적 제제의 사용방법, 보관방법 또는 이들 모두를 제공하는 지시사항을 더 포함할 수 있다. In one embodiment of the present invention, the product may further include instructions for providing a method of use, a method of storage, or both of the stable pharmaceutical formulation.
본 발명의 일 구현예에서, 상기 제품은 상업적 및 사용자 관점에서 필요한 기타 도구, 예를 들어 바늘, 주사기 등을 포함할 수 있다.In one embodiment of the present invention, the product may include other tools necessary from a commercial and user point of view, for example, needles, syringes, and the like.
이하 본 발명에서 사용되는 용어를 다음과 같이 정의한다.Hereinafter, terms used in the present invention are defined as follows.
본 발명에서 사용되는 용어 "결합 분자"는 키메라, 인간화 또는 인간 단일클론 항체와 같은 단일클론 항체를 포함하는 온전한(intact) 이뮤노글로불린(immunoglobulin), 또는 항원에 결합하는 이뮤노글로불린인 항원-결합 단편을 포함한다. 예를 들면 SARS-CoV-2의 스파이크 단백질(spike protein)과 결합에 있어서, 온전한(intact) 이뮤노글로불린과 경쟁하는 이뮤노글로불린 단편을 포함하는 가변성 도메인, 효소, 수용체, 단백질을 뜻한다. 구조와는 상관없이 항원-결합 단편은 온전한(intact) 이뮤노글로불린에 의해 인식된 동일한 항원과 결합된다. 상기 항원-결합 단편은 항체의 아미노산 서열의 2개 이상의 연속기, 20개 이상의 연속 아미노산 잔기, 25개 이상의 연속 아미노산 잔기, 30개 이상의 연속 아미노산 잔기, 35개 이상의 연속 아미노산 잔기, 40개 이상의 연속 아미노산 잔기, 50개 이상의 연속 아미노산 잔기, 60개 이상의 연속 아미노산 잔기, 70개 이상의 연속 아미노산 잔기, 80개 이상의 연속 아미노산 잔기, 90개 이상의 연속 아미노산 잔기, 100개 이상의 연속 아미노산 잔기, 125개 이상의 연속 아미노산 잔기, 150개 이상의 연속 아미노산 잔기, 175개 이상 연속 아미노산 잔기, 200개 이상의 연속 아미노산 잔기, 또는 250개 이상의 연속 아미노산 잔기의 아미노산 서열을 포함하는 펩티드 또는 폴리펩티드를 포함할 수 있다. As used herein, the term “binding molecule” refers to an intact immunoglobulin, including monoclonal antibodies, such as chimeric, humanized or human monoclonal antibodies, or antigen-binding, which is an immunoglobulin that binds to an antigen. Includes fragments. For example, in binding to the spike protein of SARS-CoV-2, it refers to a variable domain, enzyme, receptor, or protein comprising an immunoglobulin fragment that competes with an intact immunoglobulin. Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact immunoglobulin. The antigen-binding fragment comprises at least two contiguous groups of the amino acid sequence of the antibody, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues , at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, a peptide or polypeptide comprising an amino acid sequence of at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues.
본 발명에 있어, 용어 "항원-결합 단편"은 특히 Fab, F(ab'), F(ab')2, Fv, dAb, Fd, 상보성 결정 영역(CDR) 단편, 단일-쇄 항체(scFv), 2가(bivalent) 단일-쇄 항체, 단일-쇄 파지 항체, 디아바디(diabody), 트리아바디, 테트라바디, 폴리펩티드로의 특정 항원에 결합하기에 충분한 이뮤노글로불린의 하나 이상의 단편을 함유하는 폴리펩티드 등을 포함한다. 상기 단편은 합성으로 또는 완전한 이뮤노글로불린의 효소적 또는 화학적 분해에 의해 생성되거나, 재조합 DNA 기술에 의해 유전공학적으로 생성될 수 있다. 생성 방법은 당업계에 잘 알려져 있다.In the context of the present invention, the term "antigen-binding fragment" particularly refers to Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv). , bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind a particular antigen to the polypeptide. etc. The fragments may be produced synthetically or by enzymatic or chemical digestion of complete immunoglobulins, or may be genetically engineered by recombinant DNA techniques. Methods of production are well known in the art.
본원에 기재된 상기 각 특징들은 조합되어 사용될 수 있으며, 상기 각 특징들이 특허청구범위의 서로 다른 종속항에 기재된다는 사실은 이들이 조합되어 사용될 수 없음을 나타내는 것은 아니다.Each of the features described herein may be used in combination, and the fact that each of the features is recited in different dependent claims of the claims does not indicate that they cannot be used in combination.
본 발명에 따른 안정한 약제학적 제제는 가속 조건 및 가혹 조건과 같은 온도 조건에서 장기간 보관 안정성이 매우 뛰어나며, 빛, 얼림/녹임, 흔들림과 같은 물리적 스트레스 조건에서도 우수한 안정성을 유지할 수 있다.The stable pharmaceutical formulation according to the present invention has excellent long-term storage stability under temperature conditions such as accelerated conditions and severe conditions, and can maintain excellent stability even under physical stress conditions such as light, freezing/thawing, and shaking.
도 1은 실시예 1 내지 3의 주성분 함량의 측정 결과이다.1 is a measurement result of the main component content of Examples 1 to 3.
도 2는 실시예 1 내지 3의 고분자량 성분 함량의 측정 결과이다.2 is a measurement result of the high molecular weight component content of Examples 1 to 3.
도 3은 실시예 1 내지 3의 저분자량 성분 함량의 측정 결과이다.3 is a measurement result of the content of low molecular weight components of Examples 1 to 3.
도 4는 실시예 1 내지 3의 온전한 면역 글로불린G 함량의 측정 결과이다.4 is a measurement result of the intact immunoglobulin G content of Examples 1 to 3.
도 5는 실시예 1 내지 3의 항체의 경쇄 및 중쇄 함량의 측정 결과이다.5 is a measurement result of the light chain and heavy chain content of the antibodies of Examples 1 to 3.
도 6은 실시예 1 내지 3의 전하 변형체 (메인 피크%) 함량의 측정 결과이다.6 is a measurement result of the charge variant (main peak %) content of Examples 1 to 3;
도 7은 실시예 1 내지 3의 전하 변형체 (산성 피크%) 함량의 측정 결과이다.7 is a measurement result of the charge variant (acid peak %) content of Examples 1 to 3;
도 8은 실시예 1 내지 3의 전하 변형체 (염기성 피크%) 함량의 측정 결과이다.8 is a measurement result of the charge variant (basic peak %) content of Examples 1 to 3;
도 9는 실시예 1, 4 내지 6의 점도의 측정 결과이다.9 is a measurement result of the viscosity of Examples 1, 4 to 6;
도 10은 실시예 1, 7 내지 16의 온도 5±3℃에서 주성분 함량의 측정 결과이다.10 is a measurement result of the content of the main component at a temperature of 5±3° C. in Examples 1 and 7 to 16. FIG.
도 11은 실시예 1, 7 내지 16의 온도 25±2℃에서 주성분 함량의 측정 결과이다.11 is a measurement result of the content of the main component at a temperature of 25±2° C. in Examples 1 and 7 to 16. FIG.
도 12는 실시예 1, 7 내지 16의 온도 40±2℃에서 주성분 함량의 측정 결과이다.12 is a measurement result of the content of the main component at a temperature of 40±2° C. in Examples 1 and 7 to 16. FIG.
도 13은 실시예 1, 7 내지 16의 온전한 면역 글로불린G 함량의 측정 결과이다.13 is a measurement result of intact immunoglobulin G content of Examples 1 and 7 to 16.
도 14는 실시예 1, 7 내지 16의 항체의 경쇄 및 중쇄 함량의 측정 결과이다.14 is a measurement result of the light chain and heavy chain content of the antibodies of Examples 1 and 7 to 16.
도 15은 실시예 1, 7 내지 16의 전하 변형체 (메인 피크%) 함량의 측정 결과이다.15 is a measurement result of the content of charge variants (main peak %) of Examples 1 and 7 to 16;
도 16은 실시예 1, 7 내지 16의 전하 변형체 (산성 피크%) 함량의 측정 결과이다.16 is a measurement result of the content of charge variants (acid peak %) of Examples 1 and 7 to 16;
도 17은 실시예 1, 7 내지 16의 전하 변형체 (염기성 피크%) 함량의 측정 결과이다.17 is a measurement result of the content of charge variants (basic peak %) of Examples 1 and 7 to 16;
도 18은 실시예 17 내지 19의 항체의 경쇄 및 중쇄 함량의 측정 결과이다.18 is a measurement result of the light chain and heavy chain content of the antibodies of Examples 17 to 19.
도 19는 실시예 17 내지 19의 온전한 면역 글로불린G 함량의 측정 결과이다.19 is a measurement result of the intact immunoglobulin G content of Examples 17 to 19.
도 20은 실시예 17 내지 19의 주성분 함량의 측정 결과이다.20 is a measurement result of the main component content of Examples 17 to 19.
도 21은 실시예 17 내지 19의 고분자량 성분 함량의 측정 결과이다.21 is a measurement result of the high molecular weight component content of Examples 17 to 19.
도 22는 실시예 17 내지 19의 저분자량 성분 함량의 측정 결과이다.22 is a measurement result of the content of low molecular weight components of Examples 17 to 19.
도 23은 실시예 1, 20 내지 31 및 비교예 1의 온전한 면역 글로불린G 함량의 측정 결과이다.23 is a measurement result of intact immunoglobulin G content of Examples 1, 20 to 31, and Comparative Example 1. FIG.
도 24는 실시예 1, 20 내지 31 및 비교예 1의 항체의 경쇄 및 중쇄 함량의 측정 결과이다.24 is a measurement result of the light chain and heavy chain content of the antibodies of Examples 1, 20 to 31 and Comparative Example 1.
도 25는 실시예 1, 20 내지 31 및 비교예 1의 주성분 함량의 측정 결과이다.25 is a measurement result of the main component content of Examples 1, 20 to 31, and Comparative Example 1.
이하 본 발명을 실시예를 통해 상세히 설명한다. 하기 실시예들은 본 발명의 내용을 예시하는 것일 뿐 발명의 범위가 실시예에 의해 한정되지 않는다. 본 발명에서 인용된 문헌은 본 발명의 명세서에 참조로서 통합된다.Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited by the examples. The documents cited herein are incorporated herein by reference.
아래 실험예에서 사용된 중화 결합 분자와 관련하여, 안정화제를 첨가한 완충액을 최적의 완충 능력을 보이는 pH에 맞게 제조한 뒤, 해당 용액에 SARS-CoV-2 중화 결합 분자를 첨가하고, 계면활성제를 목표 농도에 도달하도록 첨가하여 해당 제형 성분을 제조하였다.With respect to the neutralizing binding molecules used in the experimental examples below, a buffer solution containing a stabilizer was prepared at a pH that showed the optimal buffering ability, and then SARS-CoV-2 neutralizing binding molecules were added to the solution, and a surfactant was added. was added to reach the target concentration to prepare the corresponding formulation component.
또한, 아래 실험예에서 사용된 약제학적 제제의 물리적 안정성, 화학적 안정성 및 생물학적 활성의 측정방법으로서 다음과 같은 방법을 사용하였다.In addition, the following method was used as a method for measuring the physical stability, chemical stability, and biological activity of the pharmaceutical formulation used in the experimental examples below.
- 탁도(Turbidity)- Turbidity
UV-Vis 분광 광도계를 이용하여 600 nm에서의 흡광도를 측정하였다.The absorbance at 600 nm was measured using a UV-Vis spectrophotometer.
- 주성분 함량- Main ingredient content
크기 배제 고성능 액체 크로마토그래피(Size Exclusion HPLC)를 이용하여 주성분 함량(main peak; %)을 측정하였다.The main component content (%) was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC).
- 고분자량 성분 함량- High molecular weight component content
크기 배제 고성능 액체 크로마토그래피(Size Exclusion HPLC)를 이용하여 고분자량 성분의 함량(pre-peak; %)을 측정하였다.The content (pre-peak; %) of the high molecular weight component was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC).
- 저분자량 성분 함량- Low molecular weight ingredient content
크기 배제 고성능 액체 크로마토그래피(Size Exclusion HPLC)를 이용하여 저분자량 성분의 함량(post-peak; %)을 측정하였다.The content (post-peak; %) of low molecular weight components was measured using size exclusion high performance liquid chromatography (Size Exclusion HPLC).
- 온전한 면역 글로불린 G의 함량- Content of intact immunoglobulin G
비환원 Chip-based CE-SDS 분석 장비인 Labchip GXII를 이용하여 온전한 면역글로불린 G의 함량을 측정하였다.The content of intact immunoglobulin G was measured using Labchip GXII, a non-reducing chip-based CE-SDS analysis equipment.
- 항체 경쇄와 중쇄의 함량- Content of antibody light and heavy chains
환원 Chip-based CE-SDS 분석 장비인 Labchip GXII를 이용하여 항체의 경쇄와 중쇄의 함량을 측정하였다.The contents of light and heavy chains of the antibody were measured using Labchip GXII, a reduced chip-based CE-SDS analysis equipment.
- 불용성 이물 입자(Sub-visible particles)의 수- Number of insoluble foreign particles (Sub-visible particles)
마이크로 플로우 이미징 (Micro Flow Imaging; MFI) 및 광 차폐형 미립자 계수기 (모델명: HIAC 9703)를 이용하여 불용성 이물 입자의 수를 측정하였다.The number of insoluble foreign particles was measured using Micro Flow Imaging (MFI) and a light-shielding particle counter (model name: HIAC 9703).
- 산화율(Oxidation)- Oxidation
질량분석을 통한 액체 크로마토그래피(LC-MS)로 펩티드 매핑(Peptide mapping)을 통해 중쇄 Met 263의 산화율(%)을 측정하였다.The oxidation rate (%) of heavy chain Met 263 was measured through peptide mapping by liquid chromatography (LC-MS) through mass spectrometry.
- 전하 변형체 - charge variant
이온 교환 크로마토그래피 (Ion exchange chromatography)로 산성 및 염기성 피크(%)를 측정하였다.Acidic and basic peaks (%) were measured by ion exchange chromatography.
- SARS-CoV-2 RBD 결합 친화도- SARS-CoV-2 RBD binding affinity
효소결합면역흡착 분석법(Enzyme-Linked ImmunoSorbent Assay; ELISA)으로 SARS-CoV-2 RBD 결합 친화도(%)를 측정하였다.SARS-CoV-2 RBD binding affinity (%) was measured by Enzyme-Linked ImmunoSorbent Assay (ELISA).
실험예 1: L-아르기닌-HCl/소르비톨/트레할로스 안정화제의 비교Experimental Example 1: Comparison of L-arginine-HCl/sorbitol/trehalose stabilizer
실험예 1에서 사용된 약제학적 제제와 관련하여, 각 완충액을 각 pH에 맞게 제조한 뒤 아르기닌 모노하이드로클로라이드, 소르비톨 및 트레할로스를 첨가하고, 이에 항체를 첨가하고, 계면활성제를 첨가하여 표 5의 시료들을 제조하였다. 실시예 1 내지 3에 따라 제조된 약제학적 제제를 5±3℃ 온도와 50±2℃ 온도에서 보관하였고, 5±3℃ 온도에서 5일 후의 안정성 그리고 50±2℃ 온도에서 3일 후 및 5일 후의 안정성을 측정하였다. 물리적 스트레스를 위해 -40℃에서 얼림과 녹임 5회를 반복하였다. 실험의 결과는 표 6 내지 18 및 도 1 내지 8에 나타내었다.With respect to the pharmaceutical formulation used in Experimental Example 1, each buffer was prepared to suit each pH, and arginine monohydrochloride, sorbitol and trehalose were added thereto, an antibody was added thereto, and a surfactant was added to the sample of Table 5 were manufactured. The pharmaceutical formulations prepared according to Examples 1 to 3 were stored at a temperature of 5±3° C. and a temperature of 50±2° C., stability after 5 days at a temperature of 5±3° C., and after 3 days and 5 at a temperature of 50±2° C. Stability after one day was measured. For physical stress, freezing and thawing at -40°C were repeated 5 times. The results of the experiment are shown in Tables 6 to 18 and FIGS. 1 to 8 .
본 실험예 1에서 사용된 항체는 상기 표 1 내지 표 2에 기재된 No. 139번 결합 분자이다.The antibody used in Experimental Example 1 was No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
실험 결과, 하기 성분으로 제조된 제형은 가속 조건 및 가혹 조건과 같은 온도 조건에서 안정성이 매우 뛰어나며, 얼림/녹임과 같은 물리적 스트레스 조건에서도 우수한 안정성을 유지함을 확인하였다. 실험한 안정화제 중 L-아르기닌 모노하이드로클로라이드를 포함한 제형에서 주성분 함량 및 온전한 면역글로불린 G의 함량이 소르비톨 및 트레할로스를 포함하는 제형보다 높은 온도 조건에서 더 안정함을 알 수 있었다. As a result of the experiment, it was confirmed that the formulation prepared with the following ingredients had excellent stability under temperature conditions such as accelerated conditions and severe conditions, and maintained excellent stability even under physical stress conditions such as freezing/thawing. Among the tested stabilizers, it was found that the main component content and the intact immunoglobulin G content in the formulation containing L-arginine monohydrochloride were more stable under high temperature conditions than the formulation containing sorbitol and trehalose.
구분division 완충제buffer pHpH 안정화제stabilizer 계면활성제Surfactants 단백질(항체) 농도Protein (antibody) concentration
실시예 1Example 1 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌 모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 2Example 2 히스티딘 10 mMhistidine 10 mM 6.06.0 소르비톨 5% (w/v)Sorbitol 5% (w/v) 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 3Example 3 히스티딘 10 mMhistidine 10 mM 6.06.0 트레할로스 10% (w/v)Trehalose 10% (w/v) 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
하기 표 6에서 탁도 측정 결과를 나타내었다.The turbidity measurement results are shown in Table 6 below.
구분division 5±3℃0일 후 (T=0)After 5±3℃ 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예 1Example 1 0.03700.0370 0.00550.0055 0.00960.0096 0.01080.0108 0.00680.0068
실시예 2Example 2 0.01210.0121 0.01620.0162 0.01570.0157 0.04120.0412 0.00700.0070
실시예 3Example 3 0.00820.0082 0.00370.0037 0.00900.0090 0.00590.0059 0.00590.0059
표 6을 보면 5±3℃, 50±2℃ 그리고 얼림/녹임 스트레스 조건에서 실시예 1의 탁도가 0.0400 이하였다. 실시예 2의 경우 50±2℃ 조건에서 실시예 1의 탁도 보다 높았다. Referring to Table 6, the turbidity of Example 1 was 0.0400 or less under conditions of 5±3° C., 50±2° C. and freeze/thaw stress. In the case of Example 2, the turbidity of Example 1 was higher than that of 50±2° C. conditions.
하기 표 7에서 크기배제 크로마토그래피 주성분 함량 (Main peak %) 측정 결과를 나타내었다.Table 7 below shows the measurement results of the main component content (Main peak %) by size exclusion chromatography.
구분division 5±3℃0일 후 (T=0)After 5±3℃ 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예 1Example 1 99.8299.82 99.8299.82 99.6299.62 99.4599.45 99.8699.86
실시예 2Example 2 99.7499.74 99.7899.78 99.5399.53 99.3399.33 99.8899.88
실시예 3Example 3 99.7699.76 99.7899.78 99.3699.36 99.2999.29 99.8799.87
표 7을 보면 5±3℃및 50±2℃ 조건에서 실시예 1이 가장 높은 주성분 함량을 포함하였다. 실시예1 내지 3의 주성분 함량은 50±2℃ 조건에서 5일 후 98.0% 이상으로 안정함을 알 수 있었다. 얼림/녹임 스트레스에 의한 주성분 함량은 실시예 1 내지 3가 유사하며, 98.0% 이상으로 안정함을 알 수 있었다 (도 1).Referring to Table 7, Example 1 contained the highest content of main components under the conditions of 5 ± 3 °C and 50 ± 2 °C. It can be seen that the main component content of Examples 1 to 3 is stable to 98.0% or more after 5 days at 50±2° C. condition. The main component content by freezing/thawing stress was similar to Examples 1 to 3, and it was found that it was stable at 98.0% or more (FIG. 1).
하기 표 8에서 크기배제 크로마토그래피 고분자량 성분 함량 (pre-peak %) 측정 결과를 나타내었다.Table 8 below shows the results of size exclusion chromatography high molecular weight component content (pre-peak %) measurement.
구분division 5±3℃0일 후 (T=0)After 5±3℃ 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예 1Example 1 0.140.14 0.140.14 0.200.20 0.240.24 0.090.09
실시예 2Example 2 0.230.23 0.170.17 0.310.31 0.400.40 0.080.08
실시예 3Example 3 0.200.20 0.170.17 0.480.48 0.410.41 0.100.10
표 8을 보면 실시예 1의 5일 후 5±3℃ 및 3일 및 5일 후 50±2℃ 조건이 가장 낮은 고분자량 성분 함량을 나타내었다. 실시예 1 내지 3의 고분자량 성분 함량은 50±2℃ 조건에서 5일 후 1.00% 이하로 안정함을 알 수 있었다. 얼림/녹임 스트레스에 의한 고분자량 함량은 실시예 1 내지 3가 유사하며, 1.00% 이하로 안정함을 알 수 있었다 (도 2).Referring to Table 8, the conditions of 5±3° C. after 5 days and 50±2° C. after 3 days and 5 days of Example 1 showed the lowest content of high molecular weight components. It was found that the high molecular weight component content of Examples 1 to 3 was stable at 50±2° C. after 5 days at 1.00% or less. The high molecular weight content due to freezing/thawing stress was similar to Examples 1 to 3, and it was found that it was stable at 1.00% or less ( FIG. 2 ).
하기 표 9에서 크기배제 크로마토그래피 저분자량 성분 함량 (post-peak %) 측정 결과를 나타내었다.Table 9 below shows the measurement results of the low molecular weight component content (post-peak %) by size exclusion chromatography.
