To constrain costs, strategies have been developed to produce recombinant polyclonal antibodies in a single pot. This process by passes the manufacturing hurdles assocaited with individual antibody production but introduces new challenges to demonstrate reproducbiel cell grwoth, genomic stability, constant antibody ratios and integrity of the component antibodies. Merus solved this by generating Oligoclonics ™ a single PER.C6 stable cell line expressing one light common chain and three heavy cahins, producing consistent antibody mixture with three unique bidning sites at 5020 pg/cell/day for up to 67 cell divisions. The process works since in many cases, light chains are promiscuous, forming functional binding sites with a variety of heavy cahins. In this case, ligand specific heavy chains were selected form a human Fab phage library, containing a single rearranged light chain. (Maynard, Current Opinion in Chemical Engineering, 2013, 2, 405-415).

In another approach, Sympress II, randomly integrates antibody genes into the DHFR CHO DG44 cell line. After gene amplication with mehotrexate, selected high expressing clones were expanded and combined to form a polyclonal master cell bank. This process ahs been evaluated with two groups of six antibodies each, recognizing vaccinia and respiratory syncytial virus. (Maynard, Current Opinion in Chemical Engineering, 2013, 2, 405-415).

Differential scanning calorimetry (DSC) can be used to examine the stability of the tertiary structure of antibody preparations. (Brass, Pharm Acta Helv. 1996, 71(6): 395-403) 

Immunoassay (ELISA): can be used to determine the binding activites of antibody preparations. 

Biospecific Interaction Analysis:

Biosensor Technology:

–BIAcore biosensor-based analytical system: (BIAcore, Pharmacia Biosensor AB, uppsala, Sweden) is designed for functional chacterization of protein-protein, protein NA and ligand-receptor interaction in real time. The adsorption of biomolecuesl to an immobilized ligand on a sensor chip is measured in the same time and place as it occurs. BIAcore is baswed on a bisensor that uses surface plasmon resonance (SPR) to monitor the adsorption of biomolecuels on a sensor chip. This optical techniques measures changes in refractive index in the vicinity of the surface. Such changes are directly proportional to the change in adsorbed mass,. The system includes a sensor chip to which the lgiand can be immobilized in a 100 nm thick hyrophilic matrix composed of 2-3 per cent flexible desxtran, a miniaturized fluidics cartridge for the transport of analytes and reagents to the sensor chip, a SPR detector, an auto inejector and sotware for the system control and evaluation of resutls. Specific ligands are iimmobilized to the sensor chip at low concetnraiton through biotin-avidin interaction or covalently thorugh amine, thiol or aldehyde chemistry. The stable binidng allows regeneration of the sensor surface of 50-100 analytical cycles. The analysis can also be performed in tissue culture media or bacterial browth without the need for purificaiton in contrast to other techniques used for kinetics analysis. The technology was originally developed using mAb-antigen interactions but is not rapidly being transferred to ther areas of biological significance. There are many applications such as epitope mapping. For example eptiope mapping of HIV-1 core protein p24 by pairwise mapping of 30 different mAbs using the BIAcore system has been performed. Both identifciation of peptides inhibiting the mAb binding and mltiple binding of several mAbs in sequence forming a hexamolecular complex on the surface were shown. (Malmqvist “Biospecific interaction analysis using biosensor technology”)

Analysis of DNA:

DNA quantification: (Brass, Pharm Acta Helv. 1996, 71(6): 395-403)

Total DNA as a contaminant in biopharmaceutical products if of concern becasue of its potential health ris. In the past, the analytical method to quantitate DNA contamination was DNA probe hydridization whcih can detect less than 10 pg of specific DNA. Merrick (“A complete system for quantitative analysis of total DNA, prtoein impurities and relevant proteins” BFE, vol 9, no.6, 1992) discloses a threshold system which includes an instrument, computer and softwarefor quantitative asss for picogram amounts of total DNA. After denaturation of DNA in a sample, a single labeling reagent is added, containing streptaidin plus two non sequence specific DNA binding proteins with high specificity fo single stranded NA. E. coli single stranded binding protein conjugated to biotein and a monoclonal anti-DNA antibody conjugated direclty to urease. After formation of reaciton complexes in liquid phase and active cpature on the giotinylated nitrocellulose membranes, urease actiity is measured in the sensor. 

–Real Time PCR (CHO DNA): can be quantified using a real time PCR. First DNA is extracted using Qiagen’s Virus biorobot kit and then samples/controls subjected to TaqMan real time PCR using a 110 base pair segment of repetitive DNA sequence in the Cricetulus griseus genome. (Nadarajah, US 14/355818)

Ion Exchange

Cation Exchange:

–% iimpurities:

Brige (US 9,884,117) discloses a purity assay of an immunogloublin single domain using a Dionex ProPac WCX-10 weak cation exchagne columne and mobile phase consisting of citrate buffer. After loading the protein(s) on the olumn, bound materials were eluted by a sodium chloride graient. The relative amount of the specific protein variant or impurities, expressed as area %, was calculated by dividing the peak area corresponding to the specific protein or to any protein impurity by the total area of all integrated peaks. 

SDS-PAGE: 

SDS-PAGE performed under non-reducing conditions is one of the most commonly used techniques to access mAb product purity. During analysis, minor bands corresponding to LMW species can be routinely observed and quantified include H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains), HL (1 heavy chain and 1 light chain), HC (1 heavy chain) and LC (1 light chain) species, with respect to antibodies (Wang US 2019/152303)

During analysis, minor bands corresponding to LMW speceis can be routinely observed and quantified including H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains), HL (1 heay chain and 1 light chain), HC (1 heavy chain ) and LC (1 light chain species, with respect to antibodies). (Wang, US 16/223,463, published as US 2019/0194298)

For Activatable Antibodies:

Chen (“Selective antibody activation through protease-activated pro-antibodies that mask binding sites with inhibitory domains” Scientific Report (2017) discloses development of protease-activated pro-antibodies by masking the binding sites of antibodies with inhibitory domains that can be removed by proteases. The latenecy-assocatied peptide (LAP) was linked through a substrate peptide of matrix metalloproteinase 2 (MMP-2) to an anti-epidermal growth factor receptor (EGFR) antibody and anti-tumor necrosis factor alpha antibody. Results of reducing SDS-PAGE showed that all of the inhibitory domains could be removed by MMP-2 to restore the binding activities of the antiobides. 

Moore (US Patent No: 2015/0087810) discloses multispecific activatable antibodies that engage immune effector cells, also referred to as immune effector cell engaging multispecific activatable antibodies. In some embodimetns, the antibodies are designed to engage leukocytes. Moore demonstrate protease activation of the multispecific activatable antibodies with SDS-PAGE. Briefly, the samples were denatured at 70C, six ug of antibody was loaded onto a NuPAGE 10% Bis-Tris gel (Invitrogen) and proteins were separated by sie using the MOPS electrophoresis buffer. Folloiwing electrophorosis the gel was stained with Coomassie blue. The change in mobility of anti-EGFR activatable antibody and ant-EGFR multispecific activatable antibody demonstrated proteolytic activation of the multispecific activatalbe antiobdies. the lack of any change in the moility of the H chain fusions demonstrates the resistance to protease cleavage. 

