See also  Membrane Adsorbers See also filtration in conjunction with Protein A chromatography for the purification of antibodies See also  “Anion exchange” for membrane absorbers with anion exchange ligands.

Companies:  disposable depth filter system: Pall, Cuno, Millipore; Sartorius Stedim Biotech

Introduction:

Although the chromatography steps play the major role in the purificaiton of antibodies, filtraiton steps are integral to any process. These can be in the form of ultrafiltration steps in TFF mode, 0.2 um membrane filtration steps for particle reduction/bioburden control, or virus reduction filtration steps to reduce the level of viruses in the process stream. Bonnerjea (J. Chromatography B, 848 (2007) 64-78)

The first series of operations in a purification train – the harvest step – focuses on the removal of particulate impurities with the goal of delivering a particle-free sueprnatant that can be fed into the subsequent, mostly chromatographyy-based prcoesses. Typically, tangential flow microfiltraiton, depth filtration and particular centrifugation are used to clarify cell culture suspensions. These initial steps remove large particles (greater than 1-2 um diameter) and are frequently followed by the use of a polishing depth filter and one or more absolute filters to remove smaller particles and to reduce the turbidity of the feedstream. (Thommes, Process Scale Purificaiton of Antibodies 2009, pp. 293-308). 

The terms “microfiltraiton” and “ultrafiltration” reference fitlraiton parameters commonly understood in the art. In particular, the term micrfiltraiton refers to the use of a fitlraiton membrane with a proe size betwen 01.-10 um and “ultrafiltraiton” refers to the use of a filtraiton membrane with a pore size between 0.001-0.1 um. Microfiltraiotn is typically used for clarifcaiotn, sterilization, removal of micropartciculates and for cell harvests; ultrfiltraiton is typically used for separating and concentrating dissolved molecules )oe.g., protien, peptides, nucleic acids, carbohydrates and other biomolecules), for exchange buffers and for gross fractionation. (Becker et al. (US 2015/0274773). 

Filter Sizing:

Very few filtration difficulties are observed at laboratory scale and 0.2 um filters, such as syringe filters may be suficient for the small product volumes,. However, filtraiton difficulites are enhanced as processes are scaled-up. Filters should either be sized appropriately for a particular application or in the absence of data, a generic approach may be taken whereby a train of pre-filters of decreasing pore size may be used prior to the final 0.2 um filter. For filter-sizine, an adequate volumne of product is reuqired to that ideally the filtraiton operation can be continued until the filter becomes blocked. Hence, the pcoress may need ot be partially sacled-up before this data can be obtained. One pilot process oepratea t 130L fermentation scale was observed to have fitlraiton issues in 2 out of 4 batches. During a futher pilot batch, filter sizine was performed at two stages in the purificaotn process which had proved to be difficult to filter previously. Bonnerjea (J. Chromatography B, 848 (2007) 64-78)

The use of large proe size pre-filters can be efective at reducing filter area and coses. For example, filter sizing data fo r the neutralised eluate form a Protien A chromatogrpahy column showed the reuqirement for three 30” 0.45/0.2 um Sartopore 2 filters or alternatively one 30′ 0.8/0.45 um pre-filter in line with one 10” 0.45/0.2 um Sartopore 2 filter. With the larger volumes anticipated with tire increases, filter sizing should be a key consideraiton of any scale-up process. Filter performance can vary depending on supplier and material of construction, emphasising the advantages of evluating several ilter types prior to scaling up. Bonnerjea (J. Chromatography B, 848 (2007) 64-78)

Size Porosity that retains Most of Antibody:

Gagnon (US 15/120,111, published as US 2017/0057992) discloses a method of purifying IgG using an electropositive membrane (AEX membrane) having a porosity that retains at least 50% antibodies/non-adsorbed solutes with a hydrodynamic diameter greater than a selected size of about 10 nm but permits passage of non-adsorbed solutes with a hydrodynamic diameters less than the elected size. In addition, at least o portion of the contacting step the sample contains a salt at a concentraiton less than about 50 mM and a pH in the range from about 3- to 0.5 pH units of the pI of the most alkaline glycoform of the IgG or a salt at a concetnration greater than about 50 mM and a pH from about 3-9 and (b) a final contacting step by eitehr (i) an absence of excess salt  or not greater than 20 mM and (ii) a pH value in a range from about 5 to within about 0.5 pH units of the pI of the most alkaline glycoform of the IgG antibody. In one embodiment, IgG is fractionaed by precipitation in 2 M ammonium sulfate, the antibody precipitates, mostly contaminants, are eliminated with the supernatant. The IgG is resolubilized by reducing the ammonium concentration to 1 M (by the addition of water). This high salt sample is then introduced into a FFF with electropositive membranes with an average pore size corresponding to a hypothetical globular protein of 50 kDA. IgG remains retains, but the high salt largely . The high salt buffer is then displaced by a low salt buffer such as 50 mM Tris, pH 8.2 and any remaining acidic contaminants bind to the electropostive membrane surface, but the IgG does not. In anotehr embodiment, mammalina cell culture harvest containing IgG was clarified by adjsutment to pH 5.2, addition fo 1% allantoin, follwoed by 0.4% sodium caprylate acid, incubated 2 hr, 5% BioWorks TREN was added, 4 hours incubation, centrifuggation to remove contaminants and then the preparation was applied directed to a TREN bearing cellulose membrane with a pre size corresponding with a globular protein with a mass of 30 kDA. The preparation was then buffer exchanged into 50 mM Tris, 2 mM EDTA, 200 mM Histidinet, 2 M NaCL, pH to dissociate non-specific interactions, then buffer exchanged into 50 mM Tris, pH 8.2 

Ultrafiltration (UF)

Two types ulfiltration methods are commonly used for ultrafiltration (UF). In direct flow filtration (DFF) the feed is forced direclty toward the membrane. As a result, molecules larger than the pores aggregate at the membrane surface and form a gel that clobks the flow of the smaller molecules through the pores, so that the flux rate decreases rapidly as filtraiton proceeds. The protein solution is often stirred during DFF in order to keep the retained protein from aggregating and blocking the pores of the membrane. The other main UF process is tangential flow filtration (TFF) in which teh sample flows accross the surface of the membrane as pressure on the solution forces smaller molecules in the solution outwards trhough the pores of the membrane. The flow of solution across the membrane during TFF helps prevent a gel of aggregated molecules from forming on the surface of the membrane that blocks the pores and preevents smaller molecules form passing through. As a result, the flux rate for TFF drops off much more slowly as filtration proceeds than occurs during DFF. Yang (WO/2004/001007)

Depth Filtration (See outline)

High Performance Tangential Flow Filtration (HPTFF)

HPTFF has been described in which a positive charge is created on the surface of an UF membrane with a cutoff of about 10-300 kDa. When a sample of crude IgG mAb is introduced within a narrow range of pH and conductivity, IgG is repelled form the membrane surface and thus prevented form passing through the pores. The majority of contaminant speceis either binds to the surface or passes through the pores by convective mass transport and is thereby eliminated. The method also permits concentration of the antibody. (Gagnon, 14/555060)

Flocculation -Filtration

Flocculation is a smilar process to coagulation, wehre suspended partciles clump together becasue the attractive forces between them overcome any repulsive forces caused by like surface charges. Such repulsive forces can be eliminated, for example, through the addition of inorgnaic electrolytes, which shield the surface charges, or by the addition of polyelectrolytes that bind to and neutralize the surface charge. Flocculation has been used mainly for the removal of whole cells from fermentation broth. Particles are often coagulated or flocculated prior to filtration to reduce the passage of small noncoagulated particles through teh filter and to produce a more porous cake which is easier to remove. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008)

Lowering pH + Divalent cation (e.g., Ca2+ or Mg2+ or Cu2+ or Co2+ or Mn2+):

Romero (WO2008/127305) disclose a method of isolating a biomacromolecule such as an antibody by lowering the pH of the composition, adding a divalent cation and separating the antibody from the impurity. The lowering of the pH of the harvest feed prior to filtration causing flocculation of large cells and cellular debris along with precipitation of other impurities like DNA which improves mass transfer of the composition near the surface of the filter (lowering the pH also reducing plugging or filling of the pores of the filter) thus reducing transmembrane pressure across the filter. Because lowering the pH of the composition can result in coprecipitation of the biomacromolecule of interest as well as the impurity, addition of divalent cations to the pH adjusted composition is suitable for increasing the recvoery of the biomacromolecule of interest. 