구분division 5±3℃0일 후 (T=0)After 5±3℃ 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예 1Example 1 0.040.04 0.040.04 0.180.18 0.310.31 0.050.05
실시예 2Example 2 0.040.04 0.040.04 0.160.16 0.270.27 0.040.04
실시예 3Example 3 0.040.04 0.050.05 0.160.16 0.300.30 0.030.03
표 9를 보면 모든 조건에서 실시예 1 내지 3의 저분자량 함량이 0.5% 이하임을 확인하였다 (도 3).Referring to Table 9, it was confirmed that the low molecular weight content of Examples 1 to 3 was 0.5% or less under all conditions (FIG. 3).
하기 표 10에서 온전한 면역 글로불린G의 함량 (비환원 chip-based CE-SDS) 측정 결과를 나타내었다.Table 10 below shows the measurement results of intact immunoglobulin G content (non-reducing chip-based CE-SDS).
구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 94.9394.93 94.9594.95 95.1195.11 95.0895.08 94.9194.91
실시예2Example 2 95.0695.06 95.195.1 94.794.7 94.394.3 95.1495.14
실시예3Example 3 95.2395.23 95.1895.18 94.7394.73 94.5494.54 94.9694.96
(단위: %)(unit: %)
표 10을 보면 실시예 1의 5일 후 5±3℃ 및 3일 및 5일 후 50±2℃ 조건에서 가장 높은 온전한 면역 글로불린G의 함량을 나타내었다. 실시예 1 내지 3의 온전한 면역 글로불린G의 함량은 50±2℃ 조건에서 5일 후 90.0% 이상으로 안정함을 알 수 있었다. 얼림/녹임 스트레스에 의한 고분자량 함량은 실시예 1 내지 3이 유사하며, 90.0% 이상으로 안정함을 알 수 있었다 (도 4).Referring to Table 10, the highest intact immunoglobulin G content was shown in Example 1 at 5±3° C. after 5 days and at 50±2° C. after 3 and 5 days. It was found that the content of intact immunoglobulin G of Examples 1 to 3 was stable to 90.0% or more after 5 days at 50±2°C. The high molecular weight content due to freezing/thawing stress was similar to Examples 1 to 3, and it was found that it was stable at 90.0% or more (FIG. 4).
하기 표 11에서 항체 경쇄와 중쇄의 함량 (환원 chip-based CE-SDS) 측정 결과를 나타내었다.Table 11 below shows the measurement results of the antibody light chain and heavy chain content (reduced chip-based CE-SDS).
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 99.899.8 99.8199.81 99.7399.73 99.6799.67 99.899.8
실시예2Example 2 99.8199.81 99.7999.79 99.7299.72 99.6299.62 99.7999.79
실시예3Example 3 99.7999.79 99.7899.78 99.7299.72 99.6499.64 99.7899.78
(단위: %)(unit: %)
표 11을 통해 모든 조건에서 실시예 1 내지 3의 항체 경쇄와 중쇄 함량이 98.0 % 이상임을 확인하였다 (도 5).From Table 11, it was confirmed that the antibody light chain and heavy chain contents of Examples 1 to 3 were 98.0% or more under all conditions (FIG. 5).
하기 표 12에서 전하 변형체 (메인 피크 %) 측정 결과를 나타내었다.Table 12 below shows the measurement results of the charge variant (main peak %).
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 6565 65.4865.48 64.6764.67 63.3563.35 65.5665.56
실시예2Example 2 65.1265.12 65.3965.39 64.2164.21 62.6762.67 65.3965.39
실시예3Example 3 65.1465.14 64.9264.92 64.0364.03 62.9262.92 65.2565.25
표 12를 통해 모든 조건에서 실시예 1 내지 3의 전하 변형체 (메인 피크 %) 함량이 유사한 수준임을 확인하였다 (도 6).Through Table 12, it was confirmed that the contents of the charge variants (main peak %) of Examples 1 to 3 were at a similar level under all conditions ( FIG. 6 ).
하기 표 13에서 전하 변형체 (산성 피크 %) 측정 결과를 나타내었다.Table 13 below shows the measurement results of the charge variant (acid peak %).
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 7.667.66 7.567.56 10.6610.66 14.8314.83 7.227.22
실시예2Example 2 7.657.65 7.797.79 11.4611.46 15.9815.98 7.557.55
실시예3Example 3 7.717.71 7.847.84 11.6411.64 15.8415.84 7.517.51
표 13을 통해 모든 조건에서 실시예 1 내지 3의 전하 변형체 (산성 피크 %) 함량이 유사한 수준임 확인하였다 (도 7).Through Table 13, it was confirmed that the contents of the charge variants (acid peak %) of Examples 1 to 3 were at a similar level under all conditions ( FIG. 7 ).
하기 표 14에서 전하 변형체 (염기성 피크 %) 측정 결과를 나타내었다.Table 14 below shows the measurement results of charge variants (basic peak %).
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 27.3327.33 26.9726.97 24.6724.67 21.8121.81 27.2327.23
실시예2Example 2 27.2227.22 26.8326.83 24.3324.33 21.3421.34 27.0727.07
실시예3Example 3 27.1427.14 27.2527.25 24.3424.34 21.2321.23 27.2427.24
표 14를 통해 모든 조건에서 실시예 1 내지 3의 전하 변형체 (염기성 피크 %) 함량이 유사한 수준임을 확인하였다(도 8).Through Table 14, it was confirmed that the contents of the charge variants (basic peak %) of Examples 1 to 3 were at a similar level under all conditions ( FIG. 8 ).
하기 표 15에서 MFI 로 측정한 불용성 이물 입자의 수(10.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 15 below shows the measurement results of the number of insoluble foreign particles (10.00 μm≤, <100.00 μm) measured by MFI.
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 33 2626 1111 22 33
실시예2Example 2 55 1010 55 1010 22
실시예3Example 3 33 22 1010 22 1818
표 15를 보면 실시예 1 내지 3의 불용성 이물 입자의 수가 모든 조건에서 6000개 이하로 안정함을 알 수 있었다. Referring to Table 15, it can be seen that the number of insoluble foreign particles of Examples 1 to 3 is stable to 6000 or less under all conditions.
하기 표 16에서 MFI 로 측정한 불용성 이물 입자의 수(25.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 16 below shows the measurement results of the number of insoluble foreign particles (25.00 μm≤, <100.00 μm) measured by MFI.
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 00 00 33 00 22
실시예2Example 2 22 22 33 22 00
실시예3Example 3 00 00 00 00 00
표 16을 보면 실시예 1 내지 3의 불용성 이물 입자의 수가 모든 조건에서 600개 이하로 안정함을 알 수 있었다.Looking at Table 16, it can be seen that the number of insoluble foreign particles of Examples 1 to 3 is stable at 600 or less under all conditions.
하기 표 17에서 HIAC으로 측정한 불용성 이물 입자의 수(10.00≤(um)) 측정 결과를 나타내었다.Table 17 below shows the measurement results of the number of insoluble foreign particles (10.00≤(um)) measured by HIAC.
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 22 00 00 22 00
실시예2Example 2 77 00 33 00 00
실시예3Example 3 00 00 00 33 00
표 17을 보면 실시예 1 내지 3의 불용성 이물 입자의 수가 모든 조건에서 10개 이하로 안정함을 알 수 있다. Referring to Table 17, it can be seen that the number of insoluble foreign particles of Examples 1 to 3 is stable to 10 or less under all conditions.
하기 표 18에서 HIAC으로 측정한 불용성 이물 입자의 수(25.00≤(um)) 측정 결과를 나타내었다.In Table 18, the number of insoluble foreign particles (25.00≤(um)) measured by HIAC is shown.
　구분division 5±3℃0일후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일후5±3℃ after 5 days 50±2℃3일후50±2℃ after 3 days 50±2℃5일후50±2℃ after 5 days 얼림/녹임 스트레스Freeze/Thaw Stress
실시예1Example 1 22 00 00 00 00
실시예2Example 2 00 00 00 00 00
실시예3Example 3 00 00 00 22 00
표 18을 보면 실시예 1 내지 3의 불용성 이물 입자의 수가 모든 조건에서 5개 이하로 안정함을 알 수 있었다.Referring to Table 18, it was found that the number of insoluble foreign particles of Examples 1 to 3 was stable to 5 or less under all conditions.
실험예 2: 고농도 항체 단백질 제제의 비교Experimental Example 2: Comparison of high-concentration antibody protein preparations
실험예 2에서 사용된 약제학적 제제와 관련하여, 각 완충액을 각 pH에 맞게 제조한 뒤 아르기닌 모노하이드로클로라이드를 첨가하고, 이에 항체를 첨가 후 농축하였다. 그 후 계면활성제를 첨가하여 표 19의 시료들을 제조하였다. 실시예 4 내지 6은 셀트리온 연구소에서 농축한 항체를 첨가했으며 실시예 1은 실험예 1에 따라 최종적으로 선정된 제제이다. 제조된 약제학적 제제를 5±3℃ 온도와 40±2℃ 온도 및 75±5% 상대 습도에서 보관하였고, 5±3℃ 온도에서 2주 및 4주 후의 안정성 그리고 40±2℃ 온도 및 75±5% 상대 습도에서 2주 후 및 4주 후의 안정성을 측정하였다. 물리적 스트레스를 위해 상온 조건에서 3000 rpm으로 4시간의 흔들림 스트레스를 주었으며 결과는 표 20 내지 27 및 도 9에 나타내었다. With respect to the pharmaceutical formulation used in Experimental Example 2, each buffer was prepared to suit each pH, arginine monohydrochloride was added thereto, and the antibody was added thereto and then concentrated. Then, a surfactant was added to prepare the samples in Table 19. Examples 4 to 6 added the antibody concentrated in Celltrion Research Institute, and Example 1 is a formulation finally selected according to Experimental Example 1. The prepared pharmaceutical formulation was stored at 5±3°C temperature, 40±2°C temperature and 75±5% relative humidity, and stability after 2 and 4 weeks at 5±3°C temperature and 40±2°C temperature and 75± Stability after 2 weeks and 4 weeks at 5% relative humidity was measured. For physical stress, shaking stress was applied at 3000 rpm at room temperature for 4 hours, and the results are shown in Tables 20 to 27 and FIG. 9 .
본 실험예 2에 사용된 항체는 상기 표 1 내지 표 2에 기재된 No. 139번 결합 분자이다.Antibodies used in Experimental Example 2 were No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
구분division 완충제buffer pHpH 안정화제stabilizer 계면활성제Surfactants 단백질(항체) 농도Protein (antibody) concentration
실시예 4Example 4 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 100mg/mL100mg/mL
실시예 5Example 5 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 150mg/mL150mg/mL
실시예 6Example 6 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 200mg/mL200mg/mL
실시예 1Example 1 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
하기 표 20에서 점도 (단위: cP) 측정 결과를 나타내었다.The viscosity (unit: cP) measurement results are shown in Table 20 below.
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks 흔들림 스트레스shake stress
실시예 4Example 4 2.62.6 2.62.6 2.62.6 2.62.6 2.62.6 2.62.6
실시예 5Example 5 6.06.0 6.06.0 6.06.0 5.85.8 5.95.9 6.06.0
실시예 6Example 6 13.813.8 12.912.9 12.912.9 12.912.9 13.013.0 12.112.1
실시예 1Example 1 1.71.7 1.71.7 1.61.6 1.71.7 1.71.7 1.71.7
표 20에서 실시예 1 및 4 내지 6의 점도는 모든 실험 조건에서 변화하지 않았으며 피하주사가 가능한 15.0 cP 이하였다 (도 9).In Table 20, the viscosity of Examples 1 and 4 to 6 did not change in all experimental conditions and was 15.0 cP or less for subcutaneous injection (FIG. 9).
하기 표 21에서 SARS-CoV-2 RBD 결합 친화도 (ELISA) 측정 결과를 나타내었다.Table 21 below shows the measurement results of SARS-CoV-2 RBD binding affinity (ELISA).
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks
실시예 4Example 4 9999 9898 101101 9393 9191
실시예 5Example 5 105105 102102 107107 9797 9999
실시예 6Example 6 9797 9393 9292 9999 9797
실시예 1Example 1 104104 103103 9797 100100 101101
표 21에서 실시예 1 및 4 내지 6의 SARS-CoV-2 RBD 결합 친화도는 모든 실험 조건에서 변화하지 않았으며 유사하였다. In Table 21, SARS-CoV-2 RBD binding affinities of Examples 1 and 4 to 6 were similar and did not change in all experimental conditions.
하기 표 22에서 크기배제 크로마토그래피 주성분 함량 (Main peak %) 측정 결과를 나타내었다.Table 22 below shows the measurement results of the main component content (Main peak %) by size exclusion chromatography.
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks 흔들림 스트레스shake stress
실시예 4Example 4 99.4499.44 99.4099.40 98.7498.74 99.3899.38 98.5798.57 99.4699.46
실시예 5Example 5 99.3599.35 99.3199.31 98.5598.55 99.2399.23 97.8497.84 99.3699.36
실시예 6Example 6 99.2999.29 99.1999.19 98.3998.39 99.0099.00 98.0798.07 99.2099.20
실시예 1Example 1 99.4899.48 99.4699.46 99.1199.11 99.5099.50 98.8398.83 99.4999.49
표 22를 보면 실시예 1 및 4 내지 6의 5±3℃ 조건에서의 주성분 함량은 99% 이상으로 안정함을 확인할 수 있었다. 그리고 40±2℃ 및 75±5% 상대 습도 조건에서 4주 후 주성분 함량은 97% 이상으로 안정함을 확인할 수 있었다. 흔들림 스트레스 조건에서 실시예 1 및 4 내지 6의 주성분 함량은 99% 이상으로 안정함을 확인할 수 있었다. Referring to Table 22, it was confirmed that the content of the main component in Examples 1 and 4 to 6 at 5±3° C. was stable at 99% or more. And it was confirmed that the main component content was stable at 97% or more after 4 weeks under the conditions of 40±2℃ and 75±5% relative humidity. It was confirmed that the main component content of Examples 1 and 4 to 6 was stable at 99% or more under shaking stress conditions.
하기 표 23에서 크기배제 크로마토그래피 고분자량 성분(pre-peak %) 측정 결과를 나타내었다.Table 23 below shows the measurement results of high molecular weight components (pre-peak %) by size exclusion chromatography.
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks 흔들림 스트레스shake stress
실시예 4Example 4 0.410.41 0.470.47 0.800.80 0.480.48 0.830.83 0.400.40
실시예 5Example 5 0.500.50 0.560.56 1.021.02 0.630.63 1.531.53 0.510.51
실시예 6Example 6 0.550.55 0.670.67 1.191.19 0.880.88 1.311.31 0.650.65
실시예 1Example 1 0.370.37 0.400.40 0.530.53 0.360.36 0.650.65 0.380.38
표 23을 보면 실시예 1 및 4 내지 6의 고분자량 성분 함량은 4주 후 5±3℃, 40±2℃ 및 75±5% 상대 습도 조건 그리고 흔들림 스트레스 조건에서 2.0% 이하임을 확인하였고, 단백질 농도가 가장 낮은 실시예 2의 고분자량 성분이 가장 낮았다. Referring to Table 23, it was confirmed that the high molecular weight component content of Examples 1 and 4 to 6 was 2.0% or less at 5±3° C., 40±2° C. and 75±5% relative humidity conditions and shaking stress after 4 weeks, and protein The high molecular weight component of Example 2, which had the lowest concentration, had the lowest concentration.
하기 표 24에서 크기배제 크로마토그래피 저분자량 성분(post-peak %) 측정 결과를 나타내었다.Table 24 below shows the results of measurement of low molecular weight components (post-peak %) by size exclusion chromatography.
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks 흔들림 스트레스shake stress
실시예 4Example 4 0.160.16 0.130.13 0.470.47 0.140.14 0.600.60 0.140.14
실시예 5Example 5 0.150.15 0.130.13 0.430.43 0.140.14 0.640.64 0.130.13
실시예 6Example 6 0.160.16 0.150.15 0.420.42 0.110.11 0.620.62 0.150.15
실시예 1Example 1 0.150.15 0.140.14 0.360.36 0.140.14 0.520.52 0.130.13
표 24에서 실시예 4 내지 6의 저분자량 성분 함량은 모든 조건에서 1.0% 이하임을 확인하였다. 모든 실시예의 저분자량 성분 함량은 실시예 1의 결과와 유사한 수준임을 알 수 있었다. In Table 24, it was confirmed that the low molecular weight component content of Examples 4 to 6 was 1.0% or less under all conditions. It can be seen that the content of low molecular weight components in all examples is at a level similar to that of Example 1.
하기 표 25에서 HIAC으로 측정한 불용성 이물 입자의 수(10.00≤(um)) 측정 결과를 나타내었다.Table 25 below shows the measurement results of the number of insoluble foreign particles (10.00≤(um)) measured by HIAC.
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks 흔들림 스트레스shake stress
실시예 4Example 4 88 1212 2020 1010 55 4040
실시예 5Example 5 3333 2525 3030 2727 3030 4343
실시예 6Example 6 3838 2222 143143 3535 6767 1212
실시예 1Example 1 00 55 3535 22 3535 9898
표 25와 같이 실시예 4 내지 6의 불용성 이물 입자(10.00≤(um))의 수는 모든 실험 조건에서 200개 이하로 안정적이며, 실시예 1의 결과와 유사한 수준임을 알 수 있었다. As shown in Table 25, the number of insoluble foreign particles (10.00≤(um)) of Examples 4 to 6 was stable to 200 or less under all experimental conditions, and it was found that the result was similar to the result of Example 1.
하기 표 26에서 HIAC으로 측정한 불용성 이물 입자의 수(25.00≤(um)) 측정 결과를 나타내었다.Table 26 below shows the measurement results of the number of insoluble foreign particles (25.00≤(um)) measured by HIAC.
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks 흔들림 스트레스shake stress
실시예 4Example 4 00 00 55 00 00 00
실시예 5Example 5 00 00 22 00 22 00
실시예 6Example 6 00 00 00 00 22 00
실시예 1Example 1 00 22 00 00 00 00
표 26과 같이 실시예 4 내지 6의 불용성 이물 입자(25.00≤(um))의 수는 모든 실험 조건에서 10개 이하로 안정적이며, 실시예 1의 결과와 유사한 수준임을 알 수 있었다. As shown in Table 26, the number of insoluble foreign particles (25.00≤(um)) of Examples 4 to 6 was stable to 10 or less under all experimental conditions, and it was found to be at a level similar to the result of Example 1.
하기 표 27에서 산화율 Oxidation (Met 263) 측정 결과를 나타내었다.Table 27 below shows the oxidation rate Oxidation (Met 263) measurement results.
　구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주후5±3℃ after 2 weeks 40±2℃2주후40±2℃ after 2 weeks 5±3℃4주후5±3℃ after 4 weeks 40±2℃4주후40±2℃ after 4 weeks
실시예 4Example 4 2.52.5 2.42.4 3.93.9 2.52.5 4.74.7
실시예 5Example 5 2.42.4 2.22.2 3.83.8 2.72.7 5.05.0
실시예 6Example 6 2.52.5 2.42.4 5.15.1 8.78.7 5.25.2
실시예 1Example 1 2.32.3 2.42.4 3.43.4 2.42.4 4.34.3
(단위: %)(unit: %)
표 27을 보면 실시예 4 내지 6의 산화율은 모든 조건에서 10% 이하로 안정적임을 알 수 있었다. Referring to Table 27, it could be seen that the oxidation rates of Examples 4 to 6 were stable at 10% or less under all conditions.
실험예 3: 완충제 농도, pH 범위, 안정화제 농도, 계면활성제 농도, 단백질 농도의 비교Experimental Example 3: Comparison of buffer concentration, pH range, stabilizer concentration, surfactant concentration, and protein concentration
실험예 3에서 사용된 약제학적 제제와 관련하여, 각 완충액을 각 pH에 맞게 제조한 뒤 아르기닌 모노하이드로클로라이드를 첨가하고, 이에 항체를 첨가 후 농축하였다. 그 후 계면활성제를 첨가하여 표 28의 시료들을 제조하였다. 실시예 1은 실험예 1에 따라 최종적으로 선정된 제제이다. 제조된 약학적 제제를 5±3℃ 온도와 25±2℃ 온도 및 60±5% 상대 습도 그리고 40±2℃ 온도 및 75±5% 상대 습도에서 보관하였고, 각 온도 조건에서 3주, 6주 및 9주간 후의 안정성을 측정하였다. 물리적 스트레스를 위해 상온 조건에서 3000 rpm으로 4시간의 흔들림 스트레스를 주었으며 얼림/녹임 스트레스는 -40℃ 조건에서 얼림과 녹임 5회 반복, 빛 스트레스는 가시광선 조도 1000 lux의 세기로 72시간 노출시켰다. 결과는 표 29 내지 43 및 도 10 내지 17에 나타내었다.With respect to the pharmaceutical formulation used in Experimental Example 3, each buffer was prepared to suit each pH, arginine monohydrochloride was added thereto, and the antibody was added thereto and then concentrated. After that, a surfactant was added to prepare the samples in Table 28. Example 1 is a formulation finally selected according to Experimental Example 1. The prepared pharmaceutical formulations were stored at 5±3°C temperature, 25±2°C temperature, 60±5% relative humidity, 40±2°C temperature and 75±5% relative humidity, and each temperature condition for 3 weeks and 6 weeks and stability after 9 weeks. For physical stress, shaking stress was applied at 3000 rpm at room temperature for 4 hours, freezing/thawing stress was repeated 5 times at -40 ° C. The results are shown in Tables 29 to 43 and FIGS. 10 to 17 .
본 실험예 3에서 사용된 항체는 상기 표 1 내지 표 2에 기재된 No. 139번 결합 분자이다.The antibody used in Experimental Example 3 was No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
구분division 완충제buffer pHpH 안정화제stabilizer 계면활성제Surfactants 단백질(항체)농도Protein (antibody) concentration
