REGENERATION AND MULTIPLE USE OF DEPTH FILTERS

Abstract

Herein is reported a method for the purification or production of a therapeutic polypeptide using the same depth filter multiple times, i.e. a depth filter which has been used before and has been regenerated.

Reported herein is a method for purifying or producing a therapeutic polypeptide, characterized in that the method comprises the following steps: a) filtering an aqueous composition containing said therapeutic polypeptide and impurities through a depth filter, recovering the flow-through and thereby obtaining said purified therapeutic polypeptide, b) contacting said depth filter with a regeneration solution and thereby regenerating the depth filter, and c) repeating steps a) and b) one or more times.

Claims

1. A method for purifying a therapeutic polypeptide, characterized in that the method comprises the following steps: a) filtering an aqueous composition containing said therapeutic polypeptide and impurities through a depth filter, recovering the flow-through and thereby obtaining said purified therapeutic polypeptide, b) contacting said depth filter with an acidic solution and thereby regenerating the depth filter, and c) repeating steps a) and b) one or more times.

2. A method for producing a therapeutic polypeptide, characterized in that the method comprises the following steps: a) filtering an aqueous composition containing said therapeutic polypeptide and impurities through a depth filter, recovering the flow-through and thereby obtaining said therapeutic polypeptide, b) contacting said depth filter (after step a)) with an acidic solution and thereby regenerating the depth filter, and c) repeating steps a) and b) one or more times.

3. The method according to any one of claims 1 to 2, wherein the acidic solution of step b) has a pH value between and including 1 to 3.

4. The method according to claim 1 or 3, wherein the acidic solution of step b) is a solution comprising phosphoric acid.

5. The method according to any one of claims 1 to 4, wherein the acidic solution of step b) is a solution comprising phosphoric acid and acetic acid.

6. The method according to any one of claims 1 to 5, wherein the acidic solution of step b) comprises phosphoric acid in a concentration of about 0.1 M to about 0.8 M, or about 0.2 M to about 0.7 M, or about 0.4 M to 0.6 M.

7. The method according to any one of claims 1 to 6, wherein the acidic solution of step b) comprises acetic acid in a concentration of about 10 mM to 2 M or about 20 mM to 1.5 M or about 50 mM to 1 M, or about 80 mM to 800 mM.

8. The method according to any one of claims 1 to 7, wherein the acidic solution of step b) comprises phosphoric acid in a concentration of about 300 mM and acetic acid in a concentration of about 167 mM.

9. The method according to any of claims 1 to 8, wherein the depth filter comprises silica.

10. The method according to any one of claims 1 to 9, wherein the method reduces the enzymatic hydrolysis activity rate.

11. The method according to any one of claims 1 to 10, wherein the depth filter comprises material that is selected from the group of (i) polyacrylic fiber and silica; (ii) cellulose fibers, diatomaceous earth, and perlite, and (iii) cellulose fiber and charged surface groups.

12. The method according to any one of claims 1 to 11, wherein the depth filter is selected from the group consisting of an X0SP depth filter, or a PDD1 depth filter, or a VR02 depth filter.

13. The method according to any one of claims 1 to 12, wherein the depth filter is contacted with the regeneration solution of step b) for about 20 min or more, 30 min or more, 40 min or more, 50 min or more or 60 min or more.

14. Use of an acidic solution for regeneration of a depth filter that is being used at least two times in the purification of a therapeutic polypeptide.

15. Use of an alkaline solution for regeneration of a depth filter that is being used at least two times in the purification of a therapeutic polypeptide.

Description

DESCRIPTION OF THE FIGURES

[0365] FIG. 1 Reference values (no regeneration) for hydrolytic activity measured by LEAP (see Examples 15, 21); Filter: PDD1; Therapeutic polypeptide: Crovalimab; Marker (thin vertical line): mass throughput of 1320 g/m.sup.2 (binding capacity limit).