Conditions:

Water as DF medium: 

Graunhofer (US2009/0291062) discloses aqueous formultations of proteins such as antibodies such as adalimumab in water based on a diafiltration process using water as a diafiltration medium. The resulting aqueous formultaiton has a significant decrease in the overall percentage of excipients and low conductivity. 

Applying gas to permeate side of UF

Bolton (WO2010/111378) discloses a method of generating a highly concentrated antibody/protein solution by circulating the first antibody solution through an UF device and applying a flow of gas to the permeate side of the porous membrane and then collecting the second permeate solution. Exposure of the permeate side of the membrane to a flow of gas such as air allows evaporation of the absorbed protein free solvent into the air so as to remove the solvent which concentrates the antibody solution, while the membrane prevents the antibody from being exposed to air which can damage proteins. In one embodiment, the UF device is connected to a retentate tank in a lopp so that the first protein solution is allowed to return back to the retentate tank which allows for continous reciruclaiton of the solution. When the protein solution inside the apparatus achieves a desired concentraiton or desired feed pressure, the reciruclation may be ramped down and the highly concentrated protein solution may be collected. 

Stabilizing Agents

–Salt of Surfactant: 

A major drawback in the applicaiton of UF membranes in bioseparations is protein fouling. Flux loss occurs over a long term period, and flux does not recover even when filtration is resumed after depressurization. Chen (J. Membrane Sci. 67(2-3): 249-261, 1992) discloses that the small anionic surfactant (AOT) provides a reduction of protein deposition by altering the electrostatic interaction between the protein and membrane surface. When used in conjunction with nonionic surfactants or when polyethyelene oxide segments are added to their backbond, the anionic surfactants showed significant flux improvement and fouling resistance compared with that of the single AOT or the nonionic surfactant. 

In order to inhibit aggregation and loss of biological activity when producing a highly concentration solution of MAbs by UF, a stabilizing additive such as a polyol, and/or a viscoity reducing agent such as a salt or surfactant is typically added to the composition containing the antibodies (US6,171,586, US2002/0045571).

–Addition of Amino Acids

–Glycine

Van Holten (US6,096,872) discloses methods of nanofiltration and ultra filtration of anti-D immunogloulin in high ionic buffer with an excipient such as polysorbate 80. The high ionic strengh buffer can be 150 mM NaCl-glycine. In one embodiment, a human IgG product was suspended in glycine buffer and filtered through an UFG.

 –Additional of basic amino acids: 

Hongo (US 13/260419, now US 9,056,896) teaches removing small viruses from antibody solution with a virus removing membrane using a monoclonal antibody solution supplemented with a basic amino acid such as arginine, histinde, lysine. The basic amino acid is thought to have the effect of decreasing the potential of the antibody surface and thus suppressing electrostatic interaction with the negative charge of the virus removing membrane. The basic amino acid also has an effect of suppressing antibody-antibody hydrophobic interaction at a pH range near the isolelectirc point of antibodies where they tend to associate with each other through hydrophobic interaction since the electrostatic repulsion between antibodies decreases.f

–Acetate or Histidine: Yang (WO/2004/001007) discloses that antibodies and histidine or acetate at a concentration in the range of 3-48 mM can be concentrated efficiently by UF to a high concentration with retention of biological activity and relatively little aggregation, even in the absence of a stabilizing or viscoscity reducing additive such as a surfactant, a polyol, a saccharide, a salt or high buffer concentration:

Temperature:

Bolton “Acheiving high mass throughput of therapeutic proteins through parvovirus retentive filters” Biotech. Progress 26(6) 1671-1677 (2010) discloses an optimum temperature of 35C for maximizing throughput through the Virosart CPV and Virsesolve Pro filters in methods of using parvovirus retentive filters . 