실시예 7Example 7 히스티딘 5 mMhistidine 5 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 8Example 8 히스티딘 15 mMHistidine 15 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 9Example 9 히스티딘 10 mMhistidine 10 mM 5.75.7 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 10Example 10 히스티딘 10 mMhistidine 10 mM 6.36.3 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 11Example 11 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 100 mML-Arginine monohydrochloride 100 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 12Example 12 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 200 mML-Arginine monohydrochloride 200 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 13Example 13 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.03% (w/v)Polysorbate 800.03% (w/v) 60mg/mL60mg/mL
실시예 14Example 14 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.07% (w/v)Polysorbate 800.07% (w/v) 60mg/mL60mg/mL
실시예 15Example 15 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 40mg/mL40mg/mL
실시예 16Example 16 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 80mg/mL80mg/mL
실시예 1Example 1 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
하기 표 29에서 탁도 측정 결과를 나타내었다.The turbidity measurement results are shown in Table 29 below.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 0.01140.0114 0.00190.0019 0.00590.0059 0.00390.0039 0.01000.0100 0.00660.0066 0.00950.0095 0.00960.0096 0.00630.0063 0.01040.0104 0.00990.0099 0.01140.0114 0.01210.0121
실시예 8Example 8 0.01210.0121 0.01030.0103 0.00860.0086 0.00760.0076 0.00960.0096 0.01440.0144 0.00750.0075 0.00800.0080 0.00890.0089 0.01480.0148 0.00340.0034 0.01030.0103 0.02310.0231
실시예 9Example 9 0.00710.0071 0.00640.0064 0.00500.0050 0.00820.0082 0.01030.0103 0.00890.0089 0.00640.0064 0.01000.0100 0.01260.0126 0.00900.0090 0.00700.0070 0.01090.0109 0.01710.0171
실시예 10Example 10 0.00970.0097 0.00950.0095 0.00700.0070 0.00720.0072 0.01000.0100 0.01080.0108 0.00850.0085 0.01020.0102 0.00720.0072 0.01110.0111 0.00480.0048 0.00570.0057 0.00990.0099
실시예 11Example 11 0.01710.0171 0.00410.0041 0.00620.0062 0.01020.0102 0.01170.0117 0.01250.0125 0.00740.0074 0.00680.0068 0.01140.0114 0.00730.0073 0.00470.0047 0.00890.0089 0.01180.0118
실시예 12Example 12 0.01560.0156 0.00440.0044 0.00610.0061 0.00970.0097 0.01050.0105 0.00720.0072 0.00680.0068 0.00750.0075 0.01050.0105 0.01120.0112 0.00270.0027 0.00650.0065 0.00890.0089
실시예 13Example 13 0.00670.0067 0.00640.0064 0.00440.0044 0.01240.0124 0.01510.0151 0.00630.0063 0.00780.0078 0.00720.0072 0.00690.0069 0.00830.0083 0.00810.0081 0.00770.0077 0.01620.0162
실시예 14Example 14 0.00590.0059 0.00600.0060 0.00660.0066 0.00390.0039 0.00690.0069 0.01060.0106 0.01120.0112 0.00800.0080 0.00930.0093 0.00920.0092 0.00420.0042 0.00660.0066 0.00930.0093
실시예 15Example 15 0.00400.0040 0.00710.0071 0.00630.0063 0.00300.0030 0.00610.0061 0.00590.0059 0.00540.0054 0.00420.0042 0.00430.0043 0.00510.0051 0.00670.0067 0.00470.0047 0.02130.0213
실시예 16Example 16 0.00530.0053 0.00720.0072 0.00730.0073 0.00430.0043 0.01030.0103 0.00870.0087 0.01340.0134 0.01200.0120 0.00800.0080 0.00850.0085 0.01280.0128 0.00700.0070 0.01500.0150
실시예 1Example 1 0.00660.0066 0.00990.0099 0.00420.0042 0.00550.0055 0.01070.0107 0.00860.0086 0.00830.0083 0.00660.0066 0.01650.0165 0.00900.0090 0.00360.0036 0.00990.0099 0.01960.0196
표 29를 보면, 실시예 7 내지 16의 탁도는 모든 조건에서 0.0400이하로 안정적이며, 실시예 1과 차이가 없는 것을 알 수 있었다. Looking at Table 29, it can be seen that the turbidity of Examples 7 to 16 is stable at 0.0400 or less under all conditions, and there is no difference from Example 1.
하기 표 30에서 크기배제 크로마토그래피 주성분 함량 (Main peak %) 측정 결과를 나타내었다.Table 30 below shows the measurement results of the main component content (Main peak %) by size exclusion chromatography.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 99.4799.47 99.5099.50 99.3699.36 98.9698.96 99.4499.44 99.2899.28 98.5398.53 99.4299.42 99.1999.19 98.2498.24 99.4699.46 99.3999.39 99.1299.12
실시예 8Example 8 99.4599.45 99.5399.53 99.4199.41 98.9998.99 99.4499.44 99.2999.29 98.5498.54 99.4599.45 99.1499.14 98.2098.20 99.4899.48 99.4399.43 99.0999.09
실시예 9Example 9 99.5399.53 99.5499.54 99.4699.46 98.7698.76 99.4599.45 99.3699.36 98.4598.45 99.4799.47 99.1599.15 97.8297.82 99.2999.29 99.4699.46 99.1999.19
실시예 10Example 10 99.2699.26 99.4699.46 99.2999.29 98.8798.87 99.3799.37 99.1499.14 98.4398.43 99.3899.38 99.0599.05 98.0998.09 99.4099.40 99.3099.30 99.1599.15
실시예 11Example 11 99.3799.37 99.4799.47 99.3499.34 98.9098.90 99.4499.44 99.2599.25 98.5098.50 99.4499.44 99.1599.15 98.1798.17 99.4599.45 99.3699.36 99.1599.15
실시예 12Example 12 99.4599.45 99.4999.49 99.4299.42 98.9498.94 99.4699.46 99.2999.29 98.5398.53 99.4499.44 99.2199.21 98.1998.19 99.4999.49 99.4199.41 99.1799.17
실시예 13Example 13 99.4899.48 99.4599.45 99.3999.39 98.9698.96 99.4299.42 99.2899.28 98.5298.52 99.3799.37 99.1999.19 98.1998.19 99.4299.42 99.4199.41 99.0899.08
실시예 14Example 14 99.4799.47 99.5099.50 99.3799.37 98.9498.94 99.4599.45 99.2899.28 98.5298.52 99.4299.42 99.1199.11 98.2198.21 99.4199.41 99.4099.40 99.0899.08
실시예 15Example 15 99.4899.48 99.3899.38 99.3899.38 98.9598.95 99.5099.50 99.3899.38 98.6898.68 99.4199.41 99.2899.28 98.3598.35 99.4899.48 99.4599.45 99.2699.26
실시예 16Example 16 99.3799.37 99.3099.30 99.2599.25 98.7098.70 99.3999.39 99.1899.18 98.3798.37 99.3999.39 99.0699.06 98.1198.11 99.4699.46 99.3799.37 98.9998.99
실시예 1Example 1 99.4799.47 99.4599.45 99.3999.39 98.9798.97 99.4399.43 99.2399.23 98.5598.55 99.3799.37 99.1899.18 98.2398.23 99.3899.38 99.3899.38 98.9198.91
표 30과 같이 실시예 7 내지 16의 주성분 함량은 5±3℃ 온도 조건에서 99% 이상으로 안정적이며, 40±2℃ 온도 및 75±5% 상대 습도 조건에서 97% 이상으로 안정적임을 알 수 있었다. 또한 실시예 1의 결과와 유사한 수준임을 알 수 있었다 (도 10, 도 11 및 12).As shown in Table 30, it was found that the main component content of Examples 7 to 16 was stable to 99% or more at 5±3° C. temperature conditions, and was stable to 97% or more at 40±2° C. temperature and 75±5% relative humidity conditions. . It was also found that the level was similar to that of Example 1 ( FIGS. 10 , 11 and 12 ).
하기 표 31에서 크기배제 크로마토그래피 고분자량 성분(pre-peak %) 측정 결과를 나타내었다.Table 31 below shows the measurement results of high molecular weight components (pre-peak %) by size exclusion chromatography.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 0.480.48 0.450.45 0.530.53 0.680.68 0.510.51 0.600.60 0.850.85 0.520.52 0.610.61 0.810.81 0.470.47 0.570.57 0.790.79
실시예 8Example 8 0.480.48 0.410.41 0.490.49 0.630.63 0.510.51 0.590.59 0.820.82 0.460.46 0.650.65 0.800.80 0.440.44 0.530.53 0.820.82
실시예 9Example 9 0.410.41 0.400.40 0.450.45 0.660.66 0.500.50 0.520.52 0.780.78 0.460.46 0.630.63 0.890.89 0.640.64 0.500.50 0.680.68
실시예 10Example 10 0.660.66 0.490.49 0.600.60 0.770.77 0.590.59 0.730.73 0.970.97 0.550.55 0.740.74 0.930.93 0.520.52 0.660.66 0.750.75
실시예 11Example 11 0.570.57 0.470.47 0.550.55 0.710.71 0.520.52 0.630.63 0.880.88 0.490.49 0.640.64 0.870.87 0.470.47 0.590.59 0.740.74
실시예 12Example 12 0.490.49 0.450.45 0.490.49 0.670.67 0.490.49 0.580.58 0.850.85 0.470.47 0.580.58 0.810.81 0.440.44 0.540.54 0.720.72
실시예 13Example 13 0.460.46 0.490.49 0.520.52 0.680.68 0.540.54 0.600.60 0.850.85 0.530.53 0.620.62 0.830.83 0.500.50 0.540.54 0.790.79
실시예 14Example 14 0.460.46 0.450.45 0.520.52 0.700.70 0.520.52 0.600.60 0.870.87 0.510.51 0.680.68 0.840.84 0.520.52 0.570.57 0.800.80
실시예 15Example 15 0.460.46 0.510.51 0.470.47 0.610.61 0.460.46 0.510.51 0.690.69 0.500.50 0.540.54 0.730.73 0.440.44 0.510.51 0.650.65
실시예 16Example 16 0.540.54 0.590.59 0.570.57 0.800.80 0.560.56 0.700.70 1.011.01 0.550.55 0.730.73 0.950.95 0.480.48 0.580.58 0.910.91
실시예 1Example 1 0.470.47 0.500.50 0.510.51 0.660.66 0.520.52 0.640.64 0.830.83 0.520.52 0.630.63 0.810.81 0.530.53 0.570.57 0.840.84
표 31과 같이 실시예 7 내지 16의 고분자량 성분 함량은 5±3℃ 온도 조건에서 1.0% 이하로 안정적이며, 40±2℃ 온도 및 75±5% 상대 습도 조건에서 2.0% 이하로 안정적임을 알 수 있었다. 또한 실시예 1의 결과와 유사한 수준임을 알 수 있었다. As shown in Table 31, the high molecular weight component content of Examples 7 to 16 is stable to 1.0% or less at 5±3° C. temperature conditions, and is stable to 2.0% or less at 40±2° C. temperature and 75±5% relative humidity conditions. could In addition, it was found that the level was similar to the result of Example 1.
하기 표 32에서 크기배제 크로마토그래피 저분자량 성분(post-peak %) 측정 결과를 나타내었다.Table 32 below shows the measurement results of low molecular weight components (post-peak %) by size exclusion chromatography.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 0.050.05 0.050.05 0.120.12 0.370.37 0.040.04 0.120.12 0.620.62 0.050.05 0.200.20 0.960.96 0.070.07 0.040.04 0.090.09
실시예 8Example 8 0.070.07 0.060.06 0.100.10 0.370.37 0.050.05 0.120.12 0.640.64 0.100.10 0.210.21 0.990.99 0.080.08 0.050.05 0.090.09
실시예 9Example 9 0.060.06 0.060.06 0.090.09 0.580.58 0.050.05 0.120.12 0.770.77 0.060.06 0.220.22 1.291.29 0.070.07 0.040.04 0.130.13
실시예 10Example 10 0.070.07 0.050.05 0.110.11 0.360.36 0.050.05 0.130.13 0.600.60 0.080.08 0.210.21 0.990.99 0.080.08 0.040.04 0.100.10
실시예 11Example 11 0.060.06 0.060.06 0.110.11 0.390.39 0.040.04 0.120.12 0.620.62 0.070.07 0.210.21 0.950.95 0.080.08 0.060.06 0.110.11
실시예 12Example 12 0.070.07 0.050.05 0.090.09 0.380.38 0.040.04 0.120.12 0.630.63 0.090.09 0.200.20 1.001.00 0.080.08 0.050.05 0.110.11
실시예 13Example 13 0.060.06 0.060.06 0.100.10 0.370.37 0.040.04 0.120.12 0.620.62 0.100.10 0.190.19 0.980.98 0.070.07 0.050.05 0.140.14
실시예 14Example 14 0.080.08 0.050.05 0.100.10 0.360.36 0.040.04 0.120.12 0.610.61 0.070.07 0.210.21 0.950.95 0.070.07 0.040.04 0.120.12
실시예 15Example 15 0.060.06 0.120.12 0.150.15 0.430.43 0.040.04 0.120.12 0.620.62 0.080.08 0.180.18 0.920.92 0.080.08 0.040.04 0.090.09
실시예 16Example 16 0.100.10 0.120.12 0.180.18 0.500.50 0.040.04 0.120.12 0.630.63 0.070.07 0.210.21 0.950.95 0.060.06 0.050.05 0.110.11
실시예 1Example 1 0.060.06 0.060.06 0.110.11 0.370.37 0.050.05 0.140.14 0.620.62 0.110.11 0.190.19 0.960.96 0.090.09 0.040.04 0.240.24
표 32와 같이 실시예 7 내지 16의 저분자량 성분 함량은 5±3℃ 온도 조건에서 0.5% 이하로 안정적이며, 40±2℃ 온도 및 75±5% 상대 습도 조건에서 1.5% 이하로 안정적임을 알 수 있었다. 또한 실시예 1의 결과와 유사한 수준임을 알 수 있었다. As shown in Table 32, it can be seen that the low molecular weight component content of Examples 7 to 16 is stable to 0.5% or less at 5±3° C. temperature conditions, and is stable to 1.5% or less at 40±2° C. temperature and 75±5% relative humidity conditions. could In addition, it was found that the level was similar to the result of Example 1.
하기 표 33에서 온전한 면역 글로불린G의 함량 (비환원 chip-based CE-SDS) 측정 결과를 나타내었다.Table 33 below shows the measurement results of the intact immunoglobulin G content (non-reducing chip-based CE-SDS).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 98.1598.15 97.8697.86 98.0998.09 97.8097.80 98.4198.41 98.6998.69 98.6798.67 97.5297.52 97.5097.50 96.5996.59 97.8697.86 98.7798.77 98.6298.62
실시예 8Example 8 98.1798.17 97.9097.90 97.9097.90 97.6897.68 98.4398.43 98.7498.74 98.7598.75 97.6197.61 97.3297.32 96.5896.58 97.7497.74 98.9598.95 98.8098.80
실시예 9Example 9 98.2498.24 97.9997.99 97.4697.46 97.4697.46 98.4398.43 98.7798.77 98.6898.68 97.6297.62 97.5297.52 96.2596.25 97.7497.74 98.9198.91 98.5998.59
실시예 10Example 10 98.2298.22 98.0198.01 98.0498.04 97.6197.61 98.4098.40 98.6398.63 98.6398.63 97.6297.62 97.3897.38 96.3796.37 97.9697.96 98.8798.87 98.2298.22
실시예 11Example 11 98.1998.19 98.0998.09 97.9397.93 97.7297.72 98.3398.33 98.8098.80 98.7598.75 97.5197.51 97.3897.38 96.5496.54 97.8297.82 98.9598.95 98.7598.75
실시예 12Example 12 98.2598.25 97.8497.84 97.9897.98 97.5097.50 98.3298.32 98.7498.74 98.7198.71 97.5797.57 97.4697.46 96.6096.60 97.8497.84 98.8598.85 98.7398.73
실시예 13Example 13 98.2198.21 97.5997.59 97.8497.84 97.5497.54 98.3898.38 98.6898.68 98.7198.71 97.5197.51 97.2597.25 96.3796.37 97.9397.93 98.8798.87 98.5998.59
실시예 14Example 14 98.2198.21 97.6797.67 97.7297.72 97.5497.54 98.4398.43 98.6498.64 98.6398.63 97.4697.46 97.4097.40 96.4496.44 97.9897.98 98.8098.80 98.4798.47
실시예 15Example 15 98.2698.26 97.8597.85 98.0998.09 97.6497.64 98.3898.38 98.6398.63 98.7298.72 97.5097.50 97.3597.35 96.9896.98 97.8797.87 99.0099.00 98.7298.72
실시예 16Example 16 98.2698.26 97.9597.95 97.3997.39 97.4397.43 98.3398.33 98.7498.74 98.6798.67 97.4697.46 97.4897.48 96.1396.13 97.6797.67 98.8898.88 98.7698.76
실시예 1Example 1 98.1598.15 97.8997.89 97.8697.86 97.8697.86 98.4498.44 98.7798.77 98.7498.74 97.6097.60 97.4697.46 96.6096.60 97.8397.83 98.9198.91 98.6398.63
(단위: %)(unit: %)
표 33에서 보듯이, 실시예 7 내지 16의 온전한 면역 글로불린G의 함량은 모든 조건에서 실시예 1의 결과와 유사한 수준임을 알 수 있었다 (도 13).As shown in Table 33, it was found that the intact immunoglobulin G content of Examples 7 to 16 was at a level similar to that of Example 1 under all conditions (FIG. 13).
하기 표 34에서 항체 경쇄와 중쇄의 함량 (환원 chip-based CE-SDS) 측정 결과를 나타내었다.Table 34 below shows the measurement results of the antibody light chain and heavy chain content (reduced chip-based CE-SDS).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 99.7599.75 99.6399.63 99.6099.60 99.3399.33 99.6899.68 99.6699.66 99.6199.61 99.7399.73 99.5299.52 99.0399.03 99.7099.70 99.7999.79 99.5799.57
실시예 8Example 8 99.7499.74 99.6099.60 99.5299.52 99.3399.33 99.6899.68 99.6099.60 99.5899.58 99.6199.61 99.5499.54 99.0199.01 99.7099.70 99.8199.81 99.6699.66
실시예 9Example 9 99.7599.75 99.5899.58 99.6899.68 99.4199.41 99.6999.69 99.5799.57 99.5699.56 99.6499.64 99.6099.60 99.1099.10 99.6899.68 99.8099.80 99.5099.50
실시예 10Example 10 99.7499.74 99.6799.67 99.5099.50 99.3699.36 99.7099.70 99.4999.49 99.5499.54 99.6399.63 99.5599.55 98.9198.91 99.6999.69 99.7999.79 99.5499.54
실시예 11Example 11 99.7499.74 99.6499.64 99.5799.57 99.3999.39 99.7099.70 99.5299.52 99.6199.61 99.6599.65 99.5799.57 99.0299.02 99.6699.66 99.8199.81 99.6599.65
실시예 12Example 12 99.7399.73 99.7299.72 99.5599.55 99.4099.40 99.6499.64 99.5599.55 99.6199.61 99.6299.62 99.5199.51 99.0799.07 99.7099.70 99.8099.80 99.5599.55
실시예 13Example 13 99.7299.72 99.5399.53 99.5899.58 99.3599.35 99.6999.69 99.5699.56 99.5999.59 99.6099.60 99.5599.55 99.0199.01 99.6699.66 99.8099.80 99.5899.58
실시예 14Example 14 99.7299.72 99.6699.66 99.5499.54 99.2999.29 99.6999.69 99.5499.54 99.5999.59 99.6599.65 99.5499.54 99.0799.07 99.6999.69 99.8099.80 99.6199.61
실시예 15Example 15 99.7099.70 99.6199.61 99.5999.59 99.2699.26 99.6899.68 99.5399.53 99.6099.60 99.6499.64 99.5399.53 99.0299.02 99.6699.66 99.8199.81 99.5199.51
실시예 16Example 16 99.7199.71 99.2299.22 99.4499.44 99.3599.35 99.7199.71 99.5699.56 99.5999.59 99.6399.63 99.5399.53 99.0699.06 99.6799.67 99.8199.81 99.4899.48
실시예 1Example 1 99.7299.72 99.5599.55 99.4999.49 99.3699.36 99.7199.71 99.5699.56 99.6099.60 99.6699.66 99.5599.55 99.0399.03 99.6899.68 99.8199.81 99.5999.59
(단위: %)(unit: %)
표 34에서 보듯이, 실시예 7 내지 16의 항체 경쇄와 중쇄의 함량은 모든 조건에서 실시예 1의 결과와 유사한 수준임을 알 수 있었다 (도 14).As shown in Table 34, the content of the antibody light chain and heavy chain of Examples 7 to 16 was found to be at a level similar to the result of Example 1 under all conditions (FIG. 14).
하기 표 35에서 전하 변형체 (메인 피크 %) 측정 결과를 나타내었다.Table 35 below shows the measurement results of the charge variant (main peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 62.29 62.29 62.36 62.36 62.29 62.29 57.88 57.88 63.32 63.32 62.39 62.39 54.34 54.34 62.61 62.61 62.06 62.06 50.73 50.73 61.53 61.53 63.01 63.01 61.81 61.81
실시예 8Example 8 62.34 62.34 62.61 62.61 62.52 62.52 58.48 58.48 63.37 63.37 63.00 63.00 54.59 54.59 62.91 62.91 63.12 63.12 49.88 49.88 61.51 61.51 63.07 63.07 61.81 61.81
실시예 9Example 9 62.33 62.33 62.62 62.62 62.03 62.03 57.44 57.44 62.97 62.97 62.12 62.12 52.83 52.83 62.53 62.53 62.34 62.34 49.59 49.59 61.50 61.50 63.07 63.07 61.91 61.91
실시예 10Example 10 61.52 61.52 62.45 62.45 62.25 62.25 58.87 58.87 62.98 62.98 63.11 63.11 54.95 54.95 62.59 62.59 62.81 62.81 50.84 50.84 61.65 61.65 63.14 63.14 61.27 61.27
실시예 11Example 11 61.46 61.46 62.30 62.30 62.16 62.16 58.15 58.15 62.92 62.92 62.83 62.83 54.32 54.32 62.61 62.61 62.70 62.70 50.25 50.25 61.43 61.43 62.44 62.44 61.72 61.72
실시예 12Example 12 61.48 61.48 62.45 62.45 62.32 62.32 58.67 58.67 62.95 62.95 62.65 62.65 54.95 54.95 62.81 62.81 62.61 62.61 50.87 50.87 61.43 61.43 63.19 63.19 61.72 61.72
실시예 13Example 13 61.56 61.56 62.29 62.29 62.30 62.30 58.38 58.38 62.80 62.80 62.49 62.49 54.71 54.71 62.90 62.90 62.82 62.82 50.80 50.80 61.41 61.41 63.20 63.20 61.60 61.60
실시예 14Example 14 61.64 61.64 62.35 62.35 62.36 62.36 58.52 58.52 62.98 62.98 62.65 62.65 54.67 54.67 62.82 62.82 62.84 62.84 51.03 51.03 61.99 61.99 63.18 63.18 61.81 61.81
실시예 15Example 15 61.54 61.54 60.68 60.68 61.79 61.79 57.83 57.83 63.10 63.10 62.31 62.31 54.56 54.56 62.82 62.82 62.59 62.59 50.57 50.57 61.53 61.53 63.21 63.21 62.08 62.08
실시예 16Example 16 61.56 61.56 62.10 62.10 62.25 62.25 58.21 58.21 62.87 62.87 62.36 62.36 55.00 55.00 62.82 62.82 62.76 62.76 50.65 50.65 61.59 61.59 63.46 63.46 61.59 61.59
