Systems, compositions, and methods for transplantation

Abstract

Systems and methods for purification and concentration of autologous alpha-2-macroglobulin (A2M) from whole blood are provided. Also provided are diagnostic methods for identifying sites in the synovial joints, spine, tendons or ligaments for treatment of pain, degeneration, or inflammation with autologous A2M. Methods for utilizing autologous A2M in combination with other autologous treatments (e.g. platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half life of the protein in joints and spine disc or epidural space.

1 .- 202 . (canceled) 
     
     
         203 . A system for enriching alpha-2-macroglobulin (A2M) from a biological sample from a mammalian subject comprising:
 (a) a centrifuge, a pump, or a combination thereof; and   (b) a flow filtration module comprising an inlet, an outlet, and one or more filters, wherein the one or more filters are fluidly connected between the inlet and outlet, wherein a filter of the one or more filters has a pore size with a molecular weight cut-off of at most 500 kDa; wherein a flow of the biological sample from the inlet to the outlet and through the one or more filters produces an A2M enriched composition, wherein the A2M enriched composition comprises an A2M polypeptide at a concentration that is at least 1.1 times higher than the concentration of A2M polypeptide present in the biological sample.   
     
     
         204 . The system of  claim 203 , wherein the system comprises a pump, wherein the pump is in fluid connection with the flow filtration module, wherein the pump is coupled to the flow filtration module upstream of the inlet or downstream of the outlet, wherein activation of the pump produces a flow of the biological sample from the inlet to the outlet and through the one or more filters to produce the A2M enriched composition. 
     
     
         205 . The system of  claim 204 , wherein the one or more filters comprises two or more filters fluidly connected in series between the inlet and the outlet. 
     
     
         206 . The system of  claim 205 , wherein a filter of the two or more filters has a pore size of at most 1 micron, wherein the filter that has a pore size of at most 1 micron is upstream of the filter that has a pore size with a molecular weight cut-off of at most 500 kDa. 
     
     
         207 . The system of  claim 206 , wherein a retentate of the filter that has a pore size with a molecular weight cut-off of at most 500 kDa comprises the A2M enriched composition. 
     
     
         208 . The system of claim Error! Reference source not found., wherein a permeate from the filter that has a pore size of at most 1 micron flows through the filter that has a pore size with a molecular weight cut-off of at most 500 kDa. 
     
     
         209 . The system of  claim 203 , wherein the system comprises a centrifuge, wherein a flow of a supernatant of the biological sample from the mammalian subject obtained by centrifuging the biological sample from the mammalian subject with the centrifuge passes through the one or more filters to produce the A2M enriched composition. 
     
     
         210 . The system of  claim 209 , wherein the A2M enriched composition is a retentate of the one or more filters. 
     
     
         211 . The system of  claim 203 , wherein the flow filtration module is a dead end or tangential flow filtration module. 
     
     
         212 . The system of  claim 203 , wherein the one or more filters comprises a hollow fiber tangential flow filter. 
     
     
         213 . The system of  claim 203 , wherein the mammalian subject is a human. 
     
     
         214 . The system of  claim 203 , wherein the biological sample from the mammalian subject is plasma, blood or bone marrow aspirate. 
     
     
         215 . The system of  claim 203 , wherein the A2M enriched composition substantially lacks cells, particles, and other molecules larger than 1 μm. 
     
     
         216 . The system of  claim 203 , wherein the A2M enriched composition comprises less amount of a non-A2M protein with a molecular weight of less than 500 kDa than in the biological sample from the mammalian subject, wherein the non-A2M protein with a molecular weight of less than 500 kDa is fibrinogen. 
     
     
         217 . The system of  claim 203 , wherein the A2M enriched composition comprises a non-A2M protein with a molecular weight of less than 100 kDa, wherein the concentration of the non-A2M protein with a molecular weight of less than 100 kDa is less than the concentration of the non-A2M protein with a molecular weight of less than 100 kDa in the biological sample, wherein the non-A2M protein with a molecular weight of less than 100 kDa is C X C motif chemokine receptor 2 (CXCR2) or ATP Binding Cassette Subfamily F Member 1 (ABCF1). 
     
     
         218 . The system of  claim 203 , wherein the A2M enriched composition is not coagulated, is substantially free of white blood cells and is substantially non-immunogenic to the mammalian subject. 
     
     
         219 . The system of  claim 203 , wherein the A2M enriched composition comprises an A2M polypeptide at a concentration that is at least 3.5 times higher than the concentration of A2M polypeptide present in the biological sample from the mammalian subject. 
     
     
         220 . The system of  claim 203 , wherein centrifugal force from the centrifuge, activation of the pump, or a combination thereof flows the biological sample from the mammalian subject from the inlet to the outlet and through the one or more filters. 
     
     
         221 . The system of  claim 203 , further comprising a sample loading module operable to introduce the biological sample from the mammalian subject into the system. 
     
     
         222 . A method for enriching alpha-2-macroglobulin (A2M) from a biological sample from a mammalian subject, the method comprising flowing the biological sample from the mammalian subject through the system of  claim 203 .