Size exclusion chromatography (SEC): 

High performance size exclusion chromatography such as the TSK G3000SWXl SEC column can be used to determined relative levels of mAb monomer (Nadarajah, US 14/355818)

Boschetti (WO/2003/042233) discloses using SELDI to identify and/or quantify non-degraded target polypeptides and distinguish impurities during the expression of target polypeptides.

O’Connor (US 2016/0251441) discloses injecting a gest sample onto a TosoHaas G300SWXL column and eluting isocratically with 0.1 M disodium phosphate containing 0.1 M sodium sulfate and 0.05% sodium azide, pH 6.8 at a flow rate of 1.0 ml/minute. The eluted prtoein was detected using UV absorbance at 280 nm. Peaks eluting elarier than the nomer peak were recorded as percent aggreate and peaks eluting after the monomer peak were recorded as percent other. 

SEC-MS:

Haberger (MABS, 2016, 8(2) 331-339) discloses detetermining HMW aggregates such as dimers as well as fragment variants in a CrossMap preparation using ultra-pressue liquid chromatography size exclusion separation combined with native electrospray ionization mass spectrometry. 

Dixit “LC-MS characterization and purity assessment of a prototype bispecific antibody” mAbs 5-6, 711-722 (2013) discloses a liquid chromatography mass spectometry 9LC-MS) based method for evaluating heterodimeric purity of a prototype antibody containing two different heavy chains and two identical light chains. The LC-MS based assay used an advanced ESI-Q-TOF mass spectromer that evalutes heterodimeric purity of MAb1, a prototype ehterodimeric antibody after deglycosylation by detecting and quantifying homodimeric and half-antibody (H+L chain) impurities. MAb1 was based on the IgG1 trastuzumab and is an Fc heterodimer composed of two different half antibodies sharing a common light chain. H chains have different amino acids at 6 sites in the Fc region (resulting in a 172 Da mass difference). MAb1 and the independently expressed homodimeric standards (contianing only the A or B heavy chains plus a common light chain were first characterized by two complentary LC-MS techniques, intact protein mass analysis after deglycosylation and LC-MS peptide mapping of Lys-C digests prepared form reduced and alkylated samples. (1) First, the homodimeric standards were purified by siz-exclusion chromatogrpahy (SEC) HPLC to separate out half antibodies and enrich full lenght homodimeric antibody in the sample. (2) Deglycosylation was performed prior to intact protein mass analysis to reduce sample heterogeneity and simplify detection and quantificiton of heterodimer and homdimers. Although half-antibodies can be detected and quantified by purely seperation based methods such as SDS-PAGE, evaluation of half anitobdies in conjunction with homodimer evaluation will save time and resources. 

Mass Spectrometry (MS) (see also MS in Diagnostic Techniques)

Light Scattering: (See outline)

RP-HPLC: 

O’Connor (US 2016/0251441) discloses intecting a sample onto a PLRP-S column. Mobile phase A was 0.05% TFA in water and mobile phase B was 0.05% TFA in acetonitrile. The protein was eluted using a gradient of 34-40% TFA in acetonitril (mobile phase b) form 5-12 minutes and was monitored using UV absorbance at 280 nm. The reproted fragment percent was the total area of all the fragments divdied by the total peak area of all the fragments and the intact antibdoy. 

Septic Systems

Many rural and suburban areas rely on septic tanks which are containers into which sewage flows. The solid material settles and is subject to microbial decomposition. The liquid is allowed to overflow and is distributed through a series of perforated piedes into the surrounding soil. As long as hoses are far enough arpat, the reduction of organic material is sufficient to reduce the concentrations of organic matter being released to levels that can be aacomodated without causing environmental harm.

Sewage Treatment Plants

Sewage that is piped to wastewater treatment plants is subjected to physical, microbial and soemtimes chemical processes. The aim is to reduce organic matter content and to lessen the biological oxygen demand. Most major cities have extensive sewage treatment plants. The border cities of Tijuana in Mexico and San Diego have developed an international agreement for jointly treating their wastewater. Sweage form Tijuana is piped across the border to a treatment plant on the US side of the border.

Primary phase:

This pahase involves physical separation of solid materials, largely through settling. The sludge can be treated in anaerobic digestors. Here, anaerobic bacteria and archaea further decompose the solid wastes, producing a stable solid material and methane that can be collected and used as a fuel.

Secondary phase:

In this phase, the liquid portion of the waste is subjected to microbial decomposition that is largely aerobic. Air may be be forced into tanks and the wastewater rigorously mixed to ensure aerobic conditions. An active microbial community devleops that degrades the organic compounds int he wastewater. A sludge forms that is allowed to settle.

Most of the sludge is removed for further treatment, but a portion containing high numbers of microbes is reintrudocued into the aerated treatment tank along with the next batch of wastewater to be treated. This ocmponent is called activated sludge because the microbes are already adapted to degrading the organic compounds in the wastewater.  The activated sludge process geenrally reduces the biological oxygen demand by 85-90%. It also greatly reduces the number of pathogens, which are largely out-competed by the nonpathogens and tend to settle in the sludge. The concentrations of Salmonella, Shigella, enteroviruses and other pathgogens are generally 9-99% lower in the effluent water that is discharged from the plant.

Tertiary phase (optional);

Sometimes prior to release, the water from the secondary treatment tanks is subjected to tertiary treatment. In this phase additional inorganic substances like ammonia, nitrate, phosphate and elimination of pathogens is done by chemical, physical or biological processes. Tertiary treatment removes nutrients that could support algal blooms and is important if the water is going to be released into a prstine laike. The teriary phage may also involve disinfection of the wasterwater as by filtration or chlorine treatment to eliminate pathogens.

Particular Techniques to Decontaminate Water

Oxidation: is one of the useful methods of waste water treatment, In this hydroxyl free radial (OH) is generated, which is highly reactive, non-selective oxidate which can destroy even the recalcitrant pollutants. Hydroxyl free radial generation is highly accelerated by combining ozone (O3), Hydrogen peroxide (H2O2), Titanium dioxide (TiO2), heterogeneous photo-catalysis, UV radiation or high electron beam radiation. (Joshi “Advances in Wastewater Treatment -A review” 2012.

Solar Water Disinfection: can be done by simply placing contaminated water in a transparent plastic bottles and leaving them in the sun for 6 hours. Ultraveiolet light kills bacteria and prarasites and inactivates viruses. It has been used primarily in impoversihed nations.

Red Tides & Alga Blooms:

The poisonous and destructive red times that occur frequently in coastal areas are often assocaited with dinoflgellates, whose pigments color the water. The blooms are most often triggered by excess nutrients form agricultural or other human activity. Red tides have a profound, determental effect on the fishing industry. Some 20 species of dinoflagellates produce powerful neurotoxins that inhibit the diaphragm, causing respiratory failure in many vertegrates. When the toxic dinoflagellates are abundant, many fishes, birds adn marine mammals may die. Humans consuming affected fish or shellfish will also be intesting neurotoxins that accumulate in the tissues of these animals.