–Zeta (surface) potential: 

Hongo (US 13/260419) disclose that if the zeta potential Ei1 (mV) of a monoclonal antibody in solution minus the zeta potential Em (mV) of the virus removing membrane is more than 20mV, the electrostatic interaction between antibody and membrane is strong, which id disadvantageous for filtration. Yet if the zeta pootential Ei0 (mV) of the monoclonal antibody in the solution (pH=4 and ionic strenght of 0.1 mM) is less than 10 mV, the soothing effect of the antibody on base potential is weak, and an improvement of filtration rate is not achieved. Thus a most beneficial zeta potential Ei1 (mv) of a monoclonal antibody in solution satisfies the following conditions: (a) 0 mV≤Ei1-Em≤20mv, with respect to the zeta potential (Em (mV) of the virus removing membrane and (b) 10mV≤Ei0-Ei1≤40mV, with respect to the zeta potential Ei0 (mV) of the monoclonal antibody in the solution (pH=4 and ionic strenght of 0.1 mM) containing the monoclonal antibody. 

Particular Contaminants which are Removed

DNA: 

Charlton, (Bioseparation, 8, 1999, 281-291) discloses using positively charged filters (anion exchangers) for removal of DNA from solutions containing antibodies at various ionic strenght (salt concentrations). If the filter is operating as a true anion exchangers, then increasing the ionic strenght of the buffer results in reduced adsorption of the DNA. The ooposite effect is observed at low ionic strenght where hydrophobic interactions are playing a role in adsorption. Similarly, the enhanced DNA adsorption observed at high salt concentration is partially reversed by teh addition of a non-ionic surfactant such as polysorbate 80. 

Viruses:

–Parvovirus filtration:

–—Prefiltration:

——Depth filters or IEX membrane adsorptive pre-filters or mixed mode 

Brown (“increasing parvovirus filter throughput of monoclonal antibodies using ion exchange membrane adsorptive pre-filtration” Biotechnolgy and Bioengineering, 106(4), 2010) dscloses pre-filtraiton using Ion exchange membrane adsorbers can imporve parvovirus filter throput of mAbs. 

Olsen (US 2018/0072769) discloses flowing a fluid including a recombinant antibody through a pre-filer before the fluid is flowed thorugh a virus filter.  Examples of pre-filters include a Sartorius Virosart Mas pre-filter, a Millipore pre-filter, a Sartopore 2 pre-filter, a Sartobind STIC pre-filter, a Sartobind Q pre-filter, a Sartobind HIC Phenyl pre-filter, a Sartobind S pre-filter, Millipore Viresolve Pro Shield pre-filter, CUNO delipid pre-filter and Millipore XOHC pre-filter. In some embodimetns, a virus filter includes a polyamide membrane, a cation exchange based based membrane, an anion exchange based membe or.a HIC based membrane. Additional examples of pre-filters are known in the art. Olsen also disclos eusing a depth filter having anionic and hydrophobic propters (used CUNO delipid filter) to purify Alexion 1210 (also called BNJ441). Significant improvement in aggregate removal and particulate content was observed when depth filtration was performed immediately prior to virus filtration. A depth filter having anionic and hydrophobic properties resulted in significant removal of host cell protein and soluble protein aggregates. 

—-additives:

—–non-ionic surfactant:

Brown, (US 14/007,610, published as US 2014/0309403) discloses a method of reducign fouling of an ultrafiltration membrane by the addition of a non-ionic surfactn such as plysorbate 20 during the filtration of a solution that contains an antibody and parvovirus particles.   

Multiple UF/DF: 

Konstantinov (US2006/0149042) discloses a process of subjecting a supernatant to an initial UF, then adjusting the conductivity of the retentate, such as by diafiltration with water for injection (WFI), diluent or buffer, and then subjecting the solution to a second UF. The process increases the concentration of cell culture supernatant containing macromolecules and an orgnaic polymer with higher yields.

Pore Size (MWCO)

–Second Mebrane smaller pore size:

Chtourou (US 7,186,410) discloses a process for preparing human immunoglobulin concnetrates form plasma using an AEX at alklaine pH and further comprising concentrating the immunoglobulin by UF and sterilization by filtering it through nanometric filters of a porosity decreasing fromm 100 to 15 nanometers. 