[0366] FIG. 2 Hydrolytic activity measured by LEAP for three different settings: 1) grey dotted: Application of water and buffer between the filtration cycles (see Example 9), 2) black dotted: pre-treatment and application of an alkaline solution between the filtration cycles (see Example 10), 3) grey: Application of an acidic solution (see Example 11); Filter: PDD1; Therapeutic polypeptide: Crovalimab; Marker (thin vertical line): mass throughput of 1320 g/m.sup.2 (binding capacity limit).

[0367] FIG. 3 Yield of mainpeak/main product for three different settings: 1) grey dotted: Application of water and buffer between the filtration cycles (see Example 9), 2) black dotted: pre-treatment and application of an alkaline solution between the filtration cycles (see Example 10), 3) grey: Application of an acidic solution (see Example 11); Filter: PDD1; Therapeutic polypeptide: Crovalimab.

[0368] FIG. 4 Reference values (no regeneration) for hydrolytic activity measured by LEAP (see Examples 12, 21); Filter: X0SP; Therapeutic polypeptide: Crovalimab; Marker (thin vertical line): mass throughput of 2640 g/m.sup.2 (binding capacity limit).

[0369] FIG. 5 Hydrolytic activity measured by LEAP for three different settings: 1) grey dotted: Application of water and buffer between the filtration cycles (see Example 6), 2) black dotted: pre-treatment and application of an alkaline solution between the regeneration cycles (see Example 7), 3) grey: Application of an acidic solution between the filtration cycles (see Example 8); Filter: X0SP; Therapeutic polypeptide: Crovalimab; Marker (thin vertical line): mass throughput of 2640 g/m.sup.2 (binding capacity limit).

[0370] FIG. 6 Yield of mainpeak/main product for three different settings: 1) grey dotted: Application of water and buffer between the filtration cycles (see Example 6), 2) black dotted: pre-treatment and application of an alkaline solution between the regeneration cycles (see Example 7), 3) grey: Application of an acidic solution between the filtration cycles (see Example 8); Filter: X0SP; Therapeutic polypeptide: Crovalimab.

[0371] FIG. 7 Reference values (no regeneration) for hydrolytic activity measured by LEAP (see Examples 18, 21); Filter: VR02; Therapeutic polypeptide: Crovalimab; Marker (thin vertical line): mass throughput of 660 g/m.sup.2 (binding capacity limit).

[0372] FIG. 8 Hydrolytic activity measured by LEAP for two different settings: 1) grey dotted: Application of water and buffer between the filtration cycles (see Example 19), 2) black dotted Application of an acidic solution between the filtration cycles (see Example 20); Filter: VR02; Therapeutic polypeptide: Crovalimab; Marker (thin vertical line): mass throughput of 660 g/m.sup.2 (binding capacity limit)

[0373] FIG. 9 Yield of mainpeak/main product for two different settings: 1) grey dotted: Application of water and buffer between the filtration cycles (see Example 19), 2) black dotted Application of an acidic solution between the filtration cycles (see Example 20); Filter: VR02; Therapeutic polypeptide: Crovalimab.

EXAMPLES

Overview

[0374]