Most dinoflogellates are photosynthetic unicells with two flagella. Dinoflagellates live in both marine and freshwater environments. Some dinoflagellates are luminous and contribute to the twikling or flashing effects one sees int eh sea at night, especially in the tropics, while other species contribute to the harmful algal blooms which can also lead to human disease. Plates made of a cellulose like material, often encrusted with silica, encase the dinoflagellate cells.

Companies: Abreos Biosciences

Links of interest: Mimo DB

Definitions

Anti-Idiotype antibody: binds to the idiotype of another antibody, usually an antibody drug. 

Idiotype: is the sepcific combination of idiotopes present within an an antibodies CDRs. A single idiotope, is a specific region within an antibodies Fv region which binds to the paratope (antigenic epitope binding site) of a different antibody. Thus, an idiotope can be considered almost synonymous with an antigenic determinant of an antibody. 

Mimotopes: are peptides with affinities to given targets. they are reaily obtained through biopanning against combinatorial peptide libraries constructed by phage display and other display technologies such as mRNA display, ribosome display, bacterial display and yeast display. Mimotopes have been used to infer the protein interation sites and for developing new diagnostics, therapeutics and vaccines. (Humang “MimoDB 2.0: a mimotope database and beyond” Nucleic Acids Research, 2012, 40, D271-D277 (November 2011). 

A mimetope is a determinant which is recognized by the same binding molecule, such as an antibody, as a particular “eptiope” but which has a different composition form the “epitope”. For example, a binding molecule can be an antibody which recognizes (i.e., bind to) and eptiope comprising a linear sequence of amino acids. A “mimetope” of this epitope comprises a different linear sequen of amino acids but which is still recognzied by the same antibody. ( Messmer, US Patent Application No: 15/944,099, published as US 2018/0291059)

Phage Display of Peptides

Phage display of foreign peptides is an established technique routinely used. Phage peptide libaries can be constructed by fusing DNA containing a degenerate region to a gene encoding a coat protein usually gene III or gene VIII. This allows the peptide to be expressed as an N or C terminal fusion on the surface of the M13 bacteriophage (page) coat protein. A large number of random peptide libraries displayed on bacteriophage are now available, some are disulfide constrained by inerting two cystein residues. A typical library size ranges from 6-45 amino acid residues. Selection of peptides of interest from the library that bind to a target molecule can then be performed by “panning”. (Casey, “Phage display of peptides in ligand selection for use in affinity chromatography”, Methods in Molecular Biology, 421, Affinity Chromatography, Methods and Protocls, Second Edition. )

Identification of peptide mimotope ligands for antibodies:

Messmer (“Identification of peptide mimotope ligands for natalizumab” Scientific Reprots, 8, 14473 (2018) discloses the use of phage display libaries to identify peptide ligands they call Veritopes™ that bind natalizumab, a therapeutic mAb indicated for use in multiple sclerosis. PKNPSKF was identified as a novel natalizumab-binding motif and peptides containing this motif demonstrated utility as capture reagents in ELISAS. A peptide containing the motif was also shown to be competitive with the natural ligand (alpha4-integrin) and a neutralizing anti-idiotype antibody for natalizumab binding. 

Peptides and Mimotopes for Affinity chromatoraphy

Peptide ligand affinity chromatography:

The advantages of peptide ligands for use in affinity chromatography include the relative low cost of high quality stables peptides and instead of having to preapre an affinity column using the whole recombinant antien, peptides that represent, for example, the antibody-binding site can be used. (Casey, “Phage display of peptides in ligand selection for use in affinity chromatography”, Methods in Molecular Biology, 421, Affinity Chromatography, Methods and Protocls, Second Edition. )

Messmer (US Patent Application No: 15/944,099, published as US 2018/0291059) discloses slecting mimetope peptides from phage display libraries , some of which contain cysteines flanking the peptide mimetope sequence to increase stability of the peptide through disulfide bonding formation. After three rounds of election with multiple phage display libaries, indiviudal phage plaques are isolated and sequenced. All clones are individually amplified, purified and their ability to specifically bind natalizumab-coated wells were assesed. The phage clone demonstrating specific, but low affinity binding to natalizumab is chemically synthesized with an N-temrinal acetyl modificaiton and a disulfide bridge between cysteins 2 and 1-0 or cysteins 8 and 16. The mimeotpe can be immobilzied onto beads and used for purificaiton of natalizumab. 

Yang (J. Chromatography A, 1216 (2009) 910-918) disclose the use of hexamer peptides (HWRGWV, HYFKFD and HFRRHL) to selectively absorbe and isoalte human IgG.

Peptide mimotopes: 

Casey, (“Phage display of peptides in ligand selection for use in affinity chromatography”, Methods in Molecular Biology, 421, Affinity Chromatography, Methods and Protocls, Second Edition. ) discloses methods to isolate a peptide mimotope from a pahge displayed random peptide library by isolation of a peptide that can mimic the sahpe of the antigen epitope and can be used to select antibodies that bind to this particular egion of the antigen. Casey also describes a process of purifying antibodies from human serum that bind to this peptide mimic.

Jensen (J of Immunological Methods, 284 (2004) 45-54) describes efficient purificaiton of unique antibodies using peptide affinity-matrix columns. Phage display was used to identify peptide ligands with unique specificity for mAbs. One peptide was linked to beaded agarose and demonstrated excellent performance as a peptide affintiy chromatogrpahy amtrix. 

Sahin (WO 2017/008844) discloses peptide mimotopes of the CD3 T cell co-receptor epsilon chain and uses of the mimotope for anti-CD3alpha antibody purificaiton. 

Simith (J of Chromatography B, 766 (2001) 13-26) describe  a polyvalent, lytic pahge display system displaying a random peptide library to discover novel mimotopes reactive with a therapeutic mAb. The novel synethic peptide was linked to beaded agarose and the performance as a mimotope affinity chromatogrpahy matrix was evaluated.

Complications

Although pahge display libraries provide a huge resource for eptiope analysis and identificaiton of mimotopes, using the sequences derived directly as a useful affinity ligands for antibody purificaiton purposes is not trivial. This seems to be due, at least in part, to the fact that the phage derived sequences have relatively low affinity and the reaction kinetics are such that binding of the antibody to the ligand cannot occur during the short time that the antibody is in contact with the matrix. (Murray, Analytical Biochemistry 296, 9-17 (2001). 

Coupling to the resin

Mimope peptide and carrier protein conjugates have been synthesized as coating antigens in immunoassay via the covalent coupling of amino or carboxyl groups of the peptide and the carrierty protein by using an active ester or by applying gltaraldehyde methods. However, these chemical coupling methods likely block the active side of mimotpe peptides, resulting in the loss or alteration of mimicking activities. (Zu, Talanta 146 (2016) 394-400). 

Abtides

Abtides refers to peptides that mimic the binding specificity of a larger molecule such as an antibody or receptor. 

Alvarex (US 5,885,577) discloses a process of identifying abtides by screening peptide libraries in a two-step prcoess. In the first screening step an antibody or receptor was used as the first target ligand. This step identifies peptide sequences termed “epitopes” or “mimetopes” which specifically bind the target ligand. An eptiope or mimetope is then used as a second target ligand in a second screening step to idenity a peptide sequence that are known as “abtides”. 