실시예 1Example 1 61.67 61.67 62.55 62.55 62.44 62.44 58.44 58.44 62.98 62.98 62.74 62.74 54.48 54.48 62.65 62.65 62.68 62.68 50.47 50.47 61.74 61.74 62.93 62.93 61.91 61.91
표 35에서 보듯이, 실시예 7 내지 16의 전하 변형체 (메인 피크) 함량은 모든 조건에서 실시예 1의 결과와 유사한 수준임을 알 수 있었다 (도 15).As shown in Table 35, it was found that the contents of the charge variants (main peak) of Examples 7 to 16 were similar to the results of Example 1 under all conditions ( FIG. 15 ).
하기 표 36에서 전하 변형체 (산성 피크 %) 측정 결과를 나타내었다.Table 36 below shows the measurement results of the charge variant (acid peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 14.06 14.06 14.27 14.27 14.70 14.70 20.71 20.71 13.57 13.57 15.38 15.38 28.31 28.31 14.12 14.12 15.87 15.87 32.85 32.85 14.36 14.36 14.12 14.12 15.06 15.06
실시예 8Example 8 14.13 14.13 14.12 14.12 14.95 14.95 21.54 21.54 13.68 13.68 15.66 15.66 29.11 29.11 14.16 14.16 15.82 15.82 34.29 34.29 14.48 14.48 14.22 14.22 15.12 15.12
실시예 9Example 9 14.08 14.08 13.97 13.97 14.75 14.75 21.37 21.37 13.91 13.91 15.49 15.49 29.22 29.22 14.11 14.11 15.64 15.64 34.21 34.21 14.48 14.48 13.87 13.87 14.50 14.50
실시예 10Example 10 14.28 14.28 14.33 14.33 15.35 15.35 22.06 22.06 14.16 14.16 15.83 15.83 29.44 29.44 14.33 14.33 16.50 16.50 33.97 33.97 14.57 14.57 14.27 14.27 16.12 16.12
실시예 11Example 11 14.36 14.36 14.41 14.41 15.07 15.07 21.62 21.62 14.16 14.16 15.65 15.65 29.11 29.11 14.20 14.20 15.74 15.74 33.90 33.90 14.75 14.75 15.07 15.07 15.08 15.08
실시예 12Example 12 14.35 14.35 14.28 14.28 14.89 14.89 21.06 21.06 14.15 14.15 15.61 15.61 28.20 28.20 14.01 14.01 15.69 15.69 32.92 32.92 14.48 14.48 13.98 13.98 14.93 14.93
실시예 13Example 13 14.42 14.42 14.36 14.36 15.04 15.04 21.45 21.45 14.22 14.22 15.98 15.98 28.61 28.61 13.98 13.98 15.76 15.76 33.07 33.07 14.58 14.58 14.06 14.06 15.20 15.20
실시예 14Example 14 14.85 14.85 14.36 14.36 14.95 14.95 21.40 21.40 14.19 14.19 15.96 15.96 28.65 28.65 14.07 14.07 15.69 15.69 33.15 33.15 15.07 15.07 14.04 14.04 15.01 15.01
실시예 15Example 15 14.42 14.42 16.27 16.27 15.32 15.32 22.04 22.04 14.08 14.08 16.07 16.07 28.53 28.53 14.14 14.14 15.54 15.54 33.47 33.47 14.53 14.53 13.95 13.95 14.83 14.83
실시예 16Example 16 14.35 14.35 14.68 14.68 15.15 15.15 22.03 22.03 14.25 14.25 16.33 16.33 28.58 28.58 14.13 14.13 15.88 15.88 33.34 33.34 14.49 14.49 13.74 13.74 15.17 15.17
실시예 1Example 1 14.13 14.13 14.17 14.17 14.81 14.81 21.20 21.20 14.02 14.02 15.72 15.72 28.94 28.94 14.16 14.16 15.75 15.75 33.64 33.64 14.33 14.33 14.15 14.15 15.23 15.23
표 36에서 보듯이, 실시예 7 내지 16의 전하 변형체 (산성 피크) 함량은 모든 조건에서 실시예 1의 결과와 유사한 수준임을 알 수 있었다 (도 16).As shown in Table 36, it was found that the contents of the charge variants (acid peak) of Examples 7 to 16 were similar to the results of Example 1 under all conditions ( FIG. 16 ).
하기 표 37에서 전하 변형체 (염기성 피크 %) 측정 결과를 나타내었다.Table 37 below shows the measurement results of charge variants (basic peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 23.66 23.66 23.37 23.37 23.01 23.01 21.41 21.41 23.10 23.10 22.23 22.23 17.36 17.36 23.26 23.26 22.08 22.08 16.42 16.42 24.11 24.11 22.86 22.86 23.13 23.13
실시예 8Example 8 23.53 23.53 23.27 23.27 22.53 22.53 19.99 19.99 22.95 22.95 21.34 21.34 16.30 16.30 22.93 22.93 21.06 21.06 15.83 15.83 24.02 24.02 22.71 22.71 23.09 23.09
실시예 9Example 9 23.58 23.58 23.41 23.41 23.22 23.22 21.18 21.18 23.12 23.12 22.39 22.39 17.94 17.94 23.36 23.36 22.02 22.02 16.19 16.19 24.01 24.01 23.07 23.07 23.59 23.59
실시예 10Example 10 24.20 24.20 23.21 23.21 22.41 22.41 19.07 19.07 22.86 22.86 21.06 21.06 15.61 15.61 23.07 23.07 20.69 20.69 15.18 15.18 23.78 23.78 22.59 22.59 22.62 22.62
실시예 11Example 11 24.18 24.18 23.29 23.29 22.77 22.77 20.24 20.24 22.92 22.92 21.53 21.53 16.57 16.57 23.19 23.19 21.57 21.57 15.85 15.85 23.83 23.83 22.47 22.47 23.21 23.21
실시예 12Example 12 24.16 24.16 23.27 23.27 22.79 22.79 20.28 20.28 22.89 22.89 21.74 21.74 16.85 16.85 23.18 23.18 21.69 21.69 16.20 16.20 24.09 24.09 22.83 22.83 23.35 23.35
실시예 13Example 13 24.03 24.03 23.36 23.36 22.66 22.66 20.17 20.17 22.98 22.98 21.53 21.53 16.68 16.68 23.12 23.12 21.41 21.41 16.13 16.13 24.02 24.02 22.74 22.74 23.19 23.19
실시예 14Example 14 23.50 23.50 23.29 23.29 22.69 22.69 20.09 20.09 22.84 22.84 21.38 21.38 16.68 16.68 23.11 23.11 21.47 21.47 15.82 15.82 22.94 22.94 22.78 22.78 23.18 23.18
실시예 15Example 15 24.05 24.05 23.01 23.01 22.88 22.88 20.12 20.12 22.82 22.82 21.63 21.63 16.91 16.91 23.04 23.04 21.86 21.86 15.96 15.96 23.95 23.95 22.85 22.85 23.10 23.10
실시예 16Example 16 24.10 24.10 23.22 23.22 22.61 22.61 19.75 19.75 22.89 22.89 21.30 21.30 16.41 16.41 23.04 23.04 21.36 21.36 16.01 16.01 23.92 23.92 22.80 22.80 23.24 23.24
실시예 1Example 1 24.19 24.19 23.28 23.28 22.76 22.76 20.36 20.36 22.99 22.99 21.53 21.53 16.58 16.58 23.19 23.19 21.58 21.58 15.89 15.89 23.93 23.93 22.91 22.91 22.86 22.86
표 37에서 보듯이, 실시예 7 내지 16의 전하 변형체 (염기성 피크) 함량은 모든 조건에서 실시예 1의 결과와 유사한 수준임을 알 수 있었다 (도 17).As shown in Table 37, it was found that the contents of the charge variants (basic peak) of Examples 7 to 16 were similar to the results of Example 1 under all conditions (FIG. 17).
하기 표 38에서 산화율 Oxidation (Met 263) 측정 결과를 나타내었다.Table 38 below shows the oxidation rate oxidation (Met 263) measurement results.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 빛스트레스light stress
실시예 7Example 7 2.42.4 2.72.7 3.43.4 6.86.8 4.44.4
실시예 8Example 8 2.72.7 2.82.8 3.43.4 7.07.0 4.94.9
실시예 9Example 9 2.42.4 2.52.5 2.72.7 4.44.4 4.44.4
실시예 10Example 10 2.42.4 2.82.8 3.93.9 8.48.4 4.84.8
실시예 11Example 11 2.32.3 3.53.5 3.33.3 7.57.5 5.35.3
실시예 12Example 12 2.32.3 2.72.7 5.45.4 8.38.3 5.05.0
실시예 13Example 13 2.42.4 2.72.7 4.74.7 8.08.0 5.15.1
실시예 14Example 14 2.42.4 2.82.8 3.83.8 7.77.7 4.54.5
실시예 15Example 15 2.42.4 2.82.8 3.73.7 7.97.9 4.34.3
실시예 16Example 16 2.42.4 2.82.8 4.04.0 8.48.4 5.25.2
실시예 1Example 1 2.42.4 2.72.7 3.53.5 6.86.8 4.94.9
(단위: %)(unit: %)
표 38에서 실시예 7 내지 16의 산화율은 모든 조건에서 실시예 1의 결과와 유사한 수준임을 알 수 있었다. In Table 38, it can be seen that the oxidation rates of Examples 7 to 16 are at a level similar to the results of Example 1 under all conditions.
하기 표 39에서 SARS-CoV-2 RBD 결합 친화도 (ELISA) 측정 결과를 나타내었다.Table 39 below shows the measurement results of SARS-CoV-2 RBD binding affinity (ELISA).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 빛스트레스light stress
실시예 7Example 7 101101 9999 9898 9797 106106 9494 9696 108108 102102 105105 107107
실시예 8Example 8 100100 9797 106106 9595 104104 9696 101101 100100 105105 102102 107107
실시예 9Example 9 104104 107107 103103 9898 106106 105105 9494 107107 112112 114114 100100
실시예 10Example 10 101101 102102 9898 101101 9696 9696 9999 9898 107107 104104 106106
실시예 11Example 11 104104 100100 106106 101101 101101 9999 9999 103103 9999 9898 102102
실시예 12Example 12 9797 100100 102102 101101 9999 105105 9999 108108 107107 113113 9797
실시예 13Example 13 111111 9494 110110 9898 106106 9696 9696 113113 103103 104104 9999
실시예 14Example 14 108108 106106 106106 102102 104104 8686 101101 100100 9898 9898 104104
실시예 15Example 15 103103 109109 118118 9797 108108 9393 9696 103103 100100 113113 110110
실시예 16Example 16 109109 101101 9090 9191 100100 9292 109109 9999 101101 108108 107107
실시예 1Example 1 100100 102102 9797 102102 100100 101101 106106 103103 9797 100100 110110
표 39에서 실시예 7 내지 16의 SARS-CoV-2 RBD 결합 친화도는 모든 조건에서 실시예 1의 결과와 유사한 수준임을 알 수 있었다. In Table 39, it can be seen that the SARS-CoV-2 RBD binding affinity of Examples 7 to 16 is at a level similar to that of Example 1 under all conditions.
하기 표 40에서 MFI 로 측정한 불용성 이물 입자의 수(10.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 40 below shows the measurement results of the number of insoluble foreign particles (10.00 μm≤, <100.00 μm) measured by MFI.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 33 1111 2121 33 2828 1111 9595 1313 3838 1010 3333 2121 88
실시예 8Example 8 1717 1616 5757 1515 2929 2020 6262 3939 1010 1717 33 2929 1515
실시예 9Example 9 33 55 88 1313 22 1313 6464 22 2525 88 1010 33 88
실시예 10Example 10 55 2020 1313 3939 2323 2020 3333 8484 3333 1818 00 1111 77
실시예 11Example 11 77 2929 1818 1313 1313 2323 3131 33 1818 2626 88 55 77
실시예 12Example 12 1010 33 77 44 22 1515 2626 1111 33 2020 1313 2020 2525
실시예 13Example 13 77 2626 1111 1515 2121 55 4848 2929 2323 3939 33 1111 2020
실시예 14Example 14 1010 3333 1111 1010 88 77 3434 55 1616 2525 22 223223 22
실시예 15Example 15 55 4646 4949 1515 33 2121 6161 77 1818 2020 33 2020 5757
실시예 16Example 16 2020 2929 1313 2828 4848 3838 9595 4949 4343 6767 1313 110110 3333
실시예 1Example 1 88 22 1010 1616 77 1010 2929 2020 1515 2121 33 7070 1818
표 40에서 실시예 7 내지 16의 불용성 이물 입자의 수(10.00㎛≤, <100.00㎛)는 모든 조건에서 300개 이하로 안정적이며, 실시예 1의 결과와 유사한 수준임을 알 수 있었다. In Table 40, it can be seen that the number of insoluble foreign particles of Examples 7 to 16 (10.00 μm≤, <100.00 μm) is stable to 300 or less under all conditions, and is at a level similar to the result of Example 1.
하기 표 41에서 MFI 로 측정한 불용성 이물 입자의 수(25.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 41 below shows the measurement results of the number of insoluble foreign particles (25.00 µm≤, <100.00 µm) measured by MFI.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 22 55 00 00 22 77 22 88 55 00 00 00 22
실시예 8Example 8 1212 77 33 55 33 22 55 00 00 22 22 22 00
실시예 9Example 9 00 00 22 00 22 22 77 00 00 22 22 00 22
실시예 10Example 10 00 22 22 33 55 22 33 00 22 33 00 33 00
실시예 11Example 11 22 00 88 33 22 55 33 22 00 22 33 00 00
실시예 12Example 12 22 00 22 22 00 55 00 00 22 22 00 00 00
실시예 13Example 13 22 33 00 00 22 00 00 22 22 33 22 33 00
실시예 14Example 14 33 55 22 00 22 00 77 00 22 55 00 88 00
실시예 15Example 15 22 55 77 77 22 33 77 22 33 00 00 55 00
실시예 16Example 16 33 77 22 22 1010 33 1313 22 77 1313 88 88 00
실시예 1Example 1 00 00 22 00 00 22 00 00 33 33 22 33 22
표 41에서 실시예 7 내지 16의 불용성 이물 입자의 수(25.00㎛≤, <100.00㎛)는 모든 조건에서 30개 이하로 안정적이며, 실시예 1의 결과와 유사한 수준임을 알 수 있었다. In Table 41, it can be seen that the number of insoluble foreign particles of Examples 7 to 16 (25.00 μm≤, <100.00 μm) is stable to 30 or less under all conditions, and is at a level similar to the result of Example 1.
하기 표 42에서 HIAC으로 측정한 불용성 이물 입자의 수(10.00≤(um)) 측정 결과를 나타내었다.Table 42 below shows the measurement results of the number of insoluble foreign particles (10.00≤(um)) measured by HIAC.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 8585 1010 1313 88 3232 1717 55 7272 1515 00 88 88 55
실시예 8Example 8 33 1818 88 00 1212 55 2727 55 55 88 1010 77 2020
실시예 9Example 9 22 55 55 33 1313 22 1717 55 33 33 1717 33 00
실시예 10Example 10 22 00 22 1010 77 88 55 2525 77 22 3535 22 00
실시예 11Example 11 1010 1717 00 88 00 55 2525 22 55 00 1818 22 00
실시예 12Example 12 22 33 77 55 22 33 1818 00 22 1515 2525 88 1313
실시예 13Example 13 22 22 55 77 55 55 2727 1313 88 1010 2020 88 00
실시예 14Example 14 55 22 33 33 33 22 1313 00 33 1515 1818 88 22
실시예 15Example 15 55 1313 88 1313 55 1212 4040 1010 55 2020 5353 1818 2020
실시예 16Example 16 2222 1313 1313 3535 2323 1717 3232 1515 2020 2525 1313 2020 2525
실시예 1Example 1 22 55 33 33 88 55 33 33 00 88 1010 22 2020
표 42에서 실시예 7 내지 16의 불용성 이물 입자의 수(10.00≤(um))는 모든 조건에서 100개 이하로 안정적이며, 실시예 1의 결과와 유사한 수준임을 알 수 있었다. In Table 42, it can be seen that the number of insoluble foreign particles (10.00≤(um)) of Examples 7 to 16 is stable to 100 or less under all conditions, and is at a level similar to the result of Example 1.
하기 표 43에서 HIAC으로 측정한 불용성 이물 입자의 수(25.00≤(um)) 측정 결과를 나타내었다.Table 43 below shows the measurement results of the number of insoluble foreign particles (25.00≤(um)) measured by HIAC.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃3주 후5±3℃ after 3 weeks 25±2℃3주 후25±2℃ after 3 weeks 40±2℃3주 후40±2℃ after 3 weeks 5±3℃6주 후5±3℃ after 6 weeks 25±2℃6주 후25±2℃ after 6 weeks 40±2℃6주 후40±2℃ after 6 weeks 5±3℃9주 후5±3℃ after 9 weeks 25±2℃9주 후25±2℃ after 9 weeks 40±2℃9주 후40±2℃ after 9 weeks 흔들림 스트레스shake stress 얼림/녹임스트레스Freezing/thawing stress 빛스트레스light stress
실시예 7Example 7 4848 33 33 55 22 55 22 88 00 00 00 22 22
실시예 8Example 8 00 77 33 00 33 33 22 22 55 00 00 22 00
실시예 9Example 9 00 00 33 00 1313 00 22 22 22 00 22 22 00
실시예 10Example 10 22 00 00 33 55 00 00 22 00 00 22 00 00
실시예 11Example 11 1010 55 00 22 00 33 77 00 33 00 22 00 00
실시예 12Example 12 00 00 22 00 00 00 22 00 00 22 88 33 00
실시예 13Example 13 00 22 00 55 22 33 00 00 22 00 00 33 00
실시예 14Example 14 22 00 22 00 22 22 00 00 22 00 00 00 00
실시예 15Example 15 00 00 33 33 33 33 22 22 00 00 00 00 00
실시예 16Example 16 00 00 00 1010 00 00 22 00 00 00 00 33 00
실시예 1Example 1 22 00 22 00 33 00 22 22 00 00 00 00 33
표 43에서 실시예 7 내지 16의 불용성 이물 입자의 수(25.00≤(um))는 모든 조건에서 50개 이하로 안정적이며, 실시예 1의 결과와 유사한 수준임을 알 수 있었다. In Table 43, it can be seen that the number of insoluble foreign particles (25.00≤(um)) of Examples 7 to 16 is stable to 50 or less under all conditions, and is at a level similar to the result of Example 1.
실험예 4: 실시예 1의 장기간 안정성 평가Experimental Example 4: Long-term stability evaluation of Example 1
실시예 1의 장기간　안정성　평가를ICH(International Conference on Harmonisation )에서 제공한 가이드라인(Guideline Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological 또는 Biological Products and ICH Guideline Q1A (R2): Stability Testing of New Drug Substances and Drug Products)에　따라　실시하였다．　Guideline Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological or Biological Products and ICH Guideline Q1A (R2): Stability Testing of New Drug Substances and Drug Products).
상기 실험예 1의 방법으로 제조한 실시예 1의 안정한 액체 약제학적 제제 16 mL을 5±3℃/주변 상대 습도에서 밀폐 용기에 보관하였다. 상기 온도 및 습도에서 1개월, 2개월 및 3개월 후 안정성을 측정하였다.16 mL of the stable liquid pharmaceutical formulation of Example 1 prepared by the method of Experimental Example 1 was stored in an airtight container at 5±3° C./ambient relative humidity. Stability was measured after 1 month, 2 months and 3 months at the above temperature and humidity.
실시예 1의 장기간 안정성을 확인하기 위하여, 외관 분석, 항체 농도 측정, 환원 CE-SDS를 이용한 항체 경쇄화 중쇄 함량 및 비환원 CE-SDS를 이용한 온전한 면역 글로불린 G의 함량 측정, SEC-HPLC를 이용한 항체 주성분, 고분자량 성분 및 저분자량 성분 측정, IEC-HPLC를 이용한 전하 변형체 (메인 피크, 산성 피크 및 염기성 피크) 측정 그리고 SARS-CoV-2 RBD 결합 친화도 측정을 수행하였으며 그 결과를 표 44에서 50에 나타내었다.In order to confirm the long-term stability of Example 1, appearance analysis, antibody concentration measurement, antibody light chain heavy chain content using reduced CE-SDS and intact immunoglobulin G content using non-reduced CE-SDS measurement, SEC-HPLC Measurement of antibody main component, high molecular weight component and low molecular weight component, charge variant (main peak, acidic peak, and basic peak) measurement using IEC-HPLC, and SARS-CoV-2 RBD binding affinity measurement were performed. The results are shown in Table 44 50 is shown.
하기 표 44에서 외관 분석 결과를 나타내었다.The results of the appearance analysis are shown in Table 44 below.
구분division 5±3℃0개월 후5±3℃ after 0 months 5±3℃1개월 후5±3℃ after 1 month 5±3℃2개월 후5±3℃ after 2 months 5±3℃3개월 후5±3℃ after 3 months
실시예 1Example 1 유백색milk white 유백색milk white 유백색milk white 유백색milk white
상기 표 44에서 나타난 바와 같이 실시예 1은 5±3℃에서 3 개월 후 외관상의 변화 없이 외관이 유지되었으므로 안정하다는 것을 알 수 있었다.As shown in Table 44, Example 1 was found to be stable because the appearance was maintained without any change in appearance after 3 months at 5 ± 3 ℃.
하기 표 45에서 항체 농도 (단위: mg/mL) 측정 결과를 나타내었다.Table 45 below shows the measurement results of antibody concentration (unit: mg/mL).
구분division 5±3℃0개월 후5±3℃ after 0 months 5±3℃1개월 후5±3℃ after 1 month 5±3℃2개월 후5±3℃ after 2 months 5±3℃3개월 후5±3℃ after 3 months
실시예 1Example 1 61mg/mL61mg/mL 61mg/mL61mg/mL 60mg/mL60mg/mL 60mg/mL60mg/mL
상기 표 45에서 나타난 바와 같이 실시예 1은 5±3℃에서 3 개월 후 항체 농도의 변화 없이 일정하게 유지되었으므로 안정하다는 것을 알 수 있었다.As shown in Table 45, Example 1 was found to be stable because it was kept constant without a change in the antibody concentration after 3 months at 5 ± 3 ℃.
하기 표 46에서 항체 경쇄와 중쇄 함량 (환원 CE-SDS) 측정 결과를 나타내었다.Table 46 below shows the measurement results of the antibody light chain and heavy chain content (reduced CE-SDS).
구분division 5±3℃0개월 후5±3℃ after 0 months 5±3℃1개월 후5±3℃ after 1 month 5±3℃2개월 후5±3℃ after 2 months 5±3℃3개월 후5±3℃ after 3 months
실시예 1Example 1 98.5198.51 98.8198.81 98.7498.74 98.6798.67
상기 표 46에서 나타난 바와 같이 실시예 1은 5±3℃에서 3 개월 후 항체 경쇄와 중쇄 함량의 변화 없이 일정하게 유지되었으므로 안정하다는 것을 알 수 있었다.As shown in Table 46, Example 1 was found to be stable because it was kept constant without change in the antibody light chain and heavy chain content after 3 months at 5 ± 3 ℃.
하기 표 47에서 온전한 면역 글로불린 G 함량(비환원 CE-SDS) 측정 결과를 나타내었다.Table 47 below shows the measurement results of intact immunoglobulin G content (non-reduced CE-SDS).
구분division 5±3℃0개월 후5±3℃ after 0 months 5±3℃1개월 후5±3℃ after 1 month 5±3℃2개월 후5±3℃ after 2 months 5±3℃3개월 후5±3℃ after 3 months
실시예 1Example 1 89.2189.21 90.8090.80 90.0090.00 90.5890.58
(단위: %)(unit: %)
상기 표 47에서 나타난 바와 같이 실시예 1은 5±3℃에서 3 개월 후 온전한 면역 글로불린 G 함량의 변화 없이 일정하게 유지되었으므로 안정하다는 것을 알 수 있었다.As shown in Table 47, Example 1 was found to be stable since it was maintained at 5±3° C. for 3 months without change in the intact immunoglobulin G content.