TABLE-US-00001 Example antibody Filter type Regeneration 1 bispecific anti-GPRC5D X0SP 1M NaCl, 0.5M NaOH antibody (no pre-treatment) 2 bispecific anti-CD20/TfR X0SP 1M NaCl, 0.5M NaOH antibody (no pre-treatment) 3 bispecific anti-CD20/TfR X0SP 167 mM acetic acid, 300 antibody mM phosphoric acid (no pre-treatment) 4 antibody-IL2 fusion X0SP 167 mM acetic acid, 300 polypeptide mM phosphoric acid (no pre-treatment) 5 antibody-IL2 fusion PDD1 167 mM acetic acid, 300 polypeptide mM phosphoric acid (no pre-treatment) 6 anti-C5 antibody X0SP Water and Buffer (no pre- treatment) 7 anti-C5 antibody X0SP 1M NaOH for pre- treatment and regeneration 8 anti-C5 antibody X0SP 0.5M phosphoric acid (no pre-treatment) 9 anti-C5 antibody PDD1 Water and Buffer (no pre- treatment) 10 anti-C5 antibody PDD1 1M NaOH for pre- treatment and regeneration 11 anti-C5 antibody PDD1 0.5M phosphoric acid (no pre-treatment) 12 anti-C5 antibody X0SP without (reference) 13 anti-C5 antibody X0SP 167 mM acetic acid, 300 mM phosphoric acid (no pre-treatment 14 anti-C5 antibody X0SP 1M NaOH pre-treatment, Water and Buffer regeneration 15 anti-C5 antibody PDD1 without (reference) 16 anti-C5 antibody PDD1 167 mM acetic acid, 300 mM phosphoric acid (no pre-treatment) 17 anti-C5 antibody PDD1 167 mM acetic acid, 300 mM phosphoric acid, Buffer (w/o water flush; no pre-treatment) 18 anti-C5 antibody VR02 without (reference) 19 anti-C5 antibody VR02 Water and Buffer (no pre- treatment) 20 anti-C5 antibody VR02 167 mM acetic acid, 300 mM phosphoric acid (no pre-treatment)

[0375] Materials and Methods

[0376] Antibodies:

[0377] General information regarding the nucleotide sequences of human immunoglobulins light and heavy chains is given in: Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). Amino acids of antibody chains are numbered and referred to according to numbering according to Kabat (Kabat, E. A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

[0378] The current invention is exemplified using a number of exemplary antibodies, including: an anti-CD20/TfR bispecific antibody as reported in WO 2017/055542 and therein in SEQ ID NO: 01 to 03 and 10; an anti-C5 antibody (Crovalimab) as reported in WO2017/104779 and therein in SEQ ID NO: 106 to 111; a fusion protein comprising an inert antibody conjugated at the C-terminus of the heavy chain to a human IL-2 (interleukin 2) as reported in WO2015/118016 and therein in SEQ ID NO: 19 and 50; an anti-GPRC5D/anti-CD3 bispecific antibody in 2+1 format as reported in WO2021/018859A2.

[0379] Synthetic Depth Filter Media:

[0380] Herein the synthetic depth filter Millistak+® HC Pro X0SP is used. It is commercially available from MilliporeSigma (Bedford, MA). The X0SP depth filter has a nominal pore size rating of 0.1 microns and is intended for secondary clarification applications.

[0381] A cellulose/diatomaceous earth-comprising (also containing silica) PDD1 depth filter (Pall PDD1 SUPRAcap™-50 (SC050PDD1)), was also used.

[0382] A cellulose-comprising (also containing silica) VR02 depth filter (Zeta Plus™ Biocap VR02) was also used.

[0383] Depth Filtration Device:

[0384] All testing was performed using 22 cm2, 23 cm.sup.2 or 25 cm2 μPod1 scale devices.

[0385] The depth filter devices were fabricated using two layers of the depth filtration media encapsulated in single-use, over-molded device housing.

[0386] Recombinant DNA Techniques:

[0387] Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular Cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. The molecular biological reagents were used according to the manufacturer's instructions.

[0388] Gene Synthesis:

[0389] Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The long gene segments, which were flanked by singular restriction endonuclease cleavage sites, were assembled by annealing and ligating oligonucleotides including PCR amplification and subsequently cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. Gene synthesis fragments were ordered according to given specifications at Geneart (Regensburg, Germany).

[0390] DNA Sequence Determination:

[0391] DNA sequences were determined by double strand sequencing performed at MediGenomix GmbH (Martinsried, Germany) or SequiServe GmbH (Vaterstetten, Germany).

[0392] DNA and protein sequence analysis and sequence data management: The GCG's (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infomax's Vector NT1 Advance suite version 8.0 was used for sequence creation, mapping, analysis, annotation and illustration.

[0393] Expression Vectors:

[0394] For the expression of the described bispecific antibodies, expression plasmids for transient expression (e.g. in HEK293 cells) based either on a cDNA organization with or without a CMV-intron A promoter or on a genomic organization with a CMV promoter can be applied.

