Methods and systems for analysis of samples containing particles used for gene delivery

Abstract

Embodiments disclosed include systems, devices, and methods for analysis of samples containing particles used for gene delivery to determine a quality of the sample and/or an indication that the gene delivery particles are in a full, partial, and/or empty state. The present disclosure also relates to determining a protein and/or NA content in samples with known proportions of gene delivery particles in a full, partial, and/or empty state and based on the determination, establish a relationship between NA content and proportions of gene delivery particles in a full state. The present disclosure also relates to using such an established relationship to predict a proportion of the gene delivery particles in a full, partial, and/or empty state in test samples having the gene delivery particles in an unknown state.

What is claimed is: 
     
         1 . A method, comprising:
 introducing a sample containing a plurality of viral vectors in a conductive medium into a capillary, a first end of the capillary being ionically coupled to a first running buffer having a first pH, a second end of the capillary being ionically coupled to a second running buffer having a second pH such that a pH gradient is formed along the capillary;   separating the plurality of viral vectors in the sample by applying a voltage between the first running buffer and the second running buffer;   incubating, after separating, the plurality of viral vectors with a selective binder configured to bind to a portion of a nucleic acid (NA) included in a subset of viral vectors from the plurality of viral vectors; and   detecting a signal associated with the selective binder to determine at least one of (i) a quantity of the NA included in the subset of viral vectors from the plurality of viral vectors to which the selective binder is bound, or (ii) or an isoelectric point (pI) associated with the subset of viral vectors from the plurality of viral vectors that include the NA to which the selective binder is bound.   
     
     
         2 . The method of  claim 1 , wherein the subset of viral vectors from the plurality of viral vectors include the NA encapsulated in a carrier component made of an organic or an inorganic material. 
     
     
         3 . The method of  claim 1 , wherein the plurality of viral vectors includes capsid protein and the signal is a first signal, the method further comprising:
 incubating the plurality of viral vectors with an anti-viral capsid protein antibody configured to bind to at least a subset of the capsid protein included in the plurality of viral vectors; and   detecting a second signal associated with the anti-viral capsid antibody to determine at least one of a relative quantity or an isoelectric point (pI) associated with at least one of the capsid proteins to which the anti-viral capsid antibody is bound.   
     
     
         4 . The method of  claim 1 , wherein the signal includes at least one of a chemiluminescence signal or a fluorescence signal. 
     
     
         5 . The method of  claim 1 , wherein the detecting the signal is used to determine the relative quantity of DNA included in the plurality of viral vectors and to which the anti-DNA antibody is bound, the method further comprising:
 calculating an Area Under the Curve (AUC) statistic associated with the signal; and   determining at least one of (i) a proportion of viral vectors in the plurality of viral vectors that include a specified DNA content, or (ii) a proportion of viral vectors in the plurality of viral vectors that are devoid of the specified DNA content based on the AUC statistic.   
     
     
         6 . The method of  claim 5 , wherein the determining the at least one of (i) the proportion of viral vectors in the plurality of viral vectors that include the specified DNA content, or (ii) the proportion of viral vectors in the plurality of viral vectors that are devoid of the specified DNA content is further based on comparing the AUC statistic with a predetermined relationship between known AUC statistic measures and known percentages of viral vectors in standard samples of viral vectors that include the complete set of the specified DNA content, the each standard sample from the standard samples of viral vectors being associated with each known AUC statistic measure from the known AUC statistic measures. 
     
     
         7 . The method of  5 , wherein a viral vector from the plurality of viral vectors has the specified set of DNA content if the viral vector includes a polynucleotide having a specified nucleotide sequence that has a match to a target sequence above a predetermined threshold value. 
     
     
         8 . The method of  claim 1 , wherein the detecting the signal is used to determine the relative quantity of the subset of the DNA included in the plurality of viral vectors, the method further comprising:
 calculating a test AUC statistic associated with the signal; and   determining a proportion of viral vectors in the plurality of viral vectors that include more than a threshold percentage of an amount of DNA content based on a predetermined relationship between a percentage of viral vectors in a standard sample of viral vectors that include the amount of DNA content and an AUC statistic associated with the standard sample of viral vectors.   
     
     
         9 . The method of  claim 1 , further comprising:
 determining, based on the signal, a proportion of the plurality of viral vectors that includes at least one of a capsid or a gene-of-interest (GOD.   
     
     
         10 . The method of  claim 1 , further comprising:
 determining, based on the signal, a first proportion of the plurality of viral vectors from the plurality of viral vectors that includes a complete set of a specified DNA content, a second proportion of the viral vectors from the plurality of viral vectors that includes a partial set of the specified DNA content, and third proportion of the viral vectors from the plurality of viral vectors that lacks any of the specified DNA content.   
     
     
         11 . The method of  claim 1 , further comprising:
 immobilizing, after the separating, the plurality of viral vectors to a wall of the capillary.   
     
     
         12 . The method of  claim 1 , wherein the anti-DNA antibody is a primary antibody, further comprising:
 introducing, after the separating and the incubating with the primary antibody, a secondary antibody to the plurality of viral vectors, the secondary antibody configured to selectively bind to the primary antibody, the secondary antibody including a label configured to generate the signal.   
     