하기 표 48에서 SEC-HPLC (주성분, 고분자량 성분, 저분자량 성분 함량) 측정 결과를 나타내었다.Table 48 below shows the measurement results of SEC-HPLC (main component, high molecular weight component, low molecular weight component content).
구분division 5±3℃0개월 후5±3℃ after 0 months 5±3℃1개월 후5±3℃ after 1 month 5±3℃2개월 후5±3℃ after 2 months 5±3℃3개월 후5±3℃ after 3 months
실시예 1Example 1 주성분 함량main ingredient content 99.8299.82 99.7699.76 99.7799.77 99.7699.76
고분자량 성분high molecular weight ingredients 0.120.12 0.160.16 0.180.18 0.190.19
저분자량 성분low molecular weight ingredients 0.060.06 0.070.07 0.050.05 0.060.06
(단위: %)(unit: %)
상기 표 48에서 나타난 바와 같이 실시예 1은 5±3℃에서 3 개월 후 SEC-HPLC를 이용해 측정한 주성분, 고분자량 성분 및 저분자량 성분 함량의 변화 없이 일정하게 유지되었으므로 안정하다는 것을 알 수 있었다.As shown in Table 48, Example 1 was found to be stable because it was kept constant without changes in the main component, high molecular weight component, and low molecular weight component content measured by SEC-HPLC after 3 months at 5 ± 3 °C.
하기 표 49에서 IEC-HPLC (메인 피크, 산성 피크 및 염기성 피크) 측정 결과를 나타내었다.IEC-HPLC (main peak, acidic peak, and basic peak) measurement results are shown in Table 49 below.
구분division 5±3℃0개월 후5±3℃ after 0 months 5±3℃1개월 후5±3℃ after 1 month 5±3℃2개월 후5±3℃ after 2 months 5±3℃3개월 후5±3℃ after 3 months
실시예 1Example 1 메인 피크main peak 63.9263.92 63.8863.88 64.2564.25 63.9163.91
산성 피크acid peak 7.457.45 7.697.69 7.537.53 6.466.46
염기성 피크basic peak 63.9263.92 63.8863.88 64.2564.25 63.9163.91
(단위: %)(unit: %)
상기 표 49에서 나타난 바와 같이 실시예 1은 5±3℃에서 3 개월 후 IEC-HPLC를 이용해 측정한 메인 피크, 산성 피크 및 염기성 피크 함량의 변화 없이 일정하게 유지되었으므로 안정하다는 것을 알 수 있었다.As shown in Table 49, Example 1 was found to be stable because it was kept constant without changes in the main peak, acidic peak, and basic peak contents measured by IEC-HPLC after 3 months at 5±3°C.
하기 표 50에서 SARS-CoV-2 RBD 결합 친화도 측정 결과를 나타내었다.Table 50 below shows the results of SARS-CoV-2 RBD binding affinity measurement.
구분division 5±3℃0개월 후5±3℃ after 0 months 5±3℃1개월 후5±3℃ after 1 month 5±3℃2개월 후5±3℃ after 2 months 5±3℃3개월 후5±3℃ after 3 months
실시예 1Example 1 100100 9797 9494 9898
상기 표 50에서 나타난 바와 같이 실시예 1은 5±3℃에서 3 개월 후 SARS-CoV-2 RBD 결합 친화도가 변화 없이 일정하게 유지되었으므로 안정하다는 것을 알 수 있었다.As shown in Table 50, Example 1 was found to be stable because the SARS-CoV-2 RBD binding affinity was kept constant without change after 3 months at 5±3°C.
실험예 5: L-아르기닌-HCl/소르비톨/트레할로스 안정화제의 비교Experimental Example 5: Comparison of L-arginine-HCl/sorbitol/trehalose stabilizer
실험예 5에서 사용된 약제학적 제제와 관련하여, 각 완충액을 각 pH에 맞게 제조한 뒤 아르기닌 모노하이드로클로라이드, 소르비톨 및 트레할로스를 첨가하고, 이에 항체를 첨가하고, 계면활성제를 첨가하여 표 51의 시료들을 제조하였다. 제조된 약학적 제제를 5±3℃ 온도와 50±2℃ 온도에서 보관하였고, 5±3℃ 온도에서 5일 후의 안정성 그리고 50±2℃ 온도에서 3일 후 및 5일 후의 안정성을 측정하였다. 물리적 스트레스를 위해 -40℃에서 얼림과 녹임 5회를 반복하였고 상온에서 3000 rpm으로 4시간 동안 흔들림 스트레스를 가하였다. 실험의 결과는 표 52 내지 64 및 도 18 내지 22에 나타내었다.With respect to the pharmaceutical formulation used in Experimental Example 5, each buffer was prepared to suit each pH, and arginine monohydrochloride, sorbitol and trehalose were added thereto, an antibody was added thereto, and a surfactant was added to the sample of Table 51 were manufactured. The prepared pharmaceutical formulation was stored at a temperature of 5±3° C. and 50±2° C., and stability after 5 days at a temperature of 5±3° C. and stability after 3 days and 5 days at a temperature of 50±2° C. were measured. For physical stress, freezing and thawing were repeated 5 times at -40°C, and shaking stress was applied at room temperature at 3000 rpm for 4 hours. The results of the experiment are shown in Tables 52 to 64 and FIGS. 18 to 22 .
본 실험예 5에서 사용된 항체는 상기 표 3 내지 표 4에 기재된 No. 322번 결합 분자이다.Antibodies used in Experimental Example 5 were No. 1 described in Tables 3 to 4 above. It is the 322 bond molecule.
구분division 완충제buffer pHpH 안정화제stabilizer 계면활성제Surfactants 단백질(항체) 농도Protein (antibody) concentration
실시예 17Example 17 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 18Example 18 히스티딘 10 mMhistidine 10 mM 6.06.0 소르비톨5% (w/v)Sorbitol 5% (w/v) 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 19Example 19 히스티딘 10 mMhistidine 10 mM 6.06.0 트레할로스 10% (w/v)Trehalose 10% (w/v) 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
하기 표 52에서 탁도 측정 결과를 나타내었다.The turbidity measurement results are shown in Table 52 below.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 0.00150.0015 0.00960.0096 0.01780.0178 0.01590.0159 0.00760.0076 0.01210.0121
실시예 18Example 18 0.00650.0065 0.01180.0118 0.00730.0073 0.00730.0073 0.01020.0102 0.00400.0040
실시예 19Example 19 0.00420.0042 0.00400.0040 0.00640.0064 0.00350.0035 0.00400.0040 0.00800.0080
표 52를 보면 모든 조건에서 실시예의 탁도가 0.0400 이하로 안정함을 알 수 있었다. Looking at Table 52, it can be seen that the turbidity of the Example is stable at 0.0400 or less under all conditions.
하기 표 53에서 항체 경쇄와 중쇄 함량 (환원 chip-based CE-SDS) 측정 결과를 나타내었다.Table 53 below shows the measurement results of the antibody light chain and heavy chain content (reduced chip-based CE-SDS).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 99.8399.83 99.8599.85 99.7399.73 99.6199.61 99.8399.83 99.8399.83
실시예 18Example 18 99.8899.88 99.8499.84 99.7799.77 99.7299.72 99.8599.85 99.8599.85
실시예 19Example 19 99.8499.84 99.8599.85 99.7999.79 99.7399.73 99.8499.84 99.8399.83
(단위: %)(unit: %)
표 53을 보면 모든 조건에서 실시예 17 내지 19의 항체 경쇄와 중쇄 함량이 유사함을 알 수 있었다 (도 18).Looking at Table 53, it could be seen that the antibody light chain and heavy chain contents of Examples 17 to 19 were similar under all conditions (FIG. 18).
하기 표 54에서 온전한 면역 글로불린 G 함량 (비환원 chip-based CE-SDS) 측정 결과를 나타내었다.Table 54 below shows the measurement results of intact immunoglobulin G content (non-reducing chip-based CE-SDS).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 99.2299.22 99.1799.17 99.1499.14 99.0699.06 99.1599.15 99.1399.13
실시예 18Example 18 99.1899.18 99.1899.18 99.1899.18 99.1299.12 99.1599.15 99.1799.17
실시예 19Example 19 99.1899.18 99.1799.17 99.1899.18 99.1399.13 99.1799.17 99.1299.12
(단위: %)(unit: %)
표 54를 보면 실시예 17은 50±2℃ 조건에서 실시예 18 및 19에 비해 낮은 온전한 면역 글로불린G의 함량을 나타내었다 (도 19).Referring to Table 54, Example 17 showed a lower content of intact immunoglobulin G compared to Examples 18 and 19 at 50±2° C. ( FIG. 19 ).
하기 표 55에서 전하 변이체 (메인 피크 %) 측정 결과를 나타내었다.Table 55 below shows the measurement results of charge variants (main peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 66.90 66.90 67.27 67.27 61.70 61.70 57.84 57.84 66.45 66.45 67.14 67.14
실시예 18Example 18 67.22 67.22 67.17 67.17 61.59 61.59 57.35 57.35 67.74 67.74 67.12 67.12
실시예 19Example 19 67.08 67.08 66.99 66.99 61.46 61.46 57.35 57.35 67.20 67.20 67.13 67.13
표 55를 보면 모든 조건에서 실시예 17 내지 19의 전하 변형체 (메인 피크 %) 함량이 유사한 수준임을 알 수 있었다. Referring to Table 55, it can be seen that the contents of the charge variants (main peak %) of Examples 17 to 19 were at a similar level under all conditions.
하기 표 56에서 전하 변이체 (산성 피크 %) 측정 결과를 나타내었다.Table 56 below shows the measurement results of charge variants (acid peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 14.90 14.90 14.70 14.70 19.13 19.13 22.74 22.74 14.97 14.97 14.76 14.76
실시예 18Example 18 14.93 14.93 14.89 14.89 20.89 20.89 25.32 25.32 14.75 14.75 14.81 14.81
실시예 19Example 19 15.04 15.04 14.93 14.93 20.81 20.81 25.52 25.52 15.08 15.08 15.07 15.07
표 56을 보면 모든 조건에서 실시예 17 내지 19의 전하 변형체 (산성 피크 %) 함량이 유사한 수준임을 알 수 있었다. Referring to Table 56, it can be seen that the contents of the charge variants (acid peak %) of Examples 17 to 19 were at a similar level under all conditions.
하기 표 57에서 전하 변이체 (염기성 피크 %) 측정 결과를 나타내었다.Table 57 below shows the measurement results of charge variants (basic peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 18.21 18.21 18.03 18.03 19.17 19.17 19.42 19.42 18.59 18.59 18.10 18.10
실시예 18Example 18 17.87 17.87 17.95 17.95 17.52 17.52 17.33 17.33 17.50 17.50 18.06 18.06
실시예 19Example 19 17.87 17.87 18.07 18.07 17.73 17.73 17.13 17.13 17.71 17.71 17.80 17.80
표 57을 보면 모든 조건에서 실시예 17 내지 19의 전하 변형체 (염기성 피크 %) 함량이 유사한 수준임을 알 수 있었다. Referring to Table 57, it can be seen that the contents of the charge variants (basic peak %) of Examples 17 to 19 were at a similar level under all conditions.
하기 표 58에서 주성분 함량 (Main peak %) 측정 결과를 나타내었다.Table 58 below shows the measurement results of the main component content (Main peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 99.59 99.59 99.58 99.58 98.61 98.61 98.02 98.02 99.5499.54 99.5599.55
실시예 18Example 18 99.50 99.50 99.52 99.52 99.02 99.02 98.72 98.72 99.5399.53 99.5699.56
실시예 19Example 19 99.54 99.54 99.51 99.51 99.03 99.03 98.74 98.74 99.5299.52 99.5499.54
표 58을 보면 50±2℃ 조건에서 실시예 17이 실시예 18 및 19에 비해 더 낮은 주성분 함량을 나타내었다. 얼림/녹임 스트레스 및 흔들림 스트레스에 의한 주성분 함량은 실시예 17 내지 19가 유사하며, 99.0% 이상으로 안정함을 알 수 있었다 (도 20).Referring to Table 58, Example 17 showed a lower main component content than Examples 18 and 19 under the conditions of 50 ± 2 °C. The main component content due to freezing/thawing stress and shaking stress was similar to Examples 17 to 19, and it was found that it was stable at 99.0% or more ( FIG. 20 ).
하기 표 59에서 고분자량 성분 함량 (pre-peak %) 측정 결과를 나타내었다.Table 59 below shows the measurement results of the high molecular weight component content (pre-peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 0.37 0.37 0.39 0.39 1.10 1.10 1.56 1.56 0.420.42 0.410.41
실시예 18Example 18 0.46 0.46 0.44 0.44 0.75 0.75 0.94 0.94 0.440.44 0.410.41
실시예 19Example 19 0.43 0.43 0.45 0.45 0.73 0.73 0.91 0.91 0.440.44 0.430.43
표 59를 보면 50±2℃ 조건에서 실시예 17의 고분자량 성분이 실시예 18 및 19에 비해 높았다 (도 21).Referring to Table 59, the high molecular weight component of Example 17 was higher than that of Examples 18 and 19 at 50±2° C. ( FIG. 21 ).
하기 표 60에서 저분자량 성분 함량 (post-peak %) 측정 결과를 나타내었다.Table 60 below shows the measurement results of the low molecular weight component content (post-peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 0.04 0.04 0.03 0.03 0.29 0.29 0.42 0.42 0.040.04 0.040.04
실시예 18Example 18 0.04 0.04 0.04 0.04 0.23 0.23 0.34 0.34 0.030.03 0.030.03
실시예 19Example 19 0.02 0.02 0.04 0.04 0.24 0.24 0.35 0.35 0.040.04 0.030.03
표 60을 보면 50±2℃ 조건에서 실시예 17의 저분자량 성분이 실시예 18 및 19에 비해 높았다 (도 22).Referring to Table 60, the low molecular weight component of Example 17 was higher than that of Examples 18 and 19 under the conditions of 50±2° C. ( FIG. 22 ).
하기 표 61에서 MFI 로 측정한 불용성 이물 입자의 수(10.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 61 below shows the measurement results of the number of insoluble foreign particles (10.00 μm≤, <100.00 μm) measured by MFI.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 33 33 57 57 83 83 18 18 17 17 10 10
실시예 18Example 18 21 21 0 0 15 15 7 7 8 8 5 5
실시예 19Example 19 13 13 7 7 21 21 4 4 5 5 8 8
표 61을 보면 실시예 17 내지 19의 불용성 이물 입자의 수가 모든 조건에서 100개 이하로 안정함을 알 수 있었다. Looking at Table 61, it can be seen that the number of insoluble foreign particles of Examples 17 to 19 is stable at 100 or less under all conditions.
하기 표 62에서 MFI 로 측정한 불용성 이물 입자의 수(25.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 62 below shows the measurement results of the number of insoluble foreign particles (25.00 µm≤, <100.00 µm) measured by MFI.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 12 12 7 7 14 14 5 5 2 2 5 5
실시예 18Example 18 5 5 0 0 4 4 2 2 0 0 0 0
실시예 19Example 19 7 7 0 0 5 5 0 0 0 0 0 0
표 62를 보면 실시예 17 내지 19의 불용성 이물 입자의 수가 모든 조건에서 20개 이하로 안정함을 알 수 있었다. Looking at Table 62, it can be seen that the number of insoluble foreign particles of Examples 17 to 19 is stable at 20 or less under all conditions.
하기 표 63에서 HIAC으로 측정한 불용성 이물 입자의 수(10.00≤(um)) 측정 결과를 나타내었다.Table 63 below shows the measurement results of the number of insoluble foreign particles (10.00≤(um)) measured by HIAC.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 1717 3737 2727 22 77 00
실시예 18Example 18 88 55 00 22 77 00
실시예 19Example 19 00 00 33 00 55 88
표 63을 보면 실시예 17 내지 19의 불용성 이물 입자의 수가 모든 조건에서 100개 이하로 안정함을 알 수 있었다. Looking at Table 63, it can be seen that the number of insoluble foreign particles of Examples 17 to 19 is stable at 100 or less under all conditions.
하기 표 64에서 HIAC으로 측정한 불용성 이물 입자의 수(25.00≤(um)) 측정 결과를 나타내었다.Table 64 below shows the measurement results of the number of insoluble foreign particles (25.00≤(um)) measured by HIAC.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃5일 후5±3℃ after 5 days 50±2℃3일 후50±2℃ after 3 days 50±2℃5일 후50±2℃ after 5 days 흔들림스트레스shaking stress 얼림/녹임스트레스Freezing/thawing stress
실시예 17Example 17 00 55 00 00 00 00
실시예 18Example 18 00 00 00 00 00 00
실시예 19Example 19 00 00 22 00 00 22
표 64를 보면 실시예 17 내지 19의 불용성 이물 입자의 수가 모든 조건에서 10개 이하로 안정함을 알 수 있었다. Looking at Table 64, it can be seen that the number of insoluble foreign particles of Examples 17 to 19 is stable to 10 or less under all conditions.
실험예 6: 완충액 종류에 따른 비교; 안정화제 종류에 따른 비교; 계면활성제 종류에 따른 비교; 히스티딘 농도에 따른 비교Experimental Example 6: Comparison according to buffer type; Comparison by type of stabilizer; Comparison according to type of surfactant; Comparison according to histidine concentration
실험예 6에서 사용된 약제학적 제제와 관련하여, 각 완충액 (아세테이트, 시트레이트, 숙시네이트, 포스페이트)을 각 pH에 맞게 제조한 뒤 아르기닌 모노하이드로클로라이드, 소듐클로라이드, 수크로오스 및 글리신을 첨가하고, 이에 항체를 첨가 후 농축하였다. 그 후 계면활성제 (폴리소르베이트 20 및 80, 폴록사머 188)를 첨가(단, 비교예 1은 미첨가)하여 표 65의 시료들을 제조하였다. 실시예 1은 실험예 1에 따라 최종적으로 선정된 제제이다. 제조된 약학적 제제를 5±3℃ 온도와 40±2℃ 온도 및 75±5% 상대 습도에서 보관하였고, 각 온도 조건에서 2주, 4주 후의 안정성을 측정하였다. 물리적 스트레스를 위해 상온 조건에서 3000 rpm으로 4시간의 흔들림 스트레스를 주었다. 결과는 표 66 내지 75 및 도 23 내지 25에 나타내었다.With respect to the pharmaceutical formulation used in Experimental Example 6, each buffer solution (acetate, citrate, succinate, phosphate) was prepared according to each pH, and arginine monohydrochloride, sodium chloride, sucrose and glycine were added thereto. After addition of the antibody, it was concentrated. Thereafter, surfactants (polysorbates 20 and 80, poloxamer 188) were added (however, Comparative Example 1 was not added) to prepare the samples in Table 65. Example 1 is a formulation finally selected according to Experimental Example 1. The prepared pharmaceutical formulation was stored at 5±3° C. temperature, 40±2° C. temperature, and 75±5% relative humidity, and stability after 2 weeks and 4 weeks at each temperature condition was measured. For physical stress, shaking stress was applied at 3000 rpm for 4 hours at room temperature. The results are shown in Tables 66 to 75 and FIGS. 23 to 25 .
본 실험예 6에서 사용된 항체는 상기 표 1 내지 표 2에 기재된 No. 139번 결합 분자이다. The antibody used in Experimental Example 6 was No. 1 described in Tables 1 to 2 above. 139 is the binding molecule.
구분division 완충제buffer pHpH 안정화제stabilizer 계면활성제Surfactants 단백질(항체)농도Protein (antibody) concentration
실시예 20Example 20 아세테이트 10 mMAcetate 10 mM 5.55.5 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 21Example 21 시트레이트 10 mMCitrate 10 mM 5.55.5 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 22Example 22 시트레이트 10 mMCitrate 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 23Example 23 숙시네이트 10 mMSuccinate 10 mM 6.56.5 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 24Example 24 포스페이트 10 mMPhosphate 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 25Example 25 포스페이트 10 mMPhosphate 10 mM 6.56.5 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 26Example 26 히스티딘 10 mMhistidine 10 mM 6.06.0 염화나트륨 150 mM150 mM sodium chloride 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 27Example 27 히스티딘 10 mMhistidine 10 mM 6.06.0 수크로오스 10 % (w/v)Sucrose 10% (w/v) 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 28Example 28 히스티딘 10 mMhistidine 10 mM 6.06.0 글리신 250 mMGlycine 250 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 29Example 29 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 200.05% (w/v)Polysorbate 200.05% (w/v) 60mg/mL60mg/mL
실시예 30Example 30 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴록사머 1880.05% (w/v)Poloxamer 1880.05% (w/v) 60mg/mL60mg/mL
실시예 31Example 31 히스티딘 50 mMHistidine 50 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
실시예 1Example 1 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM 폴리소르베이트 800.05% (w/v)Polysorbate 800.05% (w/v) 60mg/mL60mg/mL
비교예 1Comparative Example 1 히스티딘 10 mMhistidine 10 mM 6.06.0 L-아르기닌모노하이드로클로라이드 150 mML-Arginine monohydrochloride 150 mM -- 60mg/mL60mg/mL
하기 표 66에서 탁도 측정 결과를 나타내었다.The turbidity measurement results are shown in Table 66 below.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후5±3℃ after 2 weeks 40±2℃2주 후40±2℃ after 2 weeks 5±3℃4주 후5±3℃ after 4 weeks 40±2℃4주 후40±2℃ after 4 weeks 흔들림스트레스shaking stress
실시예 20 Example 20 0.00980.0098 0.01240.0124 0.00720.0072 0.00980.0098 0.01120.0112 0.00960.0096
실시예 21Example 21 0.01180.0118 0.00840.0084 0.00910.0091 0.00990.0099 0.01370.0137 0.01350.0135
