Particular Type of Chromatography

Anion exchange:

–for adenovirus purification: it is usually preferred to use at least one AEX step. (Weggeman (US2009/0123989). 

Hydroxyapatite chromatograpy: has been studied for viral clearance for about 45 years. The mechanism of binding can be due to either synergistic effects of multi-site binding, strong interactions of clustered surface phosphates or both. The original eluant used almost exclusively was phosphate but sodium chloride has also been sued. A variety of other additives such as polyethylene glycol (PEG) has been employed as elution modifiers. PEG has been shown to improve aggregate separation by differentially enhancing retention of larger solutes. The tight binding of viruses to CHT can be explained at least in part by the multi-modal nature of hydroxyapatite interactions. For enveloped viruses, the significant number of phosphate groups on the lipd envelope interact strongly with the calcium atoms via a chelation mechaism as has been previously shown for both proteins and nucleic acids. In general, the much larger surface area provided by viruses would be expected to result in ighter binding as compared to much smaller bio molecules such as antibodies (US 13/178970). 

Particular Types of Viruses 

Adeno-associated virus (AAV):

AAV has become a vector of choice becasue of its safety profile. It can not replicate on its own and is not integrated directly into the host genome. The process begins by inserting a desired genetic payload sequence into a virus and producing vectors using transfected host cells. Host cells are lysed, viral particles extracted and purified to eliminate host cell contaminant and nimimiz AAV virons that are missing the therapeutic sequence. AAV manufacturing using human embryonic kidney cells in either adherent or suspension mode includes several tyipcal processing steps: cell expansion, plasmid transfection, viral vector production, cell lysis, puficaiton and fill and finish. The purificaiton process usually invlves clarifciaiton, capture through affintiy chromatography, polishing with AEX, TFF concentration/diafilgration and final filtration. Traditional chromatographic approaces used for separation of empty and full capsids are based on monoliths or columns packed with resin. A promising alternative is the use of membrane adsorbers. Used with small conductivity step changes (rather than linear gradients), Pall Mustang Q membranes can yield distinct elution peaks for both DNA-free and DNA-containing capsides. (Cameau “Overcoming obstacles in AAV viral vector manufacturing” BioProcess International, June 2021, 19(6); pp. 68-69)

AAV is a helper-dependent DNA paravovirus that belongs to the genus Dependovirus. AAV requires co-infection with an unrelated helper virus such as adenovirus, herpes virus or vaccinia in order for a productive infection to occur. In the basence of a helper virus, AAV establishes a latent state by inserting its genome into a host cell chromosome. Subsequent infetion by a helper virus rescues the integrated viral genome, which can then replicate to product infectious viral progeny. AAV has a wide host range and is able to replicate in cells from any species in the presence of the suitable helpter virus. AAAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. Wright et al. (US 2013/0072548). 

An important objective in the design of rAAV production and purificaiton systems is to implement stategies to minimize/control the generation of production related impurities such as proteins, nucleic acids, and vector related impurities, including wild-type/pseudo WT AAV species and AAV encapsidated residual DNA impurities. Removal of impurities in AAV vectors is complicated due to the wayrAAV vectors are produced. In one production process, rAAV vectors are produced by a transient transfection proces using three plasmids. Significant amounts of plasmid DNA are introduced into the ells to produce rAAV vectors. In addition, when rAAV vectors are released from the producing ells, cellular proteins and nucleic acids are co-released. Considering that the rAAV vector represents only aobut 1% of the biomass, it is very challenging to purify rAAV vectors to a level of purity which can be used as a clinical human gene therapy product. Wright et al. (US 2013/0072548).

–AEX-CEX or CEX-AEX:

Adenovirus is usually preferred to use at least one AEX step. (Weggeman (US2009/0123989).

Qu (US 16/088743, published as US 2020/0299650) discloses a method for purifying an AAV vector that includes harvesting cells which include the AAV vector, lysing the cells, treating the lysate with a nuclease to reduce nucleic acid, filtering the nucleic acid reduced lystate and subsecting the lysate to AEX followed by CEX or vice versa, followed by filtration.

Wright et al. (US 2013/0072548) discloses a method for puriyfing an AAV vector that includes a transgene encoding a therpaeutic protein which includes harvesting cells transduced with AAV, concentration the cells via TF, lysing the cells by micofluidization to form a lysate, clarificaiton of the AAV particles and then ion exchange column chromatography which can include various combinations of AEX and CEX.

—AEX/CEX – SEC –CEX/AEX:

 Wright (US Patent Application No: 16/088,743, published as US 2020/0299650) discloses that including a size exclusion column chromatography (SEC)in betweenthe CEX/AEX steps is particularly effective in removing impurities. 

–Apatite Chromatography:

Thorne (US 2015/0024467) disclsoes a process for isolating rAAV by capturing the rAAV aprticles on an aptite chromatogrpahy medium in the presence of polyethylene glycol (PEG). The method also includes upstream processing steps such as for example centrifugation, AEX and doenstream processing steps such as HIC, size exclusion chromatography and/or AEX. 

–Hydrophobic interaction chromatogrpahy (HIC): Thorne (US 2015/0024467) discloses a method for isoalting rAAV particles which includes contacting a feedstream with rAAV particles with a HIC medium in a high salt buffer, wherein the rAAV particles bind the HIC and eluting the rAAV partciels with a medium salt buffer. 

Hepatitis B surface antigen: Infection with HBV leads to the production of large virus particles. Vaccination of several million individuals world wide with HBsAg purified form the plasma of asymptomatic human carriers was used for almost two decades as an effective means of prevenitng HB associated health problems such as liver failure. However, the manufacturing process was tedious and time consuming requiring procuedures to inactivate infectious HBV that might be present in plasma. By the early 1980s advances in genetic engineering and biotechnology allowed the first HBV to be obtained by formulation of HBsAg produced in recombinant straints of yeast Sacharamyces cerevisiae followed by cell disruption and purified by several extraction and chromatographic steps. Following disuprtion of the cell, cell debris was diluted with potassium thiocyanate containing disruption buffer and the pH adjusted from 8 to an optimun vlaue of 4.5 so as to precipitate non-protein contaminants. Acid precipitation was followed by centrifugation to remove the precipitated proteins, carbohydrates and lipids. The the clarified HBsAg prepration is subjected to bach adsorption at acid pH onto diatomaceous earth matrix followed by elution form the matrix at low ionic strnght and basic pH (Hardy Journal of Biotechnology 77, 2000, 157-167). 

The HBV has a spherical shape with a lipoprotein coating mostly of HBV surface antigen (HBsAg). Recombinant HBV vaccines have been created based on teh HBsAg syntehsized in yeast foor ammalian cells. Transformed cells are cultures in indstrial scale fermentors, the antigen collected, purified and formulated into spherical particles that epxose the highly immunogenic “a” antigenic determinant region. The main purificaion step is immunoaffinity chromatography (IAC). (BioProcess International, March 2023, volue 21, number 3, Quintana “Purificaiton of hepatitis B virus surface antigen for vaccine products”. 

Filtration

Weggeman (US2009/0123989) discloses a method for the purifiction of a virus comprising a step of ultrafiltration wherein the retentate contains the virus, wherein back pressure of at least 5 kPa is applied on the permeate side. This results in an improvement over processes wherein no back pressure is applied on the permeate side. 

Filtration in combination with CEX: Mheta (US2011/0034674) discloses method of subjecting a protein/antibody mixture to CEX and an endotoxin removal step prior to passing through a virus filter. The method improves the capacity of the virus filter during protein purification.