Companies:  Haemonetics

Apheresis (cytapheresis and Plasmapheresis):

Conventional apheresis methods include centrifugation, which can separate any of the components of the blood, and membrane filtration, which can be used for plasmapheresis only. (David M. Ward “Conventional Apheresis Therapees: A REview” J. Clinical Apheresis 26: 230-238 (2011). 

Cytapheresis: incldues teh removal of excessive white blood cells (leukocytopheresis) or platelets (thrombocytapheresis) or the exchange of diseased red blood cells (erythrocytopheresis). 

Plasmapheresis is the separation of whole blood into a plasma componet and a non-plasma component where the plasma component is retained and the non-plasma component is returend to the dornor. A variety of satellit pouch plasmapheresis systems have been patented where whole blood is withdrawn form a donor and flows to a pouch containing anticoagulant. The pourch is then disconnected from the donor phelbotomy line, centrifuged, and the sueprnatant plasma fraction is expressed into a connected plasma pouch. The pouch containing teh non-plasma componetn is then reconnected to the phelbotomy system to be returend to the conor.

Therapeutic plasmapheresis and therapeutic plasma exchange (TPE) are terms that are often used synonymously. The plasma that is removed can be replaced by fresh frozen plasma (FFP), 5% albumin or similar colloidal solution, or the patient’s own plasma after a secondary online purificaotn prcoecdure. Most commonly the objective of plasmapehresis therapy (TPE) is to remove antibodies implicated in the pathogenesis of autoimmune disease. Other important targets include circulating antigen-antibody complexes that cause vasculitis in conditions such as hepatitis C, alloantibodies in transplant rejections and transfusion situations, parapropteins that casue hypervisosity or neurologic and renal damage, pooly characterized pathogenic molcules such as in focal segmental glomerulosclerosis (FSGS), low MW lipoporteins that casue premature atherogenesis in homozygous hypercholesterolemia, and other endogensous and exogenous toxins.  (David M. Ward “Conventional Apheresis Therapees: A REview” J. Clinical Apheresis 26: 230-238 (2011). 

For example, treating rheumatoid arthritis patients by apheresis utilizes an immunosorbent device to remove immune complexes. For example, the Prosorba Column is a single use device that ctonains Protein A covalenty boudn to inert silica granules. When plasma is passed thro the device the immobilized Protein A binds outthe circualting cimmune complexes. The cleaned plasma is then returend to the pateint. Smith (WO 2006/076480)

 Aparatuses/Devices:

Fell (US 5,387,187) disclsoes an aperhesis apparatus for separating blood. A phelbotomy needle draws anticoagulated whole blood from a donor into a seapration chamber that seaprates plasma from higher density blood components. The plasma is displaced to a plasma collection bag. The seapration process is terminated and the higher density blood components remaining in the separation chamber are diluted with saline solution and are returend to the donor via the phelobotomy needle. 

Lathan (US 4,086,924) described an apparatus where whole blood is withdrawn from a donor using a phlebotomy needle and pressure cuff, measns for supplying anticoagulant to the withdrawn blood and the anticoagulated withdawn whole blood is then transported by a blood pump to means for seaprating it into a plasma and a non-;lasma component such as a plasmapheresis centrifuge. 

Extracorporeal Immunoadsorption:

–Specific ligands (e.g., antibodies, Protein A) bound to Support:

In immunoadsorption (IA) a biospecific column can be used for an extracorporeal blood treatment. The IA column contains a material consisting of a matrix having at least one ligand covalently bound thereto. An example of an IA column is a protein A containing column. Otehr examples are immunoglobulin based columns for specific elimination of immunoglobulins from blood plasma. In IA a plasma filter or a contrifuge is used, which continously spearates the plasma from teh blood cells. Then the blood plasma is transported via a tube to a column, in which the target protein or componetn is bound and thereby is specifically separated form other blood plasma components. Nilsson (WO 2013/062479)

Extra-corporeal immunoadsorption (ECI) consists of a highly purified protein A that is bonded to a silica matrix. (see for example Prosorba column). Plasma is collected form the patient in a pheresis procedure and then passed over the column. circulating immune complexes and IgG bind to the protein A and thus selectively removed from plasma. The plasma can then be returend to the pateint, thus eliminating the need for a plasma exchagne. It is useful for treating Idiopathic thrombocytopenic purpura (ITP) which is characterized by rapid platelet destruction and typicallyt appears in young women and also in male patietns who are sero-positive for HIV, hemolytic uremic syndrome (HUS), and it has also benen approved for rheymatoid arthritis (RA). , Germany received FDA approval for pateints with hemophelia devleoping inhibtors, i.e., allo-antibodies directed agaisnt factor cocnetrates. (Bittermann, WO 2014/0201542). 

The molecular interaction between protein A and Ig is well characterized adn the binding site on teh Fc fragment involves residues in the CH2ZCH3 region of particularly IgG1, IgG2, IgG4 and to a lesser extent to IgG3. IA columns approved by the FDA include the protein-A silica column (Prosorba, Fresenius Medical Care, Redmont, CA) for teh treatment of refractory rheumatoid arthritis and for resistant idiopathic thrombocytopenic purpura. The prtoein-A sepharose column Immunosorba, Fresenius Medical Care, St Wendel, Saarland

Jones (US 5,782,792) discloses exposing blood plasma to a protein A immunoadsorbent which binds to IgG containing immune complexes with high affinity and is useful for the treatment of Rheumatoid arthritis).

Nilsson (WO 2013/062479) discloses a method for extracorporeal elimination of one or mroe components from blood which incldues adding whole blood or blood plasma to a blood treatment device such as a blood bag or a blood plasma bag or column containing an adsorbent, consisting of at least one matrix and at least one ligand covalently bound thereto. The antibody, protein or other component bound to the adsorbent may be eluated from the adsorbent as by chaing the pH.  

Palmer (“removal of anti-HLA antibodies by extracorporeal immunoadsorption to enable renal transplantation” 333(8628), 1989, p. 1989, Lancet) discloses that anti-HLA antibodies may occur as a result of failed blood transfusions or pregnancy and that in this respect kidneys have been successfully transplanted after anti-HLA antibodies were removed by extracorporeal immunoadsorption with staphylococcal prtoein A. Since the vast majority of anti-HLA antibodies are IgG, they can be removed by this technique. Plasma was passed through one of the two columns while the other is being regenerated. Treated plasma, depleted of IgG, is returend to the patient. 

Smith (WO 2006/076480) describes targeted apheresis where an immunosorbent apehresis cartridge containing immobilzied human IgG is used to selectively remove immune complexes and RF from the blood Purified IgG can be isoalted form blood used to preapre the apheresis cartidge becasue RF has been shown to react with altered IgG . The altered IgG is immobilzied to a support matrix such as agarose beads.

Removal of Antigens/Pathogens

Ambrus (US4,714,556) discloses an extracorporeal apparatus having hollow fiber UF membranes which have attached proteins having strong affinity for pathogenic factors/antigens in blood.

Removal of Plasma Proteins

Immuno-Affinity columns in series:

Naylor (WO 2004072647) discloses immunoaffinity chromatography coloms and affinity disks configured in series for teh revmoal/depletion of two or more proteins such as HSA, IgG figrinogen, etc. from blood serum, blood plasma, cerebrospinal fluid and/or urine samples. 

