JPWO2020086893A5 - - Google Patents
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- JPWO2020086893A5 JPWO2020086893A5 JP2021522332A JP2021522332A JPWO2020086893A5 JP WO2020086893 A5 JPWO2020086893 A5 JP WO2020086893A5 JP 2021522332 A JP2021522332 A JP 2021522332A JP 2021522332 A JP2021522332 A JP 2021522332A JP WO2020086893 A5 JPWO2020086893 A5 JP WO2020086893A5
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- 210000000234 capsid Anatomy 0.000 claims description 87
- 239000002245 particle Substances 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 39
- 241000700605 Viruses Species 0.000 claims description 27
- 235000004252 protein component Nutrition 0.000 claims description 24
- 230000003612 virological effect Effects 0.000 claims description 23
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- 241001164823 Adeno-associated virus - 7 Species 0.000 claims description 8
- 241001164825 Adeno-associated virus - 8 Species 0.000 claims description 8
- 241000713800 Feline immunodeficiency virus Species 0.000 claims description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 6
- 241001655883 Adeno-associated virus - 1 Species 0.000 claims description 6
- 241000702423 Adeno-associated virus - 2 Species 0.000 claims description 6
- 241000202702 Adeno-associated virus - 3 Species 0.000 claims description 6
- 241000580270 Adeno-associated virus - 4 Species 0.000 claims description 6
- 241001634120 Adeno-associated virus - 5 Species 0.000 claims description 6
- 241000972680 Adeno-associated virus - 6 Species 0.000 claims description 6
- 241000649045 Adeno-associated virus 10 Species 0.000 claims description 6
- 241000649046 Adeno-associated virus 11 Species 0.000 claims description 6
- 241000649047 Adeno-associated virus 12 Species 0.000 claims description 6
- 241000713704 Bovine immunodeficiency virus Species 0.000 claims description 4
- 108090000565 Capsid Proteins Proteins 0.000 claims description 4
- 102100023321 Ceruloplasmin Human genes 0.000 claims description 4
- 241000702421 Dependoparvovirus Species 0.000 claims description 4
- 241000714165 Feline leukemia virus Species 0.000 claims description 4
- 241000713858 Harvey murine sarcoma virus Species 0.000 claims description 4
- 241000725303 Human immunodeficiency virus Species 0.000 claims description 4
- 241000713862 Moloney murine sarcoma virus Species 0.000 claims description 4
- 241000125945 Protoparvovirus Species 0.000 claims description 4