실시예 22Example 22 0.01340.0134 0.00910.0091 0.01340.0134 0.01100.0110 0.00900.0090 0.01670.0167
실시예 23Example 23 0.01210.0121 0.00860.0086 0.00930.0093 0.00760.0076 0.00860.0086 0.04300.0430
실시예 24Example 24 0.01220.0122 0.00740.0074 0.00830.0083 0.00910.0091 0.00870.0087 0.01130.0113
실시예 25Example 25 0.00590.0059 0.00710.0071 0.00890.0089 0.00880.0088 0.01010.0101 0.01450.0145
실시예 26Example 26 0.01310.0131 0.01040.0104 0.01140.0114 0.00830.0083 0.01460.0146 0.02940.0294
실시예 27Example 27 0.00510.0051 0.00490.0049 0.00270.0027 0.00130.0013 0.00190.0019 0.00230.0023
실시예 28Example 28 0.00200.0020 0.00330.0033 0.00250.0025 0.00880.0088 0.00280.0028 0.00410.0041
실시예 29Example 29 0.00830.0083 0.00900.0090 0.00890.0089 0.00990.0099 0.00850.0085 0.01050.0105
실시예 30Example 30 0.00910.0091 0.00770.0077 0.00900.0090 0.01080.0108 0.00720.0072 0.01320.0132
실시예 31Example 31 0.00800.0080 0.00780.0078 0.00880.0088 0.00930.0093 0.00970.0097 0.01280.0128
실시예 1Example 1 0.00830.0083 0.00690.0069 0.01070.0107 0.00760.0076 0.00680.0068 0.01250.0125
비교예 1Comparative Example 1 0.00960.0096 0.00800.0080 0.00910.0091 0.01090.0109 0.00830.0083 0.33790.3379
표 66을 보면, 실시예 20 내지 31의 탁도는 모든 온도 보관 조건에서 0.0400이하로 안정적이며, 실시예 1과 차이가 없는 것을 알 수 있었다. 하지만 흔들림 스트레스에서 비교예 1의 탁도가 매우 크게 증가하는 것으로 보아 물리적인 흔들림 스트레스에 계면활성제의 존재가 필수적임을 알 수 있었다. Looking at Table 66, it can be seen that the turbidity of Examples 20 to 31 is stable at 0.0400 or less under all temperature storage conditions, and there is no difference from Example 1. However, it can be seen that the presence of a surfactant is essential for the physical shaking stress, as the turbidity of Comparative Example 1 is significantly increased in the shaking stress.
하기 표 67에서 온전한 면역 글로불린 G 함량(비환원 CE-SDS) 측정 결과를 나타내었다.Table 67 below shows the measurement results of intact immunoglobulin G content (non-reduced CE-SDS).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후5±3℃ after 2 weeks 40±2℃2주 후40±2℃ after 2 weeks 5±3℃4주 후5±3℃ after 4 weeks 40±2℃4주 후40±2℃ after 4 weeks 흔들림스트레스shaking stress
실시예 20 Example 20 98.66 98.66 98.2398.23 97.6197.61 97.9297.92 97.2297.22 98.59 98.59
실시예 21Example 21 98.65 98.65 98.2498.24 97.6297.62 98.0098.00 97.2697.26 98.59 98.59
실시예 22Example 22 98.61 98.61 98.2698.26 97.4997.49 97.9297.92 97.7397.73 98.63 98.63
실시예 23Example 23 98.49 98.49 98.2298.22 97.7397.73 98.0398.03 97.6997.69 98.60 98.60
실시예 24Example 24 98.64 98.64 98.2098.20 97.7097.70 97.9297.92 97.5397.53 98.55 98.55
실시예 25Example 25 98.64 98.64 98.2298.22 97.6297.62 97.9797.97 96.9996.99 98.50 98.50
실시예 26Example 26 98.61 98.61 98.2198.21 97.6997.69 98.0498.04 96.9296.92 98.58 98.58
실시예 27Example 27 98.61 98.61 98.2298.22 97.5997.59 98.0098.00 97.2897.28 98.44 98.44
실시예 28Example 28 98.64 98.64 98.1498.14 97.8897.88 98.0098.00 96.9996.99 98.54 98.54
실시예 29Example 29 98.62 98.62 98.1098.10 97.7897.78 97.9397.93 97.3997.39 98.56 98.56
실시예 30Example 30 98.57 98.57 98.1798.17 97.8797.87 98.0298.02 97.0997.09 98.57 98.57
실시예 31Example 31 98.75 98.75 97.9897.98 97.9197.91 98.0298.02 97.8097.80 98.56 98.56
실시예 1Example 1 98.59 98.59 98.1398.13 97.8797.87 97.7797.77 97.5097.50 98.50 98.50
비교예 1Comparative Example 1 98.65 98.65 98.0698.06 97.6197.61 97.9297.92 97.5297.52 98.54 98.54
(단위: %)(unit: %)
상기 표 67에서 나타난 바와 같이 실시예 20 내지 31은 5±3℃와 40±2℃ 온도 및 75±5% 상대 습도 조건에서 4주 후 온전한 면역 글로불린 G 함량이 실시예 1과 차이가 없었다. 또한 흔들림 스트레스에 의한 영향 감소 현상 없이 안정함을 알 수 있었다 (도 23).As shown in Table 67, Examples 20 to 31 did not differ from Example 1 in the intact immunoglobulin G content after 4 weeks at 5±3° C. and 40±2° C. and 75±5% relative humidity conditions. In addition, it was confirmed that there was no decrease in the effect of shaking stress (FIG. 23).
하기 표 68에서 항체 경쇄와 중쇄 함량 (환원 CE-SDS) 측정 결과를 나타내었다.Table 68 below shows the measurement results of the antibody light chain and heavy chain content (reduced CE-SDS).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후5±3℃ after 2 weeks 40±2℃2주 후40±2℃ after 2 weeks 5±3℃4주 후5±3℃ after 4 weeks 40±2℃4주 후40±2℃ after 4 weeks 흔들림스트레스shaking stress
실시예 20 Example 20 99.6099.60 99.5599.55 99.3199.31 99.5999.59 99.1899.18 99.5799.57
실시예 21Example 21 99.6099.60 99.5599.55 99.3199.31 99.5999.59 99.2499.24 99.5599.55
실시예 22Example 22 99.6199.61 99.5499.54 99.3299.32 99.6199.61 99.2999.29 99.6099.60
실시예 23Example 23 99.6199.61 99.5599.55 99.3999.39 99.5999.59 99.2999.29 99.5699.56
실시예 24Example 24 99.5999.59 99.5699.56 99.3999.39 99.5999.59 99.2699.26 99.5899.58
실시예 25Example 25 99.5799.57 99.5399.53 99.1599.15 99.5799.57 98.8798.87 99.5999.59
실시예 26Example 26 99.6399.63 99.5599.55 99.4299.42 99.6099.60 99.3599.35 99.5699.56
실시예 27Example 27 99.6199.61 99.5499.54 99.3999.39 99.5799.57 99.3499.34 99.5899.58
실시예 28Example 28 99.6299.62 99.5499.54 99.2899.28 99.5999.59 99.2199.21 99.6099.60
실시예 29Example 29 99.6099.60 99.5499.54 99.3999.39 99.5799.57 99.2899.28 99.5599.55
실시예 30Example 30 99.6099.60 99.5599.55 99.3799.37 99.5999.59 99.2899.28 99.5699.56
실시예 31Example 31 99.5999.59 99.5599.55 99.3699.36 99.5899.58 99.4099.40 99.5399.53
실시예 1Example 1 99.5199.51 99.5399.53 99.3099.30 99.5699.56 99.2699.26 99.5799.57
비교예 1Comparative Example 1 99.5799.57 99.5499.54 99.3999.39 99.5799.57 99.2899.28 99.5999.59
(단위: %)(unit: %)
상기 표 68에서 나타난 바와 같이 실시예 20 내지 31은 5±3°C와 40±2℃ 온도 및 75±5% 상대 습도 조건에서 4주 후 항체의 경쇄와 중쇄 함량의 변화 없이 일정하게 유지되었으므로 안정하다는 것을 알 수 있었다. 또한 흔들림 스트레스에 대해서도 안정함을 확인하였다 (도 24).As shown in Table 68, Examples 20 to 31 were stable because they were kept constant without change in the light chain and heavy chain contents of the antibody after 4 weeks at 5±3 ° C, 40 ± 2 ° C temperature, and 75 ± 5% relative humidity conditions. knew that it was In addition, it was confirmed that it is stable against shaking stress (FIG. 24).
하기 표 69에서 SEC-HPLC 주성분 함량 (Main peak %) 측정 결과를 나타내었다.SEC-HPLC main component content (Main peak %) measurement results are shown in Table 69 below.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후　5±3℃ after 2 weeks 40±2℃2주 후　40±2℃ after 2 weeks 5±3℃4주 후　5±3℃ after 4 weeks 40±2℃4주 후　40±2℃ after 4 weeks 흔들림스트레스　shaking stress
실시예 20 Example 20 99.6499.64 99.5899.58 98.7998.79 99.5799.57 98.3798.37 99.6199.61
실시예 21Example 21 99.6599.65 99.5799.57 98.7998.79 99.5699.56 98.4098.40 99.6199.61
실시예 22Example 22 99.6399.63 99.5599.55 98.9798.97 99.5499.54 98.6798.67 99.6299.62
실시예 23Example 23 99.6399.63 99.5599.55 98.9698.96 99.5399.53 98.6898.68 99.5899.58
실시예 24Example 24 99.6099.60 99.5199.51 98.9798.97 99.4999.49 98.6398.63 99.5899.58
실시예 25Example 25 99.5699.56 99.4399.43 98.8498.84 99.4299.42 98.4998.49 99.5499.54
실시예 26Example 26 99.5599.55 99.4599.45 98.7398.73 99.4299.42 98.3998.39 99.5399.53
실시예 27Example 27 99.5999.59 99.5399.53 99.0499.04 99.5399.53 98.5898.58 99.5699.56
실시예 28Example 28 99.5999.59 99.5399.53 99.1399.13 99.4999.49 98.8798.87 99.6199.61
실시예 29Example 29 99.5899.58 99.5399.53 99.0199.01 99.5099.50 98.6798.67 99.5999.59
실시예 30Example 30 99.5799.57 99.5499.54 99.0299.02 99.5299.52 98.7198.71 99.6399.63
실시예 31Example 31 99.6599.65 99.5299.52 99.0899.08 99.5399.53 98.7898.78 99.6599.65
실시예 1Example 1 99.5999.59 99.5499.54 98.9998.99 99.5099.50 98.5998.59 99.6199.61
비교예 1Comparative Example 1 99.6199.61 99.5699.56 99.0199.01 99.5399.53 98.7198.71 99.6099.60
표 69를 보면 실시예 20 내지 31은 5±3℃ 조건에서 4주 후 주성분 함량이 99.0% 이상으로 안정함을 확인하였다. 40±2℃ 온도 및 75±5% 상대 습도 조건에서 주성분 함량의 감소를 보이지만 4주 후 주성분 함량은 모든 실시예에서 98.0% 이상으로 안정함을 확인하였고, 실시예 1과 큰 차이가 없음을 알 수 있었다 (도 25).Referring to Table 69, it was confirmed that Examples 20 to 31 were stable with a main component content of 99.0% or more after 4 weeks at 5±3°C. Although a decrease in the main component content was seen at 40 ± 2 ° C. temperature and 75 ± 5% relative humidity conditions, it was confirmed that the main component content was stable at 98.0% or more in all examples after 4 weeks, and there was no significant difference from Example 1. could (Fig. 25).
하기 표 70에서 고분자량 성분 함량 (pre-peak %) 측정 결과를 나타내었다.Table 70 below shows the measurement results of the high molecular weight component content (pre-peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후　5±3℃ after 2 weeks 40±2℃2주 후　40±2℃ after 2 weeks 5±3℃4주 후　5±3℃ after 4 weeks 40±2℃4주 후　40±2℃ after 4 weeks 흔들림스트레스　shaking stress
실시예 20 Example 20 0.290.29 0.360.36 0.600.60 0.350.35 0.660.66 0.310.31
실시예 21Example 21 0.290.29 0.370.37 0.640.64 0.360.36 0.670.67 0.310.31
실시예 22Example 22 0.310.31 0.390.39 0.670.67 0.380.38 0.750.75 0.320.32
실시예 23Example 23 0.310.31 0.390.39 0.710.71 0.390.39 0.780.78 0.360.36
실시예 24Example 24 0.340.34 0.430.43 0.710.71 0.440.44 0.820.82 0.360.36
실시예 25Example 25 0.390.39 0.500.50 0.860.86 0.510.51 0.960.96 0.420.42
실시예 26Example 26 0.390.39 0.490.49 0.910.91 0.500.50 1.001.00 0.400.40
실시예 27Example 27 0.360.36 0.400.40 0.670.67 0.400.40 0.940.94 0.370.37
실시예 28Example 28 0.340.34 0.420.42 0.610.61 0.430.43 0.660.66 0.340.34
실시예 29Example 29 0.360.36 0.400.40 0.660.66 0.420.42 0.760.76 0.350.35
실시예 30Example 30 0.350.35 0.390.39 0.650.65 0.400.40 0.710.71 0.330.33
실시예 31Example 31 0.310.31 0.430.43 0.610.61 0.400.40 0.660.66 0.320.32
실시예 1Example 1 0.350.35 0.400.40 0.660.66 0.420.42 0.800.80 0.340.34
비교예 1Comparative Example 1 0.330.33 0.380.38 0.660.66 0.400.40 0.720.72 0.340.34
표 70을 보면 실시예 20 내지 31은 모든 온도조건에서 4주 후 그리고 흔들림 스트레스 후 고분자량 성분이 1.0% 이하로 안정함을 알 수 있었다. 또한 실시예 1의 결과와 큰 차이가 없음을 알 수 있었다. Referring to Table 70, it was found that Examples 20 to 31 were stable at 1.0% or less of high molecular weight components after 4 weeks and after shaking stress under all temperature conditions. In addition, it was found that there was no significant difference from the results of Example 1.
하기 표 71에서 저분자량 성분 함량 (post-peak %) 측정 결과를 나타내었다.Table 71 below shows the measurement results of the content of low molecular weight components (post-peak %).
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후　5±3℃ after 2 weeks 40±2℃2주 후　40±2℃ after 2 weeks 5±3℃4주 후　5±3℃ after 4 weeks 40±2℃4주 후　40±2℃ after 4 weeks 흔들림스트레스　shaking stress
실시예 20 Example 20 0.070.07 0.060.06 0.610.61 0.080.08 0.980.98 0.080.08
실시예 21Example 21 0.060.06 0.060.06 0.580.58 0.080.08 0.930.93 0.080.08
실시예 22Example 22 0.070.07 0.060.06 0.350.35 0.080.08 0.580.58 0.060.06
실시예 23Example 23 0.060.06 0.060.06 0.340.34 0.080.08 0.540.54 0.060.06
실시예 24Example 24 0.060.06 0.060.06 0.320.32 0.070.07 0.560.56 0.060.06
실시예 25Example 25 0.050.05 0.060.06 0.310.31 0.080.08 0.550.55 0.040.04
실시예 26Example 26 0.070.07 0.060.06 0.360.36 0.080.08 0.610.61 0.070.07
실시예 27Example 27 0.050.05 0.060.06 0.290.29 0.070.07 0.480.48 0.070.07
실시예 28Example 28 0.080.08 0.060.06 0.260.26 0.070.07 0.470.47 0.040.04
실시예 29Example 29 0.070.07 0.060.06 0.340.34 0.080.08 0.580.58 0.060.06
실시예 30Example 30 0.080.08 0.060.06 0.330.33 0.080.08 0.580.58 0.040.04
실시예 31Example 31 0.040.04 0.060.06 0.320.32 0.070.07 0.560.56 0.040.04
실시예 1Example 1 0.070.07 0.060.06 0.350.35 0.080.08 0.610.61 0.050.05
비교예 1Comparative Example 1 0.060.06 0.060.06 0.330.33 0.070.07 0.570.57 0.060.06
표 71을 보면 실시예 20 내지 31은 모든 온도조건에서 4주 후 그리고 흔들림 스트레스 후 저분자량 성분이 1.0% 이하로 안정함을 알 수 있었다. 또한 실시예 1의 결과와 큰 차이가 없음을 알 수 있었다.Referring to Table 71, it can be seen that Examples 20 to 31 were stable at 1.0% or less of low molecular weight components after 4 weeks and after shaking stress in all temperature conditions. In addition, it was found that there was no significant difference from the results of Example 1.
하기 표 72에서 MFI 로 측정한 불용성 이물 입자의 수(10.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 72 below shows the measurement results of the number of insoluble foreign particles (10.00 μm≤, <100.00 μm) measured by MFI.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후　5±3℃ after 2 weeks 40±2℃2주 후　40±2℃ after 2 weeks 5±3℃4주 후　5±3℃ after 4 weeks 40±2℃4주 후　40±2℃ after 4 weeks 흔들림스트레스　shaking stress
실시예 20 Example 20 44 1010 3030 1010 2525 152152
실시예 21Example 21 44 2626 22 2424 1313 13911391
실시예 22Example 22 44 2222 4141 1313 2525 592592
실시예 23Example 23 1111 22 2020 2222 1010 10501050
실시예 24Example 24 1515 44 2323 00 3333 327327
실시예 25Example 25 3737 44 1717 2626 77 4848
실시예 26Example 26 22 00 1313 33 4242 32413241
실시예 27Example 27 77 44 88 2626 1111 1616
실시예 28Example 28 44 55 88 77 1919 9797
실시예 29Example 29 1515 33 165165 1313 3737 2424
실시예 30Example 30 1111 1818 1212 2727 8686 398398
실시예 31Example 31 66 22 1111 77 2424 228228
실시예 1Example 1 66 44 22 44 2424 145145
비교예 1Comparative Example 1 3333 7474 6666 298298 497497 255093255093
표 72를 보면 비교예 1에서 계면활성제가 없을 경우 불용성 이물 입자의 수가 매우 크게 증가함을 확인하였다. 온도 보관뿐만 아니라 흔들림 스트레스에서 불용성 이물 입자의 생성을 막기 위해 계면활성제가 필요하다는 것을 확인할 수 있었다. 실시예 26의 경우 흔들림 스트레스에 의한 불용성 이물 입자의 수가 다른 실시예에 비해 상대적으로 높았다. Referring to Table 72, it was confirmed that in Comparative Example 1, in the absence of a surfactant, the number of insoluble foreign particles was significantly increased. It was confirmed that a surfactant was needed to prevent the formation of insoluble foreign particles under shaking stress as well as temperature storage. In the case of Example 26, the number of insoluble foreign particles due to shaking stress was relatively higher than in the other examples.
하기 표 73에서 MFI 로 측정한 불용성 이물 입자의 수(25.00㎛≤, <100.00㎛) 측정 결과를 나타내었다.Table 73 below shows the measurement results of the number of insoluble foreign particles (25.00 μm≤, <100.00 μm) measured by MFI.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후　5±3℃ after 2 weeks 40±2℃2주 후　40±2℃ after 2 weeks 5±3℃4주 후　5±3℃ after 4 weeks 40±2℃4주 후　40±2℃ after 4 weeks 흔들림스트레스　shaking stress
실시예 20 Example 20 22 22 44 22 44 2525
실시예 21Example 21 00 00 00 22 22 1515
실시예 22Example 22 22 22 66 00 00 2828
실시예 23Example 23 00 00 22 44 00 99
실시예 24Example 24 00 00 22 00 44 100100
실시예 25Example 25 00 22 00 55 22 00
실시예 26Example 26 22 00 00 00 22 7878
실시예 27Example 27 00 00 00 44 00 22
실시예 28Example 28 00 33 22 00 44 2121
실시예 29Example 29 00 00 44 44 44 22
실시예 30Example 30 00 22 00 44 2020 99
실시예 31Example 31 00 00 00 22 00 6868
실시예 1Example 1 00 00 00 00 00 1212
비교예 1Comparative Example 1 00 00 77 1515 4545 14511451
표 73을 보면 계면활성제가 없는 비교예 1을 제외하고 나머지 실시예 20 내지 31의 불용성 이물 입자의 수에 비추어 모든 온도조건에서 4주 후 안정함을 알 수 있었다. Looking at Table 73, it can be seen that, in view of the number of insoluble foreign particles of Examples 20 to 31, except for Comparative Example 1 without a surfactant, it is stable after 4 weeks in all temperature conditions.
하기 표 74에서 HIAC으로 측정한 불용성 이물 입자의 수(10.00≤(um)) 측정 결과를 나타내었다.In Table 74, the number of insoluble foreign particles (10.00≤(um)) measured by HIAC is shown.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후　5±3℃ after 2 weeks 40±2℃2주 후　40±2℃ after 2 weeks 5±3℃4주 후　5±3℃ after 4 weeks 40±2℃4주 후　40±2℃ after 4 weeks 흔들림스트레스　shaking stress
실시예 20 Example 20 33 00 2323 6565 22 22
실시예 21Example 21 33 22 22 22 55 00
실시예 22Example 22 22 00 55 22 33 22
실시예 23Example 23 00 00 2828 22 00 22
실시예 24Example 24 00 00 00 22 00 5050
실시예 25Example 25 7575 22 1212 88 22 00
실시예 26Example 26 00 88 1212 22 33 1818
실시예 27Example 27 77 1515 1010 2020 1818 1515
실시예 28Example 28 1818 22 77 2323 55 5555
실시예 29Example 29 00 00 22 00 55 77
실시예 30Example 30 33 33 33 22 1717 365365
실시예 31Example 31 00 22 1212 22 00 153153
실시예 1Example 1 22 00 55 00 88 9595
비교예 1Comparative Example 1 55 00 00 1313 22 2460324603
표 74를 보면 비교예 1을 제외한 실시예 20 내지 31의 불용성 이물 입자의 수가 모든 온도 보관 조건에서 100개 이하로 안정함을 알 수 있었다. Referring to Table 74, it can be seen that the number of insoluble foreign particles of Examples 20 to 31 except for Comparative Example 1 is 100 or less under all temperature storage conditions.
하기 표 75에서 HIAC으로 측정한 불용성 이물 입자의 수(25.00≤(um)) 측정 결과를 나타내었다.Table 75 below shows the measurement results of the number of insoluble foreign particles (25.00≤(um)) measured by HIAC.
구분division 5±3℃0일 후(T=0)5±3℃ after 0 days (T=0) 5±3℃2주 후　5±3℃ after 2 weeks 40±2℃2주 후　40±2℃ after 2 weeks 5±3℃4주 후　5±3℃ after 4 weeks 40±2℃4주 후　40±2℃ after 4 weeks 흔들림스트레스　shaking stress
실시예 20 Example 20 22 00 33 55 00 22
실시예 21Example 21 00 00 00 00 00 00
실시예 22Example 22 22 00 00 00 00 00
실시예 23Example 23 00 00 1010 00 00 00
실시예 24Example 24 00 00 00 00 00 3232
실시예 25Example 25 4343 00 55 00 00 00
실시예 26Example 26 00 00 1010 00 00 00
실시예 27Example 27 00 22 00 22 00 00
실시예 28Example 28 22 00 22 77 00 88
실시예 29Example 29 00 00 00 00 00 00
실시예 30Example 30 00 22 00 00 33 33
실시예 31Example 31 00 00 88 00 00 1818
실시예 1Example 1 00 00 22 00 00 4040
비교예 1Comparative Example 1 33 00 00 00 00 1619516195
표 75를 보면 비교예 1을 제외한 실시예 20 내지 31의 불용성 이물 입자의 수에 비추어 모든 온도 조건에서 안정함을 알 수 있었고, 실시예 1의 결과와 유사한 것을 알 수 있었다.Looking at Table 75, it can be seen that in view of the number of insoluble foreign particles of Examples 20 to 31 except for Comparative Example 1, it is stable in all temperature conditions, and it can be seen that the results are similar to those of Example 1.