Ofsthun (US 5,871,649) discloses an affinity membrane devide for removal of target molecules in plasma. The device is used in an extraceorporeal blood circuit and consists of hollow fiber membranes, ligand immobilized in the pore surface of the hollow fibers and a housing to encase the follow fibers. The plasma components flow into and out of the hollow fiber by means of positive and reverse filtraiton. Initially, as the blood to be treated is transported into the membrane device, the pressure of the blood inside of the hollow fiber causes plasma to pass form the lumen by convention towards the shell of the elongated housing. Then, near the outlet , the pressure inside the hollow fiber membrane is lower than the pressure of the plasma outside of the fiber. As a reslt, the plasma then flows back into the lumen. The plasma that flows back through the wall has been treated or modified with the ligands immobilized to the surface of the pores of the fiber. 

Enzyme coated

Sjoholm (WO0069869) discloses a microporous membrane that includes an immobilized biologically active substance in the form of hollow fibres enclosed within an outer casing with a first inlet and a first outlet. In one embodiment, the biologically active subsance is the enzyme asparaginase from E coli, stabilized on microparticulate polyamide grain enclosed in hollow fibre pores. The enzyme can break down asparagine, which is essential for tumor cells. 

Removal of Antibodies/Immunoglobulins

Leventhal (WO 95/31209) discloses a method for preventing or ameliorating hyperacute rejetion which occurs upon transplant of a pig organ which involves perfusion of the recipient’s plasma over  a coulumn coupled to a protein that binds to human immunoglobulin. The immunoglobulin binding protein can be Staphylococcus aureus protein A/G and anti-human immunoglobulin antibodies. 

Sowemimo-Coker (US 2012/0219633; see also US 15/665971, published as US 2017/0368477; see also US Patent Application 16/090,689, published as US 2020/0325169)) disclsoes a filter device that includes a firt container for receiving biological fluid and containing immunoglobulin binding media and a downstream luekocyte depletion filter. The immunoglobulin binding media can include a variety of functional groups (e.g., ionic, hydrophobic, acidic, basic) such as 4-MEP HyperCell or chromatography osbents with Protein A/G, hydroapatite. 

Removal of Particular Types of Cells

Efficient separations of certain types of cells from complex mixtures has important applications in blood trasnfusions, cancer therapies, auto immune diseases and diagnostics. Cell separation devices employed in these separations have been used in extracorporeal circuits to selectively isolate a specific subset of cells. Separation prcoesses may be used to remove a subset of cells (negative slection). Datar (US 6,008,040)

Leukocytes are present in blood in a wide variety of sizes. For instance, gell like aggregates may be in blood in sizes which vary up to about 200 micrometers. Luekocytes range form macrocytes and granulocytes, typically 15-20 micrometers to lymphocytes which are 5-7 micrometers and large. Datar (US 6,008,040)

Red blood cless are typically 7 micrometers in diameters. Datar (US 6,008,040)

All cells are able to deform as to pass through much msaller openings than their normal size, as in the case of flow in capillary blood vessels. Datar (US 6,008,040)

Leukocytes (white blood cells):

–Negative Selection

Bormann (US 2005/0247627) discloses a file divice for producing a leukocyte depleted plasma rich fluid where the the filter includes a first filter element that includes a porous fibrous leukocyte depletion medium having a first predtermiend critical wetting surfae tension and a second filter elements that includs a porous fibrous leukocyte depletion medium having a second CWST. 

Datar (US 6,008,040) discloses a negative selection of luekocyes by using a continously cascaded toruous flow path. One or more specific ligands may be added to the packings to interact with one or more componetns in the fluid mixture from which the components need searpated. The cascading flow channel provdies a smooth trickling flow of the fluid mixture which porviding increased surface area and decreased hydraulic pore diameters of the separation media, as well as increased interaction between the omponents containin the fluid matrix and the seapration emdia. In one embodiment, a prcoess is provided to simultaneously remove rejection antibodies by using ligands such as emlibiose which are attached to cross-linked, baeded agrose particles and attached to IgM. 

Lee (US 6,337,026) discloses a high capacity luekocyte depletion filtration media that incorporate a high specific surface area components with a matrix of fibers, yielding a filtration medium that remove leukocytes byat least 99.99%. 

Pall (US 4,925,572) discloses a device for the depletion of leukocytes which includes successfive porous elements such that each successive elment has a smaller pore diameter than that preceding it. 

See also Continuous Chromatography under Chromatography, Column arrangement.

Commercial Protein L Resins:

Capto L (GE Healthcare Life Sciences) Data file 29-0100-08 AB) combines a rigid, high flow agarose matrix with the immunoglobulin binding recombinant protein L ligand, which ahs strong affinity for the variable region of an antibody’s kappa light chain. It is thus suitable for capture of a wide range of antibody fragments such as Fabs, scFv and Dabs. The average ligand density is 10 mg/mL and particle size is 85 um. 

Capto L (Data file 29-0100-08 AC GE, 2012-2014) discloses a recombinant Protein L with 4 binding domains that binds to the variable region of the kappa light chain of immunoglobulins and their fragments. 

Tosoh Bioscience Toyopearl AF r-protein L-650F resin is based on polymethacryalte partciles. It offers a static binding capacity (SBC) fo 54 mg/mL for Fab fragments and remains stable at pH 2-12. Muller, “Intensificaiton of Fab-fragmetn Purification, Multicolumn chromatography using prepacked prtoein L columns”, BioProcess international, June 2023, 21(6)).

Introduction:

Many Gram-positive pathogenic bacteria have evolved cell-surface immunoglobulin-binding proteins, which may enable them to coat their cell surfaces with host protein and evade the immune response. The best characterized are staphylococcal protein A and streptococcal protein G, which interact with the immunoglobulin heavy chain, principally at the Fc CH2-CH3 interact, although SPG also interacts with FabCH1. Another protein, peptostreptococcal protein L (PPL) has been reported to be released form the bacterail cell wall by solubilization with proteolytic and muranolytic enzymes. It is expressed in about 10% of Peptostreptococcus magnus isolates and has been implicated in the virulence of these bacteria. (Murphy, Molecular Microbiology 12(6): 911-920, 1994).

Protein L is a cell surface protein expressed by Peptostreptoccocus magnus, which binds to the variable light chains of immunoglobulins without interfering with antigen binding. It can be used for purification of mammalian antibodies of all classes in contrast to the Ig-binding proteins protein A and protein G.

Both peptococcus and peptostreptococcus have been reported to produce Protein L, which binds to the Kappa light chain of human immunoglobulins. (Trowern, US 6,162,903). 

Antibody fragmetns such as FAB, ScFvs and nanobodies have mereged as increasinly important therapeutic and diagnostic alternatives to full lenght mAbs. Whereas Mab downstream processing is well established and easy to scale based on protein A capture, the purificaiotn of antibody fragmetns is just on the verge of standardized prcoessing. The most promising candidate for effective capture of those containing a kappa light chain is protein L affintiy chromatography in the first step of downstream prcoessing. (Muller, “Intensificaiton of Fab-fragmetn Purification, Multicolumn chromatography using prepacked prtoein L columns”, BioProcess international, June 2023, 21(6)). 