- 241000714474 Rous sarcoma virus Species 0.000 claims description 4
- 241000713311 Simian immunodeficiency virus Species 0.000 claims description 4
- 241000713675 Spumavirus Species 0.000 claims description 4
- 108700019146 Transgenes Proteins 0.000 claims description 4
- 230000003993 interaction Effects 0.000 claims description 4
- 238000004811 liquid chromatography Methods 0.000 claims description 4
- 239000013603 viral vector Substances 0.000 claims description 4
- 235000018102 proteins Nutrition 0.000 claims description 3
- 102000004169 proteins and genes Human genes 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 3
- 239000013607 AAV vector Substances 0.000 claims description 2
- 241000701242 Adenoviridae Species 0.000 claims description 2
- 241000537221 Alphabaculovirus Species 0.000 claims description 2
- 241001664176 Alpharetrovirus Species 0.000 claims description 2
- 241000702419 Ambidensovirus Species 0.000 claims description 2
- 241001219222 Amdoparvovirus Species 0.000 claims description 2
- 241001443586 Atadenovirus Species 0.000 claims description 2
- 241000701802 Aviadenovirus Species 0.000 claims description 2
- 241000701412 Baculoviridae Species 0.000 claims description 2
- 241000537222 Betabaculovirus Species 0.000 claims description 2
- 241001231757 Betaretrovirus Species 0.000 claims description 2
- 241000124740 Bocaparvovirus Species 0.000 claims description 2
- 206010006187 Breast cancer Diseases 0.000 claims description 2
- 241000965621 Brevidensovirus Species 0.000 claims description 2
- 241000701022 Cytomegalovirus Species 0.000 claims description 2
- 241000537219 Deltabaculovirus Species 0.000 claims description 2
- 241001663879 Deltaretrovirus Species 0.000 claims description 2
- 241001663878 Epsilonretrovirus Species 0.000 claims description 2
- 241000713730 Equine infectious anemia virus Species 0.000 claims description 2
- 241000283073 Equus caballus Species 0.000 claims description 2
- 241000121268 Erythroparvovirus Species 0.000 claims description 2
- 241000537223 Gammabaculovirus Species 0.000 claims description 2
- 241001663880 Gammaretrovirus Species 0.000 claims description 2
- 241000713813 Gibbon ape leukemia virus Species 0.000 claims description 2
- 241000439647 Hepandensovirus Species 0.000 claims description 2
- 241000700586 Herpesviridae Species 0.000 claims description 2
- 241001651351 Ichtadenovirus Species 0.000 claims description 2
- 241001051759 Iltovirus Species 0.000 claims description 2