—-In final Filtration step:

Falkenstein (WO2011039274) discloses antibody solutions with a concetnraiton of more than 100 g/l are prone to difficulties such as blocking of the employed filter by aggregates formed during the formulation or concentraiton process during the final filtartion step.. Falkenstein discloses a method for filtration of antibodies which have been concentrated to at least 100 g/l as by diafiltration by combination of two immediately consecutive double membrane filtration steps wtih a first filter of 3.0 um (“pre-filter) and 0.8 um (main filter) pore size and a second filter of 0.45 um (pre-filter) and 0.22 um (main filter) pore size. Such filters such as Sartoclean CA (3.0 um + 0.8 um filter cartridge) and Sartobrain P (0.45 um + 0.2 um filter cartride) were employed. Sartoclean CA mini cartiridges are available with 3.0 um/0.8 um cellulose acetate double membranes and Sartobran Sartorius is available as doulbe filtraiton unit 0.45/0.2um. 

–Second membrane larger MWCO

Becker et al. (US 2015/0274773) discloses processing a crude feed stream to thereby eliminte the need for time consuming impurity precipitation by ombining at elast two TFF unit oeprations. In one embodiment, the first TFF unit was conducted with an UF membrane having a 50 kD cut-off value and a second TFF unit having a 300 kD cut-off. A third unit with a a 50 kD cut-off can also be incorporated. In certain emobdiments, the dual stage TFF methods may also include the membranes with even larger pore sizes such as with sterilization prior to the initial/first TFF unit oepration. Sterizization of load fluids, e.g., cell culture sueprnatants containing proteins of interest vial filtration methods are well known in the art and typically include filtratioon of the sueprnatant through filter membranes having pire sizes ranging form about 0.1-45 um. Accordingly dual stage TFF membhos comprising sterilizaiton of cell culture supernatant prior to the TF unit operations by filtration through a suitable filter membrane having a pore size between 0.1-.45 um is contemplated also. 

Gonzalez (US8,772,461 and WO/2009/129226; see also US14/294460) discloses a method for concentrating a protein such as IgG by ultrafiltering the solution using a first membrane to form a first retentate solution comprising the protein, diafiltering this first retentate with an aqueous solution using the first membrane to form a second retentate solution comprising the protein at about the first concentration, formulating the second retentate having the diafiltered protein with glycine and adjusting the pH and then ultrafiltering the second retentate solution using a second membrane to form a final retentate solution. In one embodiment, the second membrane has a molecular weight cutoff of about twice the MWCO of the first membrane. 

Smider US2006/0088883) discloses that based on a calculated MW, a polypeptide of greate and lesser size can be isolated using UF through membranes of different pore sizes. As a first step, the protein mixture is UF through a membrane with a pore size that has a lower MWCO than the MW of the recombinant catlytic polypeptide or the proteolytic antibody light chain. The retentate of the UF is then UF against a membrane with a MWCO greater than the MW of the recombinatn catalytic polypeptide or the proteolytic antibody light china. The polypeptide will pass through the membrane into the filtrate which can then be processed in a next step of column chromatography.

Wang (“cascade ultrafiltration bioreactor-separator system for continous production of F(ab’2) fragment from immunoglobulin G, J. Membrane Science 2010, 351(1-2), 96-103) discloses a continous two stage UF system for fragmentation of IgG by pepsin and purification of the F(ab’)2 fragment thus generated. In the first stage, a 10 kDa MWCA membrane retains pepsin, IgG and F(ab’)2 while allowing degraded Fc sub fragments through. In the second stage, a 70 kDa mebrane retains both IgG and (F(ab’)2 while allowing pepsin through. Under this method, the pepsin concentration within the first stage is significantly higher than in the feed. The first st6age thus carrys out IgG digestion while the second stage serves to separate pepsin and (F(ab’)2.

Surface Area of Membrane

–Second Membrane Smaller Surface Area; Recirculating Wash buffer:

Teschner (US 12/789345 (now US  8,546,548 and US 13949565, now US 9,175,068 and US 14/855,686, published as US 2016-0244512; see also US/188839, published as US 2019/0085064) disclose a method for concentrating IgG by concentrating a first solution compirsing IgG to 2-10% (w/V) by UF using a first UF, diafiltering the first IgG concentrate comprising the same first UF/DF membrane. concentrating the first IgG diafiltrate using the first UF membrane, collecting this IgG concentrate, washing the first UF by recircualting a wash buffer through the UF, transfering this IgG post wash solution into a second UF/DF comprising a 2nd UF membrane and concentrating it by UF, collecting this IgG concentrate and combining it with the prior IgG concentrate. In one embodiment, the second membrane has a surface area which is less than the frist surface area such as no more than a tenth of the surface area of the first UF membrane. 