Unpredictability

Gore (WO 00/15803) disclsoes a Protein L mutant modified by an amino acid at one or more positions 39, 53 and 57 and/or by an amino acid instert between positions 59 and 60 such that the dissociation costant (dk) with respect to human immunogloubilin k-chain is 400 nM or more at pH 8.  Gore further discloses that the rate of dissociation of the Y53F PpL k chain complex was affected by pH and dissociated faster at lower pH values. This was not the case for Y64W PpL, which dissociated faster at pH 9. Of note, Gore teaches that specific substitutions at positions 39, 53 and 57 and an insertion between positions 59 and 60 markedly affected the binding affinity. Some of the other subsitutions did not result in expression of any poolypeptide, potentially due to instability of the mutated polypeptide. (see Table 3). 

Where Protein L binds:

Protein L binds to antibodies from a wide range of species including about 50% of human and 75% of mouse antibodies through the Vk region. The location of this unique binding site in the framework region of the light chain of antibodies allows protein L to bind an alternative subset of immunoglobuilins compared to protein A and G and also to bind the range of antibody fragment such as scFv, Fab and single domains, disulphide bonded Fv, a Fab fragment and a F9ab)2 fragment used in antibody engineering, if they have the correct K framework. Protein L has been found to bind to Vk of subgroups I, III and IV. (Enever 9WO/2005/033130).

Protein L binds human Fab via VL kappa 1, 3, and 4 but does not bind to VL kappa 2 and none of the VL domains of the lambda isotype. (Hermans US13/982970, published as US 2013/0337478)

Structure of Protein L:

Structure Depends on Peptostreptococcus Magnus: Strain:

Protein L (PpL) contains a plurality of VL-k-binding domains consisting of 70-80 residues. Yoshida Hyogo (US 15/660,365, published as US 2018/0016306). The number of FL-k-binding domains and the amino acid sequence of each domain are different depending on the kind of Protein L strain. For example, the number of Vl-k-binding domains in PpL of Peptostrepococcus magnus 312 strain is five, and the number of VL-k-binding domains in PpL of Peptostreptoccus mangus 3316 strain is four. Yoshida, Hyogo, (US 15/660, 373, published as US 2017/327535).

Each of the VL-k-binding domains of PpL312 are referred to as B1, B2, B3, B4 and B5 domain in the order from the N-terminal and each of the VL-kbinding domains of PpL3316 are referred to as a C1, C2, C3, C4 domain in teh order from the N-terminal Yoshida Hyogo (US 15/660,365, published as US 2018/0016306),

The gene for protein L expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus has been cloned and sequence. Teh gene translates into a protein of 719 amino acid residues. Following a signal sequence of 18 amino acids and a NH2-temrinal region (“A) of 79 residues, the molecule contains five homlogogus “B” repeats of 72-76 amino acids each. Furtehr, toward the COOH terminus, two additional repeats (“C”) are found. These are not related to the “B” repeats, but are highly homologous to each other. After the C repeats (52 amino acids each), a hydrophilic, proline-rich putative cell wall-spanning region (W”) is found, followed at the COOH temrinal end by a hydrophobic membrane anchor (“M”). Fragmetns of the gene have been expressed, and the corresponding peptides analyzed for Ig binding activity. The B repeats are found to be responsible for the interaction with Ig light chains. (Kastern, J Biological Chemistry 267 (18): 12820-12825). 

The two strains of P. magnus (312 and 3316) produce slightly different Protein L molecules. Strain 312 produces Protein L that contains five highly homologous Ig binding domains, wehreas strain 3316 produces a prtoein with four Ig-binding domains. (Housden, Biochemical Society Transactions, 31(3) June 2003). 

312 Strain

Bjorck (US 5,965,390) discloses sequences of protein L including multiples of the domains B1-B5 which bind to light chains in Ig. 

B5 domain of Strain 312:

Yoshida, Hyogo, (US 15/660, 373, published as US 2017/327535) discloses an affinity matrix based on the beta5 domain of Protein L dervied from Peptostreptococcus magnus 312 strain. Yoshida, Hyogo (US 15/660,373 published as US 2017/0327535; see also US Patnet Application No: 16/176,090, published as US 2019/0119362) also teaches the beta5 domain of Protein dervied from the 312 strain with an N-terminal deletion). 

3316 Strain:

A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof have been expressed in E coli. The four C units were shown to be responible for binding to immunoglobulin and the four D units for binding to albumin. The protein L molecule therefore binds to albumin at a site separate from that involved in binding to immunoglobulin. (Murphy, Molecular Microbiology 12(6): 911-920, 1994). 

Protein L Mutants:

Substitions positions 39 53, 57; insertion between 59 and 60:

Gore (WO 00/15803) disclsoes a Protein L mutant modified by an amino acid at one or more positions 39, 53 and 57 and/or by an amino acid instert between positions 59 and 60 such that the dissociation costant (dk) with respect to human immunogloubilin k-chain is 400 nM or more at pH 8.  Gore dislcloses due to decreased binding affinity of Y53F PpL, purication can occur under milder conditions. 

Mutations of Magnus 3316 Strain

–Substitution positions 7, 13, 22 and 29 

Majima (15/770,150, published as US 2018//0305414) discloses a high binding capacity for an immunoglobulin kappa chain having excellent alkali stalisty by modifying an amino acid sequence of Protein L dervied from Peptostreptococcus magnus 3316 strain. In particular, Majima discloses substituting at least two or more sites from positions 7, 13, 22 and 29 with a basic amino acid other than lysine (e.g., R or H) or a hydroxyl group continaing amino acid (e.g., S, T or Y) in one of the four immunoglobulin-binding domains including in Protein L dervied from Peptostreptococcus magnus 3316 strain. 

Mutations of 312 Strain: 

–N-terminal deletion:

Yoshida (US 16/176090, published as US 2019/0119362) also teaches using the B5 domain for affinity chromatography purification of an antibody with a kappa chain variable region. In one preferred embodiment, the beta 5 domain of Protein L has an N terminal deletion. 

–Substituions positions 15-18:

Yoshida Hyogo (US 15/660,365, published as US 2018/0016306) also discloses a substitution of one of the amino acid residues at positions 15-18 in one of the Vl-k-binding domains B1-B5 and C1-C4 of PpL which results shifting the pH to a neutral side for dissociation on an affinity resin of the VL-k. 

–Mutation of B1 Domain

Enevery (“engineering high affinity superantigens by pahge display” J. Mol. Biol. 347, 107-120 (2005) discloses mutants of the N-temrinal B1 domain of P. magnus strain 312 with a 14 fold increased affinity for humVk1 and a greater than tenfold increased affinity for huVkIII. 

Conditions

Binding:

Hyogo (US16183308, published as US 2019/0211082) discloses a method of purifying an antibody that contains a kappa-chain variable region using Protein L on a carrier where the antibody i is adsorbed to the matrix at a temeprature from 4-40C. 

Washing:

–Carpylate (caprylic acid)

Suda (WO 2014/141150) discloses a method of purifying an antibody by adsorbing the protein/antibody onto a Protein L resin and washing with an aliphatic carboxylate to remove contaminants prior to elution. In one embodiment the wash buffer further includes about 1-500 mM sodium acetiate. 