- 206010061598 Immunodeficiency Diseases 0.000 claims description 2
- 208000029462 Immunodeficiency disease Diseases 0.000 claims description 2
- 241000121270 Iteradensovirus Species 0.000 claims description 2
- 241000713666 Lentivirus Species 0.000 claims description 2
- 241000701043 Lymphocryptovirus Species 0.000 claims description 2
- 102100033448 Lysosomal alpha-glucosidase Human genes 0.000 claims description 2
- 241000175216 Macavirus Species 0.000 claims description 2
- 241001051756 Mardivirus Species 0.000 claims description 2
- 241000701244 Mastadenovirus Species 0.000 claims description 2
- 241001529936 Murinae Species 0.000 claims description 2
- 241000701034 Muromegalovirus Species 0.000 claims description 2
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 2
- 241000701945 Parvoviridae Species 0.000 claims description 2
- 241000712907 Retroviridae Species 0.000 claims description 2
- 241000122129 Roseolovirus Species 0.000 claims description 2
- 241000700584 Simplexvirus Species 0.000 claims description 2
- 208000000389 T-cell leukemia Diseases 0.000 claims description 2
- 208000028530 T-cell lymphoblastic leukemia/lymphoma Diseases 0.000 claims description 2
- 241000404928 Tetraparvovirus Species 0.000 claims description 2
- 208000010094 Visna Diseases 0.000 claims description 2
- 108700010877 adenoviridae proteins Proteins 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 108010028144 alpha-Glucosidases Proteins 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- 230000007813 immunodeficiency Effects 0.000 claims description 2
- 210000004185 liver Anatomy 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims description 2
- 238000006467 substitution reaction Methods 0.000 claims description 2
- 241001430294 unidentified retrovirus Species 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 230000002463 transducing effect Effects 0.000 claims 1
Description
いくつかの実施形態において、移動相Ａは、約４５分かけて、約１５％から約１００％まで増加する。
[本発明1001]
ウイルス粒子のウイルスカプシドのタンパク質成分の化学量論を決定する方法であって、
ウイルス粒子のサンプルを親水性相互作用液体クロマトグラフィー（ＨＩＬＩＣ）に供して、前記ウイルス粒子のウイルスカプシドのタンパク質成分を分離することと、
前記ウイルスカプシドのタンパク質成分の質量を決定して、ＨＩＬＩＣによって分離された前記タンパク質成分を同定することと、
前記ＨＩＬＩＣ分離からの前記ウイルスカプシドのタンパク質成分の相対存在量を決定し、それにより、ウイルス粒子のウイルスカプシドのタンパク質成分の化学量論を決定することと
を含む、前記方法。
[本発明1002]
前記ウイルス粒子が、アデノ随伴ウイルス（ＡＡＶ）粒子を含む、本発明1001の方法。
[本発明1003]
前記タンパク質カプシドのタンパク質成分が、前記ＡＡＶ粒子のＶＰ1、ＶＰ2及びＶＰ3を含む、本発明1002の方法。
[本発明1004]
前記ＶＰ1、ＶＰ2、及びＶＰ3の質量が、ＡＡＶ1カプシド、ＡＡＶ2カプシド、ＡＡＶ3カプシド、ＡＡＶ4カプシド、ＡＡＶ5カプシド、ＡＡＶ6カプシド、ＡＡＶ7カプシド、ＡＡＶ8カプシド、ＡＡＶｒｈ8カプシド、ＡＡＶ9カプシド、ＡＡＶ10カプシド、ＡＡＶ11カプシド、ＡＡＶ12カプシド、またはそのバリアントのうちの1つ以上の理論的質量と比較される、本発明1002の方法。