Claims (30)

  1. (A) 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자; (A) a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2;
    (B) 완충제; (B) a buffer;
    (C) 안정화제; 및(C) stabilizers; and
    (D) 계면활성제를 포함하는, 안정한 약제학적 제제.(D) A stable pharmaceutical formulation comprising a surfactant.
  2. 제1항에 있어서, 상기 약제학적 제제는 액상임을 특징으로 하는, 안정한 약제학적 제제.The stable pharmaceutical formulation according to claim 1, characterized in that the pharmaceutical formulation is liquid.
  3. 제1항에 있어서, The method of claim 1,
    (A) 중화 결합 분자는 사스-코로나바이러스-2 표면의 스파이크 단백질의 RBD(Receptor binding domain) 영역에 결합함을 특징으로 하는, 안정한 약제학적 제제.(A) A stable pharmaceutical formulation, characterized in that the neutralizing binding molecule binds to the receptor binding domain (RBD) region of the spike protein on the surface of SARS-coronavirus-2.
  4. 제1항에 있어서,According to claim 1,
    (A) 중화 결합 분자는 표 1 또는 표 3의 결합 분자로 이루어진 군으로부터 선택되는 어느 하나임을 특징으로 하는, 안정한 약제학적 제제.(A) A stable pharmaceutical formulation, characterized in that the neutralizing binding molecule is any one selected from the group consisting of the binding molecules of Table 1 or Table 3.
  5. 제1항에 있어서,According to claim 1,
    (A) 중화 결합 분자는 표 2 또는 표 4의 결합 분자로 이루어진 군으로부터 선택되는 어느 하나임을 특징으로 하는, 안정한 약제학적 제제.(A) A stable pharmaceutical formulation, characterized in that the neutralizing binding molecule is any one selected from the group consisting of the binding molecules of Table 2 or Table 4.
  6. 제1항 내지 제5항 중 어느 한 항에 있어서, 6. The method according to any one of claims 1 to 5,
    (A) 중화 결합 분자는 scFv 절편, scFv-Fc 절편, Fab 절편, Fv 절편, 디아바디(diabody), 키메라 항체, 인간화 항체 또는 인간 항체임을 특징으로 하는, 안정한 약제학적 제제.(A) A stable pharmaceutical formulation, characterized in that the neutralizing binding molecule is an scFv fragment, scFv-Fc fragment, Fab fragment, Fv fragment, diabody, chimeric antibody, humanized antibody or human antibody.
  7. 제1항에 있어서, According to claim 1,
    (A) 중화 결합 분자는 서열번호: 829의 CDR1 영역, 서열번호: 830의 CDR2 영역, 및 서열번호: 831의 CDR3 영역을 포함하는 경쇄 가변영역; 및(A) a neutralizing binding molecule comprising: a light chain variable region comprising a CDR1 region of SEQ ID NO: 829, a CDR2 region of SEQ ID NO: 830, and a CDR3 region of SEQ ID NO: 831; and
    서열번호: 832의 CDR1 영역, 서열번호: 833의 CDR2 영역, 및 서열번호: 834의 CDR3 영역을 포함하는 중쇄 가변영역a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 832, the CDR2 region of SEQ ID NO: 833, and the CDR3 region of SEQ ID NO: 834
    을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation comprising a.
  8. 제1항에 있어서, According to claim 1,
    (A) 중화 결합 분자는 서열번호: 2017의 폴리펩티드 서열의 경쇄 가변영역; 및(A) the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 2017; and
    서열번호: 2018의 폴리펩티드 서열의 중쇄 가변영역SEQ ID NO: 2018 heavy chain variable region of the polypeptide sequence
    을 포함하는 결합 분자와 80% 내지 99% 동일한 서열을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation, characterized in that it comprises a sequence that is 80% to 99% identical to a binding molecule comprising a.
  9. 제1항에 있어서, According to claim 1,
    (A) 중화 결합 분자는 서열번호: 2017의 폴리펩티드 서열의 경쇄 가변영역; 및(A) the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 2017; and
    서열번호: 2018의 폴리펩티드 서열의 중쇄 가변영역SEQ ID NO: 2018 heavy chain variable region of the polypeptide sequence
    을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation comprising a.
  10. 제1항에 있어서, The method of claim 1,
    (A) 중화 결합 분자는 서열번호: 2507의 CDR1 영역, 서열번호: 2508의 CDR2 영역, 및 서열번호: 2509의 CDR3 영역을 포함하는 경쇄 가변영역; 및(A) a neutralizing binding molecule comprises a light chain variable region comprising a CDR1 region of SEQ ID NO: 2507, a CDR2 region of SEQ ID NO: 2508, and a CDR3 region of SEQ ID NO: 2509; and
    서열번호: 2510의 CDR1 영역, 서열번호: 2511의 CDR2 영역, 및 서열번호: 2512의 CDR3 영역을 포함하는 중쇄 가변영역a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 2510, the CDR2 region of SEQ ID NO: 2511, and the CDR3 region of SEQ ID NO: 2512
    을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation comprising a.
  11. 제1항에 있어서, According to claim 1,
    (A) 중화 결합 분자는 서열번호: 3523의 폴리펩티드 서열의 경쇄 가변영역; 및(A) the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 3523; and
    서열번호: 3524의 폴리펩티드 서열의 중쇄 가변영역Heavy chain variable region of the polypeptide sequence of SEQ ID NO: 3524
    을 포함하는 결합 분자와 80% 내지 99% 동일한 서열을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation, characterized in that it comprises a sequence that is 80% to 99% identical to a binding molecule comprising a.
  12. 제1항에 있어서, According to claim 1,
    (A) 중화 결합 분자는 서열번호: 3523의 폴리펩티드 서열의 경쇄 가변영역; 및(A) the neutralizing binding molecule comprises a light chain variable region of the polypeptide sequence of SEQ ID NO: 3523; and
    서열번호: 3524의 폴리펩티드 서열의 중쇄 가변영역Heavy chain variable region of the polypeptide sequence of SEQ ID NO: 3524
    을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation comprising a.
  13. 제1항에 있어서, According to claim 1,
    (A) 중화 결합 분자의 농도는 1 내지 240 mg/ml임을 특징으로 하는, 안정한 약제학적 제제.(A) A stable pharmaceutical formulation, characterized in that the concentration of the neutralizing binding molecule is 1 to 240 mg/ml.
  14. 제1항에 있어서,According to claim 1,
    (B) 완충제는i) 히스티딘, 히스티딘염 또는 이들의 혼합물, ii) 아세테이트(acetate), iii) 시트레이트(citrate), iv) 숙시네이트(succinate), v) 포스페이트(phosphate), 또는 vi) 글루코네이트(gluconate)를 포함함을 특징으로 하는, 안정한 약제학적 제제.(B) the buffer is i) histidine, histidine salt or mixtures thereof, ii) acetate, iii) citrate, iv) succinate, v) phosphate, or vi) gluco A stable pharmaceutical preparation, characterized in that it contains gluconate.
  15. 제1항에 있어서,According to claim 1,
    (B) 완충제의 함량은 1 내지 100 mM임을 특징으로 하는, 안정한 약제학적 제제.(B) A stable pharmaceutical formulation, characterized in that the content of the buffer is 1 to 100 mM.
  16. 제1항에 있어서,According to claim 1,
    (C) 안정화제는 i) 금속염, ii) 당 또는 이의 유도체 및 iii) 아미노산 또는 이의 염으로 구성된 군으로부터 선택되는 어느 하나 이상을 포함함을 특징으로 하는, 안정한 약제학적 제제.(C) The stabilizing agent, characterized in that it comprises any one or more selected from the group consisting of i) a metal salt, ii) a sugar or a derivative thereof, and iii) an amino acid or a salt thereof.
  17. 제16항에 있어서,17. The method of claim 16,
    상기 i) 금속염은 염화나트륨(NaCl), 염화칼륨(KCl), 염화칼슘(CaCl) 또는 이들 중 2 이상의 혼합물을 포함함을 특징으로 하는 안정한 약제학적 제제.Wherein i) the metal salt is sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl), or a stable pharmaceutical formulation, characterized in that it comprises a mixture of two or more thereof.
  18. 제16항에 있어서,17. The method of claim 16,
    상기 ii) 당 또는 이의 유도체는 소르비톨, 만니톨, 트레할로스, 수크로오스 또는 이들 중 2 이상의 혼합물을 포함함을 특징으로 하는 안정한 약제학적 제제.wherein ii) the sugar or derivative thereof comprises sorbitol, mannitol, trehalose, sucrose, or a mixture of two or more thereof.
  19. 제16항에 있어서,17. The method of claim 16,
    상기 iii) 아미노산은 글리신, 트레오닌, 메티오닌, 아르기닌 또는 이들 중 2 이상의 혼합물을 포함함을 특징으로 하는, 안정한 약제학적 제제.wherein iii) amino acid comprises glycine, threonine, methionine, arginine or a mixture of two or more thereof.
  20. 제14항에 있어서,15. The method of claim 14,
    상기 iii) 아미노산의 염은 L-아르기닌 모노하이드로클로라이드(monohydrochloride)를 포함함을 특징으로 하는, 안정한 약제학적 제제.The iii) salt of the amino acid is L- arginine monohydrochloride (monohydrochloride), characterized in that it comprises a stable pharmaceutical formulation.
  21. 제1항에 있어서,The method of claim 1,
    (C) 안정화제의 함량은 50 내지 300 mM 또는 1 내지 20%(w/v)임을 특징으로 하는, 안정한 약제학적 제제.(C) A stable pharmaceutical formulation, characterized in that the content of the stabilizer is 50 to 300 mM or 1 to 20% (w/v).
  22. 제1항에 있어서, According to claim 1,
    (D) 계면활성제는 폴리소르베이트, 폴록사머 또는 이들의 혼합물을 포함함을 특징으로 하는, 안정한 약제학적 제제.(D) A stable pharmaceutical formulation, characterized in that the surfactant comprises a polysorbate, a poloxamer or a mixture thereof.
  23. 제21항에 있어서, 22. The method of claim 21,
    상기 폴리소르베이트는 폴리소르베이트 20, 폴리소르베이트 40, 폴리소르베이트 60, 폴리소르베이트 80 또는 이들의 혼합물을 포함함을 특징으로 하는, 안정한 약제학적 제제.Wherein the polysorbate comprises polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80 or a mixture thereof, a stable pharmaceutical formulation.
  24. 제1항에 있어서,According to claim 1,
    (D) 계면활성제의 농도는 0.01 내지 0.1 %(w/v)임을 특징으로 하는, 안정한 약제학적 제제.(D) A stable pharmaceutical formulation, characterized in that the concentration of the surfactant is 0.01 to 0.1% (w/v).
  25. 제1항에 있어서,According to claim 1,
    pH가 5.0 내지 7.0임을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation, characterized in that the pH is 5.0 to 7.0.
  26. (A) 사스-코로나바이러스-2(SARS-CoV-2) 표면의 스파이크 단백질(Spike protein, S protein)에 결합하는 중화 결합 분자 1 내지 240 mg/ml; (A) 1 to 240 mg/ml of a neutralizing binding molecule that binds to a spike protein (S protein) on the surface of SARS-CoV-2;
    (B) 완충제 1 내지 100 mM; (B) 1-100 mM buffer;
    (C) 안정화제 50 내지 300 mM 또는 1 내지 20%(w/v); 및(C) 50-300 mM or 1-20% (w/v) of a stabilizing agent; and
    (D) 계면활성제 0.01 내지 0.1 %(w/v)를 포함하는, 안정한 약제학적 제제.(D) a stable pharmaceutical formulation comprising 0.01 to 0.1% (w/v) of a surfactant.
  27. 제26항에 있어서,27. The method of claim 26,
    상기 (A) 중화 결합 분자는 서열번호: 829의 CDR1 영역, 서열번호: 830의 CDR2 영역, 및 서열번호: 831의 CDR3 영역을 포함하는 경쇄 가변영역; 및Said (A) neutralizing binding molecule comprises: a light chain variable region comprising a CDR1 region of SEQ ID NO: 829, a CDR2 region of SEQ ID NO: 830, and a CDR3 region of SEQ ID NO: 831; and
    서열번호: 832의 CDR1 영역, 서열번호: 833의 CDR2 영역, 및 서열번호: 834의 CDR3 영역을 포함하는 중쇄 가변영역a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 832, the CDR2 region of SEQ ID NO: 833, and the CDR3 region of SEQ ID NO: 834
    을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation comprising a.
  28. 제26항에 있어서,27. The method of claim 26,
    상기 (A) 중화 결합 분자는 서열번호: 2507의 CDR1 영역, 서열번호: 2508의 CDR2 영역, 및 서열번호: 2509의 CDR3 영역을 포함하는 경쇄 가변영역; 및The (A) neutralizing binding molecule comprises: a light chain variable region comprising a CDR1 region of SEQ ID NO: 2507, a CDR2 region of SEQ ID NO: 2508, and a CDR3 region of SEQ ID NO: 2509; and
    서열번호: 2510의 CDR1 영역, 서열번호: 2511의 CDR2 영역, 및 서열번호: 2512의 CDR3 영역을 포함하는 중쇄 가변영역a heavy chain variable region comprising the CDR1 region of SEQ ID NO: 2510, the CDR2 region of SEQ ID NO: 2511, and the CDR3 region of SEQ ID NO: 2512
    을 포함함을 특징으로 하는, 안정한 약제학적 제제.A stable pharmaceutical formulation comprising a.
  29. 제1항 내지 제28항 중 어느 한 항에 기재된 안정한 약제학적 제제가 충진된 유리 바이알(vial).29. A glass vial filled with the stable pharmaceutical formulation according to any one of claims 1 to 28.
  30. 제1항 내지 제28항 중 어느 한 항에 기재된 안정한 약제학적 제제가 충진된 프리-필드 시린지(pre-filled syringe).29. A pre-filled syringe filled with the stable pharmaceutical formulation according to any one of claims 1 to 28.
PCT/KR2021/010606 2020-08-11 2021-08-10 Stable pharmaceutical formulation WO2022035197A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20200100805 2020-08-11
KR10-2020-0100805 2020-08-11