Elution:

–acetic acid sodium acetate

Hyogo (US16183308, published as US 2019/0211082) discloses a method of purifying an antibody that contains a kappa-chain variable region using Protein L on a carrier where the antibody is eluted from the matrix using an acetate buffer. In some ebmodiments, the acetate buffer is at a pH value of 2.5-4.0 and the concentration of an acetate ion in the buffer is 10-500 mM.

Suda (WO 2014/141150) discloses a method of purifying an antibody by adsorbing the protein/antibody onto a Protein L resin and washing with an aliphatic carboxylate to remove contaminants prior to elution. In one embodiment the wash buffer further includes about 1-500 mM sodium acetiate. In one embodiment the eltuion buffer includes 1.8 mM sodium acetate adn about 28.2-300 mM acetic acid at about pH 2.4-3.6. 

companies: 3M filtration

See also depth filters generally

Despite low shear centrifuge designs, the centrate effluent may still contain appreciable amounts of smaller sized contaminants (e.g., aggregates and colloids). Depth filtration is most commonly used as a secondary clarification to remove the debris and to prevent plugging of downstream processes, such as chromatography. Charged depth filters are the preferred method for clarification and debris removal downstream of the primary harvest step, due to their ability to retain a large amount of contaminants using boht size exclusion and adsorption. (Low, J Chromatography B, 848 (2007) 48-63). 

Pegal “Evaluating disposable depth filtraiton platforms for MAB harvest clarification” Bioprocess International, 2011, 52-55) showed that clarifying cell culture harvest with only single use depth filtraiton can be an attractive alternative to the traditional fed batch process that includes both centrifugation and filtration. 

Types of Depth Filters

Charged Depth Filters:

Charged depth filters are available commercially from, for example, Cuno, Inc. (e.g., ZETA PLUS S series, ZETA PLUS SP series, ZETA PLUS LP series, ZETA PLUS CP series, ZETA PLUS LP BC series), EMD Millipore (e.g., DOHC, COHC, FOHC, A1HC, B1HC, XOHC) ), Sartorius AG, and Pall Corporation (e.g., SEITZ P series, SEITZ K series, SUPRADUR series, STAX series, SUPRACAP Series, SUPRAPAK series, SUPRADISC seires). (Hoang, US 15/751, 231, published as US 2018/02301800. 

Depth Filtration in Combination with Other Types of Unit Operations

Rajendran (US 14/977,869, published as US2016/0176921) discloses a method of purifying a protein such as eculiumab by capturing the protein such as by protein A affinity chromatography, followed by one or more unit operations and then flowing the protien frough a depth filter to provide a filtrate. 

Centrifugation + Depth Filtration: 

The first step in the recovery of an antibody from mammalian cell culture is harvest or removal of cells and cell debris to yield a clarified, filtered fluid suitable for chromatography. This is generally accomplished through use of centrifugation, depth filtration and steril filtration. The depth filter is employed most frequently after the centrifugation process because there is a practical lower limit to the particle size that can be removed by centrifugation. The current preferred process for initial recovery is to use a disc-stack continuous centrifiguation coupled with depth filtration. Considerably thicker than a membrane filter, depth filters are about 2-4 mm in thickness. For harvesting applications, depth filters can be applied directly with whole cell broth or in conjunction with a primary separator such as TFF or centrifugation. The whole cell broth depth filter harvest is common for bench, pilot and smaller commercial scale applications. For this, a filtration tran containing three stages of filters is usually employed. The primary stage uses a coarse or open, depth filter with a pore size range of up to 10 um and removes whole cells and large particles. The secondary stage uses a tighter depth filter and clears colloidal and submicron particles. The alst stage contains a membrane filter that is 0.2 um pore size in most cases. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).      

Singh (WO/2013/009491) disclsoes a process for clarificaiton of feeds containing target iobmolecules such as mAbs using a pimary clarificaiton depth filtraiton device without the use of a primary clarificaiton centrifugation step or a primary clarificaiton tangential flow microfiltration step. 

In combination with Protein A chromatography

–Protein A — Charged DF –Incubation:

Hoang (US 15/751231, published as US 2018/0230180) discloses a method that increases the re-oxidation of antibody molecules using a Millistak A1HC depth filtery at a throughput of 350 L/m2. There was more than two-fold decrease in % pre-peaks observed in the charged depth filtered filtrate compared to sterile filtered filtrate immediate following fitration. The DF was effective in enhacing re-oxidation of antibody molecules when performed prior to Protein A chromatography or after Protein A chormatography. Hoang disloses that incubating the fitlrate for at least four hours before being contacted with the chared dF or after DF. Hoang states that the structure and stability of antigen bining protein molecuels depends heavily on the disulfide bonds that link the two heavy chains and the heavy and light chains and that during production and purificaiton, one or more disulfide bonds can be reduced to free thiol groups. Thus reduction of the inter-chain dislfide bonds weakens the strucutral integrity of the antigen binding protein and can lead to antigen bidning protein fragments such as L and H chains as well as antigen-binding protein aggregates. Thus reducing the percentage of reduced antigen binding protein molecules with the DF step is important. 

–Protein A – Unit operations (e.g., IEX, HIC, viral inactivation, viral filtraiton, adjustment of pH, etc) –DF

Rajendran (US 14/977,869, published as US2016/0176921) discloses a method of purifying a protein such as eculiumab by capturing the protein such as by protein A affinity chromatography, followed by one or more unit operations and then flowing the protien frough a depth filter to provide a filtrate.

Sample -CEX – DF -AEX:

Kim (US 14/896,380, published as US 2016/0122384) discloses purifying an antibody with CEX followed by a multilayer filter (depth filtering) and then AEX

Conditions/Parameters

Isoelectric point: 

Zou (WO 2008/036899) discloses a emthod for removing parvovirus or fragments thereof from a therpaeutic protein such as an antibody by passing the solution through a depth filter at a pH within 1 pH unit of the isolectric point (pi) of the virus such as a pH in the range of 4.0-6. In one embodiment the DF is an electropostively charged filter suchas a Millipore AIHC filter. The method can include the step of maintaining the solution at a pH for a lenght of tiem effective to inactivate virus in the solution. 

Contacting solution with positive ion

Hoang (US 15/751231, published as US 2018/0230180) discloses aadding a positive ion to an aqueous solution with a charged depth filter. For example, copper can be added to the aquous solution or the solution can also be sparged with air or oxygen before, during and/or after contacting the solution with the cahrged dF. 

See also depth filtration for antibody purification

Companies:  3 M Purification

Depth filters refers to the use of a porous medium that is capable of retaining particulates throughout its matrix rather than just on its surface. These filters are frequently employed when the feed stream contains a higher content of particles. In such cases, depth filters can remove larger, insoluble contaminants prior to final filtraiton through a microfiltration membrane that would otherwise clog relatively quickly. Depth filters employed in bioprocessing are typically composed of a fibrous bed of cellulose or polypropylene fibers along with a filter aid (e.g., diatomaceous earth) and a binder that is used to create flat sheets of filter medium. (Yigzaw, Biotechnology progress, American Institute of Chemical Engineers, US, 22(1), 2006, 288-296).