[本発明1005]
前記ＡＡＶ粒子が、ＡＡＶ1カプシド、ＡＡＶ2カプシド、ＡＡＶ3カプシド、ＡＡＶ4カプシド、ＡＡＶ5カプシド、ＡＡＶ6カプシド、ＡＡＶ7カプシド、ＡＡＶ8カプシド、ＡＡＶｒｈ8カプシド、ＡＡＶ9カプシド、ＡＡＶ10カプシド、ＡＡＶ11カプシド、ＡＡＶ12カプシド、またはそのバリアントを含む、本発明1003の方法。
[本発明1006]
前記ＡＡＶ粒子が、ＡＡＶ1 ＩＴＲ、ＡＡＶ2 ＩＴＲ、ＡＡＶ3 ＩＴＲ、ＡＡＶ4 ＩＴＲ、ＡＡＶ5 ＩＴＲ、ＡＡＶ6 ＩＴＲ、ＡＡＶ7 ＩＴＲ、ＡＡＶ8 ＩＴＲ、ＡＡＶ9 ＩＴＲ、ＡＡＶ10 ＩＴＲ、ＡＡＶ11 ＩＴＲ、またはＡＡＶ12 ＩＴＲを含む、本発明1002の方法。
[本発明1007]
前記ＡＡＶ粒子が、組換えＡＡＶ（ｒＡＡＶ）粒子または異種導入遺伝子をコードするＡＡＶベクターである、本発明1002の方法。
[本発明1008]
前記ＡＡＶ粒子が、対象の肝臓の細胞に形質導入するために選択されたカプシドセロタイプを有する、本発明1002の方法。
[本発明1009]
前記ＡＡＶ粒子が、カプシドセロタイプＡＡＶ7、ＡＡＶ8、またはＡＡＶ9を有する、本発明1008の方法。
[本発明1010]
前記ＡＡＶ粒子が、カプシドセロタイプＡＡＶ9を有する、本発明1009の方法。
[本発明1011]
前記ＡＡＶ粒子が、カプシドセロタイプＡＡＶ9を有しており、抗ＣＤ63抗体に連結したライソゾームアルファグルコシダーゼ（ＧＡＡ）をコードするウイルスベクターである、本発明1002の方法。
[本発明1012]
前記ウイルス粒子が、異種導入遺伝子をコードするウイルスベクターを含むか、またはアデノウイルス（Ａｄｅｎｏｖｉｒｉｄａｅ）、パルボウイルス（Ｐａｒｖｏｖｉｒｉｄａｅ）、レトロウイルス（Ｒｅｔｒｏｖｉｒｉｄａｅ）、バキュロウイルス（Ｂａｃｕｌｏｖｉｒｉｄａｅ）、及びヘルペスウイルス（Ｈｅｒｐｅｓｖｉｒｉｄａｅ）からなる群から選択されるウイルス科に属する、本発明1001の方法。
[本発明1013]
前記ウイルス粒子が、アタデノウイルス、アビアデノウイルス、イクタデノウイルス、マストアデノウイルス、シアデノウイルス、アンビデンソウイルス、ブレビデンソウイルス、ヘパンデンソウイルス、イテラデンソウイルス、ペンスチルデンソウイルス、アムドパルボウイルス、アベパルボウイルス、ボカパルボウイルス、コピパルボウイルス、ディペンドパルボウイルス、エリスロパルボウイルス、プロトパルボウイルス、テトラパルボウイルス、アルファレトロウイルス、ベータレトロウイルス、デルタレトロウイルス、イプシロンレトロウイルス、ガンマレトロウイルス、レンチウイルス、スプーマウイルス、アルファバキュロウイルス、ベータバキュロウイルス、デルタバキュロウイルス、ガンマバキュロウイルス、イルトウイルス、マルディウイルス、シンプレックスウイルス、バリセロウイルス、サイトメガロウイルス、ムロメガロウイルス、プロボスシウイルス、ロゼオロウイルス、リンホクリプトウイルス、マカウイルス、ペルカウイルス、及びラディノウイルスからなる群から選択されるウイルス属に属する、本発明1012の方法。
[本発明1014]
前記レトロウイルスが、モロニーマウス肉腫ウイルス（ＭｏＭＳＶ）、ハーベイマウス肉腫ウイルス（ＨａＭｕＳＶ）、マウス乳癌ウイルス（ＭｕＭＴＶ）、テナガザル白血病ウイルス（ＧａＬＶ）、ネコ白血病ウイルス（ＦＬＶ）、スプーマウイルス、フレンドウイルス、マウス幹細胞ウイルス（ＭＳＣＶ）ラウス肉腫ウイルス（ＲＳＶ）、ヒトＴ細胞白血病ウイルス、ヒト免疫不全ウイルス（ＨＩＶ）、ネコ免疫不全ウイルス（ＦＩＶ）、ウマ免疫不全ウイルス（ＥＩＶ）、ビスナ・マエディウイルス；ヤギ関節炎・脳炎ウイルス；ウマ伝染性貧血ウイルス；ネコ免疫不全ウイルス（ＦＩＶ）；ウシ免疫不全ウイルス（ＢＩＶ）；またはサル免疫不全ウイルス（ＳＩＶ）である、本発明1012の方法。
[本発明1015]
前記ＨＩＬＩＣが、水中にトリフルオロ酢酸を含む移動相Ａまたはアセトニトリル中にトリフルオロ酢酸を含む移動相Ｂを使用する、本発明1001の方法。
[本発明1016]
前記移動相Ａまたは前記移動相Ｂが、約0．1％トリフルオロ酢酸を含む、本発明1013の方法。
[本発明1017]
前記クロマトグラフィーにおける移動相Ａの割合が、時間の経過とともに増加する、本発明1015の方法。
[本発明1018]
移動相Ａが、約45分かけて、約15％から約100％まで増加する、本発明1017の方法。
[本発明1019]
ウイルス粒子のカプシドにおけるタンパク質成分の不均一性を決定する方法であって、
前記ウイルス粒子を親水性相互作用液体クロマトグラフィー（ＨＩＬＩＣ）に供して、前記ウイルス粒子カプシドのタンパク質成分を分離することと、
前記タンパク質カプシドのタンパク質成分の質量を決定することと、
前記決定されたウイルス粒子カプシドのタンパク質成分の質量を理論的質量と比較することであって、前記理論的質量からの、前記ウイルス粒子カプシドのタンパク質成分の質量のうちの1つ以上のずれは、カプシド不均一性を示している、ことと
を含む、前記方法。
[本発明1020]
前記不均一性が、セロタイプの混合、バリアントカプシド、カプシドアミノ酸の置換、切断カプシド、または改変カプシドのうちの1つ以上を含む、本発明1019の方法。
In some embodiments, mobile phase A is increased from about 15% to about 100% over about 45 minutes.