First Tank for dilution of Precipitation – First and Second Filtration Units adapted to return first retentate and second retentation or second permeate to frist tank having the precipitate for a final dilution factor

(Menyawi, US 17/054,018, published as US 2021/0246162) disclsoes a clsoed system for extrating a prtoein of interest which includes a first tank adapted to dilute a precipitate to a first dilution factor to form a suspension, receiving the suspension into a first filtraiton unit to produce a first permeate enriched with the protien of itnerest and a first rententate depleted of the protien of interest wherein the first filtraiotn unit si adapted to retun the first retentate to the first tank and a second tank in connected with the first filtraiton unit for recovering the first permeate enriched iwth the protein of interest as well as a second filtraiton unit for concentrating the first permeate in the second tnak which is adapted to produce a seecond retentate enriched with the protien of itnerest and a second permeate depleted of the protien of itnerest wherien the seocnd unit is optionally adapted to return the second retentate and/or the the second permeate to teh frist tank. The clsoed sytem reduces cost of goods such as water, buffers and chemicals needed for the spearation of the protein of interest. 

Temperature

–Elevated Temeprature:

Winter (US 2006/0051347 also published as US 2007/0237762) teaches processes for concentrating proteins such as antibodies by first ultrafiltering and then diafiltering and then a second ultrafiltering whereby one or more of the first ultrafiltering, the second ultrafiltering and the diafiltering are accomplished at elevated termpatures such as 30-50C.

In conjunction with Non-Affinity A Purification Schemes          

UF/DF – AEX:

Kooke (US 14/410562) discloses a method of purifying an antibody composition using UF/DF and then AEX.

ultrafiltration/TFF: 

While diverse methods for preparing and purifying therapeutic MAbs have been developed, they typically have in common a final step of concentration by ultrafiltration that precedes formulation of the final product. TFF is also commonly used for diafiltration and concentration of MAb preparation in the final steps of preparing a highly concentrated MAb solution (WO 2004/001007).

Carbon filtration-AEX-Mixed Mode-Carbon filtration: 

Ishihara (US 13/826195) disoses mAb purification using activated carbon in a non-adsorption mode where a clarified solution is passed through an activated carbon filter (e.g., CUNO Lte., Zeta carbon filter). The resulting activated carbon eluate is applied to an AEX and the eluate is passed through a multimodal chromatography column with the eluate then being passed through an activated carbon filter. 

Carbon filtration-CEX-Activated C-AEX: see Ishihara (US 13/826195)

Non-affinity chromatography/HPTFF:

Fahrner (US 2003/0229212A1) teaches a method for purifying a target protein such as an antibody using an ion exchange chromatography step such as cation or anion exchange and/or mixed mode ion exchange chroatography followed by HPTFF.

Design of the membrane Housing

Design of the membrane housing has been knwon to have significant impact on the efficiency of a membrane based processing step. Conventional membrane housing are in the form of flat sheet cassettes and of hollow fiber and spiral bound capsules. An optimal design would maximize the membrane surface area and the flow rate, keeping the size of the moedule to a minimum so as to reduce holdup and dead spaces and make sanitization and validation easier to perform, resulting in significant savings of coset and process time. Pleated membrane configuration is an approach that allws more surface area in a relatively small membrane moedule. A deep pleat membrane confirugation provides more surface area as compared to the traditional fan pleated membrane contruction. The deep pleated design can provide as much as double surface area and hence can be oeprated at double the flow rate of conventiaonl fan pleated design. Keleenpack capsules from Pall Life Sciences are avialbe with Ultipleat (deep pleat) membrane configuration and have been shown to give good recoveries and high flux rates during clarificaiton of mAbs from CHO and hybridoma cell cultures. (Rathore & Shirke) “Recent Devleopments in Membrane-Based Separation in Biotechnology Processes: Review” Prepative Biochemistry and Biotechnolgy 41: 4, 398-421).