     
         13 . The method of  claim 1 , wherein the plurality of viral vectors includes vectors generated using at least one of lentivirus, retrovirus, adenovirus, or parvovirus. 
     
     
         14 . A method, comprising:
 introducing a plurality of samples into a plurality of capillaries, each sample from the plurality of samples including a different known proportion of Adeno-associated virus (AAV) vectors that include an amount of NA content;   separating each sample from the plurality of samples using isoelectric focusing;   labeling each sample from the plurality of samples in each capillary from the plurality of capillaries with a binder configured to bind to at least a portion of the specified DNA content;   detecting a signal associated with the binder from each sample from the plurality of samples; and   determining a relationship between the signal from each sample and the known proportion of AAV vectors that include the specified NA content in that sample.   
     
     
         15 . The method of  claim 14 , further comprising:
 determining a proportion of AAV vectors in a test sample that includes DNA content based on the relationship.   
     
     
         16 . The method of  claim 14 , wherein, the binder is a first antibody and the signal is a first signal, the method further comprising:
 labeling each sample from the plurality of samples in each capillary from the plurality of capillaries with a second antibody configured to bind to a protein of a capsid included in the AAV vectors; and   detecting a second signal associated with the second antibody from each sample from the plurality of samples, the relationship between the first signal from each sample and the known proportion of AAV vectors being determined based on the second signal obtained from each sample.   
     
     
         17 . The method of  claim 14 , further comprising:
 immobilizing, after the separating and before labeling, the viral vectors in each sample from the plurality of samples to a wall of the capillary associated with that sample.   
     
     
         18 . A non-transitory processor-readable medium storing code representing instructions to be executed by a processor, the instructions comprising code to cause the processor to:
 receive a test signal associated with a test sample containing a plurality of viral vectors, the test signal being associated with a DNA content included in at least a subset of viral vectors from the plurality of viral vectors;   extract a feature of the test signal that is configured to indicate a quantity of the DNA content of the subset of viral vectors from the plurality of viral vectors;   compare the feature against a function that describes a relationship between (i) known features obtained from a plurality of signals, each signal from the plurality of signals detected from a standard sample from a plurality of standard samples, each standard sample containing a known proportion of viral vectors that include a specified amount of DNA content, and (ii) the known proportions of viral vectors that include the specified amount of DNA content contained in each standard sample from the plurality of standard samples; and   determine, based on the comparison, a proportion of the plurality of viral vectors contained in the test sample that include a capsid and the specified DNA content.   
     
     
         19 . The non-transitory processor-readable medium of  claim 18 , wherein the feature is a first feature, and the code to cause the processor to extract the first feature includes code to cause the processor to extract a second feature of the test signal that is configured to indicate an isoelectric point (pI) associated with the DNA content included in the subset of viral vectors from the plurality of viral vectors. 
     
     
         20 . The non-transitory processor-readable medium of  claim 18 , the instructions further comprising code to cause the processor to:
 receive information associated with an identity of the plurality of viral vectors in the test sample; and   select the function based on the identity of the plurality of viral vectors.   
     
     
         21 . The non-transitory processor-readable medium of  claim 18 , wherein the identity of the plurality of viral vectors includes an indication of a type of virus used to generate the plurality of viral vectors. 
     
     
         22 . A method, comprising:
 introducing a sample containing a plurality of viral vectors in a conductive medium into a plurality of capillaries, a first end of each capillary from the plurality of capillaries being ionically coupled to a first running buffer, a second end of each capillary from the plurality of capillaries being ionically coupled to a second running buffer such that a pH gradient is formed along each capillary from the plurality of capillaries;   separating the plurality of viral vectors in the sample in each capillary by applying a voltage between the first running buffer and the second running buffer;   incubating the plurality of viral vectors in a first capillary from the plurality of capillaries with a first binder configured to selectively bind to a portion of a nucleic acid (NA) included in a first subset of the viral vectors from the plurality of viral vectors;   incubating the plurality of viral vectors in a second capillary from the plurality of capillaries with a second binder configured to selectively bind to a portion of a protein included in a second subset of viral vectors from the plurality of viral vectors;   detecting a first signal associated with the first binder to determine a quantity of the NA included in the first subset of viral vectors to which the first binder is bound in the first capillary; and   detecting a second signal associated with the second binder to determine a quantity of the protein included in the second subset of viral vectors to which the second binder is bound in the second capillary.   
     
     
         23 . The method of  claim 22 , wherein the sample introduced in the first capillary from the plurality of capillaries and the sample introduced in the second capillary from the plurality of capillaries are obtained from a common source. 
     
     
         24 . The method of  claim 22 , further comprising:
 determining a proportion of viral vectors of the plurality of viral vectors that include the portion of NA based on a comparison of the quantity of the NA included in the first subset of viral vectors and the quantity of the protein included in the second subset of viral vectors.   
     
     
         25 . The method of  claim 22 , further comprising:
 identifying, based on the first signal, a first isoelectric point (pI) associated with the first subset of viral vectors;   identifying, based on the second signal, a second pI associated with the second subset of viral vectors; and   
       determining a proportion of viral vectors of the plurality of viral vectors that include the portion of NA content based on a comparison of the first pI, the second pI, the quantity of the NA included in the first subset of viral vectors, and the quantity of the protein included in the second subset of viral vectors.