Uses for Depth Filters

Clarification: Dept filters are traditionally used in the clarification of cell culture broths, to maintain capacity on membrane filters or to protect chromatography columns or virus filters. They are typically composed of cellulose, a porous filter aid such as diatomaceous earth and an ionic charged resin binder. For harvesting applications, dept filters can be applied directly with the whole cell broth where a filtration train containing three stages of filters is often employed. The primary stage uses a coarse or open depth filter with a pore size range of up to 10 um and removes whole cells and large particles. The secondary stage uses a tigther depth filter and clears colloidal and submicron particles. The last stage contains a membrane filter that is 0.2 um pore size in most cases. Debt filtration is frequently employed after the centrifugation process because there is a practical low limit to the particle size that can be removed by centrifugation (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Remvoal of endotoxins, viruses, DNA and HCP: Withe the addition of adsorptive surfaces, depth filters are now also being used for a variety of other applications involving removal of endotoxins, viruses, DNA nd HCP. (Rathore and Shirke, Preparative Biochemistry & Biotechnology 41:398-421, 2011). 

Due to the adsorptive mechanism of depth filters, they have been extensively used as a purification tool to remove a wide range of prcoess contaminants. Positively charged dept filters have been employed for the removal of E. coli derived and other dendogenous endotoxins and viruses many times smaller than the average pore size of the filter. They have also been used to remove prions form an immunoglobulin solution. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Depth Filtration + Centriguation: Depth filters are traditionally used in the clarification of cell culture broths to maintain capacity on membrane filters or to protect chromatography columns or virus filters.  A depth filter is most frequetnly used after centrifugation because there is a practical lower limit to the particle size that can be removed by centrifugation. This type of depth filter has a pore size range of 0.1-4 um and is usually made of two distinct layers, with the upstream zone being a courser grade compared with the downstream. The larger particles are trapped in the coarse grade meida and smaller particles are trapped in the tighter media, reducing premature plugging and increasing filtration capacity.  (Liu, “Recovery and purificaiton process development for monoclonal antibody productn, mAbs 2:5, 480-499, 2010). 

Depth Filtration without Centrifugation: O’Connor (US13/880424) discloses purification of proteins such as antibodies using depth filters where the method does not include centrifuging. First, the mixture containing the protein is mixed with a solubilization buffer containing ethanolamine, arginine, EDTA, urea and DTE and then the protein is clarified with two or more depth filters. The method further includes refolding the protein with a refolding bufer having ethanolamine, EDTA and GSSG.

Composition

Cellulose + Diatomaceous earth: Depth filters are typically composed of cellulose, a porous filter aid such as diatomaceous earth and an ionic charge resin binder. A binding resin is often added to a small weight percent to covalently bind dissimilar construction materials together, giving the resultant media wet strenght and converring positive charge to the media surfaces. Because of this make up, depth filters rely on both size exclusion and adsorptive binding to effect separation. (Liu, “Recovery and purificaiton process development for monoclonal antibody production, mAbs 2:5, 480-499, 2010). 

Millipore Data Sheet, “Millistak+ Pod disposable depth filter system” 2009 discloses a depth filter system which is ideal for clarification of cell cultures which is composed of cellulose fiber and diatomaceous earth.

Posivitvely charged Depth Filters

Positively charged depth filters have been employed for the removal of endotoxins from water, virus particles that were smaller than the effective pore size of the filter, to remove DNA from a buffer solution and spiked prions from an immunoglobulin solution. Depth filters have also been sued to remove contaminant proteins in the cell culture harvest supernatant prior to Protein A chromatography for the purificaiton of antibodies. (Yigzaw, Biotechnology progress, American Institute of Chemical Engineers, US, 22(1), 2006, 288-296).

3 M purification, USA sells the Zetaplus encasulated filtraiton whystem which contains a hybrid depth filter media consisting of both size exclusion and anion exchange components. The primary application of this system is clarification of the fermnetation broth. However, published applicaitons have shown that the filter also provides capability to clear DNA and HCP from the protein A pool obtained during processing of CHO mAB product. (Preparative Biochemsitry & Biotechnology 41: 398-421, 2011). 

Inter-alpha inhibitor proteins (IαIp) are a fmialy of structurally related serine protease inhibitors found at relatively high concentraitons (400-800 mg/L) in human plasma. Unlike other inhiibtor molecules, this fmaily consists of a compbiation of polypeptide chains (light and heavy) covalently linked uniquely by a chondroitin sulfate chain. the heavy chains of Inter-alpha proteins (H1, H2 and H3) are also called Hyaluronic acid (HA) binding prtoeins. The major forms found in human plasma are inter-alpha-inhibitor (IαI).

IaIp are recoved from mateiral otherwise discarded during manufacture of important products by plasma fractionation. For example, IaIP is extracted from a Cohn Fraction I or Fraction IV-1 precipitate (Coyn, 1946), a Cohn-Oncley Fraction II+III precipitation (Oncley), a Kistler and Nischmann Precipitate A or Precpitate B precipitate (Kistler and Nischmann) or adsorbed from a Cohn-Oncley Fraction II+III suspension formed during the manufacture of IgG gamma globulins.

Bairstow (US2012/0053113) discloses a method for preparing IaIP from plasma using the steps of forming fraction II+++ precipitate, resuspending the precitate to form a fraction II+III supension and contacting the suspension with a solid phase to remove the IaIP from the suspension and extracting the IaIP form the solid phase. In particular, first proteins are precipitated from a cryo-pool plasma fraction with 6-10 alcohol at pH 7-7.5 to obtain a first precipitate and 1st supernatant, the 1st supernatant is precipitated in a second precipitation step with between 20-25 alcohol at pH of 6.7-73, resuspend the second precipitate to form a suspension and mix finely divided silocon dioxide, filter and extract the IaIp form the filter cake.

Single initial precipitation step (Fraction I+II+III+IV-1 preciptiation or Fraction I-IV-1 precipitation or initial low pH, high alcohol precipitation):

Bruckschwaiger (US13/776448) discloses an initial purificaiton step that copresicipates IgG and A1PI followed by a solubization step of this precipitate which leaves inter-alpha-trypsin inhibitor (IalIp) in the insoluble portion and the immunoglobulins in the soluble portion. The method includes the steps of precipitating immunoglobulins, A1PI, Factor H and IaIp in a first precipitation step by adding ethanol to a Cohn pool to 20-30% at a pH 5-6 to form a first precipitate and first supernatant, separating the first precipitate (containing the immunoglobulins, A1PI, IaIp, albumin, Factor H) from the supernatant. Next the first precipitate is suspended to form a first supension which is then treated as with silicon dioxide. The soluble porition of the supsnsion contains immunoglobulins while the insoluble porition contains A1pI, fibrinogen, Factor H and IaIP.

Isolation using Ion Exchange

Anion Exchange:

(Lim, WO2005/046587) discloses a process for producing a blood plasma derived IalphaIp composition using DEAE Sephadex. 

Antibodies are among the most important classes of proteins involved in the adaptive immune system. Together with T-cell receptors, they provide a robust system of defense agaisnt infeciton caused by forign antigens. Antiobides are capable of binding to a virtually infinite set of antigens.