Publications (1)

Publication Number Publication Date
WO2022035197A1 true WO2022035197A1 (en) 2022-02-17

Family

ID=80248006

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2021/010606 WO2022035197A1 (en) 2020-08-11 2021-08-10 Stable pharmaceutical formulation

Country Status (2)

Country Link
KR (1) KR20220020228A (en)
WO (1) WO2022035197A1 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022261153A1 (en) * 2021-06-08 2022-12-15 Eli Lilly And Company 2019 novel coronavirus antibody-containing pharmaceutical formulations
WO2023079086A1 (en) * 2021-11-05 2023-05-11 Astrazeneca Uk Limited Composition for treatment and prevention of covid-19
US12030927B2 (en) 2022-02-18 2024-07-09 Rq Biotechnology Limited Antibodies capable of binding to the spike protein of coronavirus SARS-CoV-2

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060120568A (en) * 2003-07-22 2006-11-27 크루셀 홀란드 비.브이. Binding molecules against sars-coronavirus and uses thereof
KR20200010103A (en) * 2018-07-19 2020-01-30 (주)셀트리온 Stable Liquid Pharmaceutical Formulation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20060120568A (en) * 2003-07-22 2006-11-27 크루셀 홀란드 비.브이. Binding molecules against sars-coronavirus and uses thereof
KR20200010103A (en) * 2018-07-19 2020-01-30 (주)셀트리온 Stable Liquid Pharmaceutical Formulation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KIM SANG IL, NOH JINSUNG, KIM SUJEONG, CHOI YOUNGGEUN, YOO DUCK KYUN, LEE YONGHEE, LEE HYUNHO, JUNG JONGTAK, KANG CHANG KYUNG, SON: "Stereotypic Neutralizing V H Clonotypes Against SARS-CoV-2 RBD in COVID-19 Patients and the Healthy Population", BIORXIV, 2 July 2020 (2020-07-02), pages 1 - 56, XP055899840, DOI: 10.1101/2020.06.26.174557 *
PARRAY HILAL AHMAD, CHIRANJIVI ADARSH KUMAR, ASTHANA SHAILENDRA, YADAV NAVEEN, SHRIVASTAVA TRIPTI, MANI SHAILENDRA, SHARMA CHANDRE: "Identification of an anti–SARS–CoV-2 receptor-binding domain–directed human monoclonal antibody from a naïve semisynthetic library", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 295, no. 36, 1 September 2020 (2020-09-01), US , pages 12814 - 12821, XP055899844, ISSN: 0021-9258, DOI: 10.1074/jbc.AC120.014918 *
SHI RUI, SHAN CHAO, DUAN XIAOMIN, CHEN ZHIHAI, LIU PEIPEI, SONG JINWEN, SONG TAO, BI XIAOSHAN, HAN CHAO, WU LIANAO, GAO GE, HU XUE: "A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2", NATURE, vol. 584, no. 7819, 6 August 2020 (2020-08-06), London, pages 120 - 124, XP055891106, ISSN: 0028-0836, DOI: 10.1038/s41586-020-2381-y *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022261153A1 (en) * 2021-06-08 2022-12-15 Eli Lilly And Company 2019 novel coronavirus antibody-containing pharmaceutical formulations
WO2023079086A1 (en) * 2021-11-05 2023-05-11 Astrazeneca Uk Limited Composition for treatment and prevention of covid-19
US12030927B2 (en) 2022-02-18 2024-07-09 Rq Biotechnology Limited Antibodies capable of binding to the spike protein of coronavirus SARS-CoV-2

Also Published As

Publication number Publication date
KR20220020228A (en) 2022-02-18

Similar Documents

Publication Publication Date Title
WO2022035197A1 (en) Stable pharmaceutical formulation
WO2021194188A1 (en) Binding molecule having neutralizing activity against sars-coronavirus-2
WO2017116205A1 (en) Persistent conjugate of triple activator activating glucagon, glp-1 and gip receptor
WO2018004260A1 (en) Stable liquid pharmaceutical preparation
WO2014158001A1 (en) Composition comprising at least two influenza a virus-neutralizing-binding molecules
WO2021060791A1 (en) Nano-perforator having improved anti-viral activity
WO2022211537A1 (en) Novel immunoactive interleukin 2 analog conjugate and method for preparing same
WO2019066586A1 (en) Long-acting conjugate of glucagon-like peptide-2 (glp-2) derivative
WO2016200189A1 (en) Rabies virus g protein epitope, and rabies virus neutralising binding molecule that binds specifically thereto
WO2022211558A1 (en) Sars-coronavirus-2-neutralizing binding molecule that binds to epitope of sars-coronavirus-2 spike protein
WO2020071865A1 (en) Therapeutic uses of glucagon and combined product comprising same
WO2019203599A1 (en) Streptococcus pneumoniae capsular polysaccharides and immunogenic conjugate thereof
WO2020017901A1 (en) Stable liquid pharmaceutical preparation
WO2016199964A1 (en) Antibody specifically binding to isolated vimentin-derived peptide, or binding fragment of peptide
WO2022019671A1 (en) Sars-coronavirus-2-neutralizing binding molecule that binds to epitope of sars-coronavirus-2 spike protein
WO2022149837A1 (en) Anti-fgfr3 antibody and use thereof
WO2021060837A1 (en) Anti-mers-cov antibody and use thereof
WO2022085905A1 (en) Antibody binding specifically to sars-cov-2 spike protein and use thereof
WO2020214013A1 (en) Therapeutic use, for hyperlipideamia, of triple agonist having activity with respect to all of glucagon, glp-1, and gip receptors, or conjugate thereof
WO2022025660A1 (en) Stable pharmaceutical preparation
WO2021235913A1 (en) Liquid formulation of long-acting conjugate of glp-2
WO2022015115A1 (en) Therapeutic use of combination containing triple agonistic long-acting conjugate or triple agonist
WO2021235907A1 (en) Liquid formulation of long-acting conjugate of glucagon derivative
WO2020138868A1 (en) Type 1 interferon neutralizing fc-fusion protein and use thereof
WO2023277620A1 (en) Therapeutic use of combination comprising triple activator having activity on all of glucagon, glp-1 and gip receptor

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21856203

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21856203

Country of ref document: EP

Kind code of ref document: A1