[Invention 1001]
A method for determining the stoichiometry of protein components of a viral capsid of a viral particle, comprising:
subjecting a sample of virus particles to hydrophilic interaction liquid chromatography (HILIC) to separate the protein components of the viral capsid of said virus particles;
determining the mass of the protein component of the viral capsid to identify the protein component separated by HILIC;
determining the relative abundance of the viral capsid protein components from the HILIC isolate, thereby determining the viral capsid protein component stoichiometry of the viral particles;
The above method, comprising
[Invention 1002]
1001. The method of invention 1001, wherein said viral particles comprise adeno-associated virus (AAV) particles.
[Invention 1003]
1003. The method of invention 1002, wherein the protein components of said protein capsid comprise VP1, VP2 and VP3 of said AAV particle.
[Invention 1004]
AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAV11 capsid, AAV12 capsid, or compared to the theoretical mass of one or more of its variants.
[Invention 1005]
wherein the AAV particle comprises an AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAV9 capsid, AAV10 capsid, AAV11 capsid, AAV12 capsid, or variants thereof; The method of the invention 1003.
[Invention 1006]
1003. The method of invention 1002, wherein the AAV particle comprises an AAV1 ITR, AAV2 ITR, AAV3 ITR, AAV4 ITR, AAV5 ITR, AAV6 ITR, AAV7 ITR, AAV8 ITR, AAV9 ITR, AAV10 ITR, AAV11 ITR, or AAV12 ITR.
[Invention 1007]
1003. The method of invention 1002, wherein said AAV particle is a recombinant AAV (rAAV) particle or an AAV vector encoding a heterologous transgene.
[Invention 1008]
1003. The method of invention 1002, wherein said AAV particles have a capsid serotype selected to transduce cells of the subject's liver.
[Invention 1009]
1008. The method of invention 1008, wherein said AAV particles have capsid serotypes AAV7, AAV8, or AAV9.
[Invention 1010]
1009. The method of the invention 1009, wherein said AAV particles have capsid serotype AAV9.
[Invention 1011]
1003. The method of invention 1002, wherein said AAV particle has capsid serotype AAV9 and is a viral vector encoding lysosomal alpha-glucosidase (GAA) linked to an anti-CD63 antibody.
[Invention 1012]
said viral particles comprise or consist of a viral vector encoding a heterologous transgene, Adenoviridae, Parvoviridae, Retroviridae, Baculoviridae, and Herpesviridae 1001. The method of the invention 1001 belonging to a virus family selected from the group.
[Invention 1013]
The virus particles are atadenovirus, aviadenovirus, ichtadenovirus, mastadenovirus, cyanadenovirus, ambidensovirus, brevidensovirus, hepandensovirus, iteradensovirus, penstildensovirus, Amdovirus. Parvovirus, abeparvovirus, bocaparvovirus, copyparvovirus, dependoparvovirus, erythroparvovirus, protoparvovirus, tetraparvovirus, alpharetrovirus, betaretrovirus, deltaretrovirus, epsilonretrovirus, gammaretrovirus , lentivirus, spumavirus, alpha baculovirus, beta baculovirus, delta baculovirus, gamma baculovirus, iltovirus, mardivirus, simplex virus, baricellovirus, cytomegalovirus, muromegalovirus, provossivirus, roseolovirus 1013. The method of the invention 1012 belonging to a virus genus selected from the group consisting of viruses, Lymphocryptoviruses, Macaviruses, Perchaviruses, and Radinoviruses.
[Invention 1014]
The retrovirus is Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spuma virus, friend virus, mouse stem cells Viruses (MSCV) Rous Sarcoma Virus (RSV), Human T-cell Leukemia Virus, Human Immunodeficiency Virus (HIV), Feline Immunodeficiency Virus (FIV), Equine Immunodeficiency Virus (EIV), Visna Maedi Virus; Equine Infectious Anemia Virus; Feline Immunodeficiency Virus (FIV); Bovine Immunodeficiency Virus (BIV); or Simian Immunodeficiency Virus (SIV).
[Invention 1015]
1001. The method of invention 1001, wherein said HILIC uses mobile phase A comprising trifluoroacetic acid in water or mobile phase B comprising trifluoroacetic acid in acetonitrile.
[Invention 1016]
1013. The method of invention 1013, wherein said mobile phase A or said mobile phase B comprises about 0.1% trifluoroacetic acid.
[Invention 1017]
1015. The method of invention 1015, wherein the proportion of mobile phase A in said chromatography increases over time.
[Invention 1018]
The method of invention 1017, wherein mobile phase A is increased from about 15% to about 100% over about 45 minutes.
[Invention 1019]
A method for determining the heterogeneity of protein components in a viral particle capsid, comprising:
subjecting the virus particles to hydrophilic interaction liquid chromatography (HILIC) to separate protein components of the virus particle capsid;
determining the mass of the protein component of said protein capsid;
comparing the determined mass of the protein component of the viral particle capsid to a theoretical mass, wherein the deviation of one or more of the masses of the protein component of the viral particle capsid from the theoretical mass is showing capsid heterogeneity and
The above method, comprising
[Invention 1020]
1019. The method of the invention 1019, wherein said heterogeneity comprises one or more of mixed serotypes, variant capsids, capsid amino acid substitutions, truncated capsids, or modified capsids.
Claims (20)
前記ウイルス粒子を含むサンプルを親水性相互作用液体クロマトグラフィー（ＨＩＬＩＣ）に供して、前記ウイルス粒子のウイルスカプシドのタンパク質成分を分離することと、
前記ウイルスカプシドのタンパク質成分の質量を決定して、ＨＩＬＩＣによって分離された前記タンパク質成分を同定することと、
前記ＨＩＬＩＣ分離からの前記ウイルスカプシドのタンパク質成分の相対存在量を決定し、それにより、前記ウイルス粒子の前記ウイルスカプシドのタンパク質成分の化学量論を決定することと
を含む、前記方法。 A method for determining the stoichiometry of protein components of intact, non-enveloped viral particles, comprising:
subjecting the sample containing the virus particles to hydrophilic interaction liquid chromatography (HILIC) to separate the protein components of the virus capsid of the virus particles;
determining the mass of the protein component of the viral capsid to identify the protein component separated by HILIC;
determining the relative abundance of said viral capsid protein components from said HILIC isolate, thereby determining the stoichiometry of said viral capsid protein components of said virus particles.
前記ウイルス粒子を含むサンプルを親水性相互作用液体クロマトグラフィー（ＨＩＬＩＣ）に供して、前記ウイルス粒子のカプシドのタンパク質成分を分離することと、
前記カプシドの前記タンパク質成分の質量を決定することと、
前記決定されたウイルス粒子のカプシドのタンパク質成分の質量を理論的質量と比較することであって、前記理論的質量からの、前記ウイルス粒子のカプシドのタンパク質成分の質量のうちの１つ以上のずれは、カプシド不均一性を示している、ことと
を含む、前記方法。 1. A method for determining the heterogeneity of protein components of the capsid of intact, non-enveloped viral particles, comprising:
subjecting the sample containing the viral particles to hydrophilic interaction liquid chromatography (HILIC) to separate the protein components of the viral particle capsid;
determining the mass of the protein component of the capsid ;
Comparing the determined mass of the protein component of the viral particle capsid to a theoretical mass, wherein one or more of the masses of the protein component of the viral particle capsid deviate from the theoretical mass. is indicative of capsid heterogeneity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022174334A JP7577719B2 (en) | 2018-10-25 | 2022-10-31 | Methods for analyzing viral capsid protein composition |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201862750583P | 2018-10-25 | 2018-10-25 | |
| US62/750,583 | 2018-10-25 | ||
| PCT/US2019/057936 WO2020086893A1 (en) | 2018-10-25 | 2019-10-24 | Methods for analysis of viral capsid protein composition |
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