[0395] Beside the antibody expression cassette the vectors contain: [0396] an origin of replication which allows replication of this plasmid in E. coli, and [0397] a ß-lactamase gene which confers ampicillin resistance in E. coli.

[0398] The transcription unit of the antibody gene is composed of the following elements: [0399] unique restriction site(s) at the 5′ end [0400] the immediate early enhancer and promoter from the human cytomegalovirus, [0401] the intron A sequence in the case of cDNA organization, [0402] a 5′-untranslated region derived from a human antibody gene, [0403] an immunoglobulin heavy chain signal sequence, [0404] the respective antibody chain encoding nucleic acid either as cDNA or with genomic exon-intron organization, [0405] a 3′ untranslated region with a polyadenylation signal sequence, [0406] a terminator sequence, and [0407] unique restriction site(s) at the 3′ end.

[0408] The fusion genes encoding the antibody chains are generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective vectors. The subcloned nucleic acid sequences are verified by DNA sequencing. For transient transfections larger quantities of the plasmids are prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).

[0409] For all constructs knob-into-hole heterodimerization technology was used with a typical knob (T366W) substitution in the first CH3 domain and the corresponding hole substitutions (T366S, L368A and Y407V) in the second CH3 domain (as well as two additional introduced cysteine residues S354C/Y349′C) (contained in the respective corresponding heavy chain (HC) sequences depicted above).

[0410] Cell Culture Techniques:

[0411] Standard cell culture techniques as described in Current Protocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.), John Wiley & Sons, Inc., are used.

[0412] Transient Transfections in HEK293 System:

[0413] The bispecific antibodies are produced by transient expression. Therefore a transfection with the respective plasmids using the HEK293 system (Invitrogen) according to the manufacturer's instruction is done. Briefly, HEK293 cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle™ 293 expression medium (Invitrogen) are transfected with a mix of the respective expression plasmids and 293Fectin™ or fectin (Invitrogen). For 2 L shake flask (Corning) HEK293 cells are seeded at a density of 1.0*10.sup.6 cells/mL in 600 mL and incubated at 120 rpm, 8% CO.sub.2. On the next day the cells are transfected at a cell density of approx. 1.5*10.sup.6 cells/mL with approx. 42 mL of a mixture of A) 20 mL Opti-MEM medium (Invitrogen) comprising 600 μg total plasmid DNA (1 μg/mL) and B) 20 ml Opti-MEM medium supplemented with 1.2 mL 293 fectin or fectin (2 μl/mL). According to the glucose consumption glucose solution is added during the course of the fermentation. The supernatant containing the secreted antibody is harvested after 5-10 days and antibodies are either directly purified from the supernatant or the supernatant is frozen and stored.

[0414] Optical Density Determination:

[0415] The protein concentration of purified antibodies and derivatives was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence according to Pace, et al., Protein Science 4 (1995) 2411-1423.

[0416] Protein Concentration Determination (yield):

[0417] Photometric Determination:

[0418] Protein concentrations were determined by UV spectroscopy using a Cary® 50 UV-Vis Spectrophotometer (Varian). Protein samples were diluted in their respective buffers and measured as duplicates. Concentrations were determined according to the following equation deriving from Lambert-Beer law: c=(A.sub.280 nm-A.sub.320 nm)/ε.Math.d. F with c protein concentration [mg/ml], A absorbance, ε extinction coefficient [ml/(mg.Math.cm)], d cell length [cm] and F dilution factor. The anti-C5 antibody-specific extinction coefficient is 1.44 ml/(mg.Math.cm), the bispecific anti-GPRC5D antibody specific extinction coefficient is 1.43 ml/(mg.Math.cm), the bispecific anti-CD20/TfR specific extinction coefficient is 1.57 ml/(mg.Math.cm) and the antibody-IL2 fusion polypeptide specific extinction coefficient is 1.25 ml/(mg.Math.cm).

[0419] Chromatographic Determination:

[0420] The concentration of the antibodies was quantitatively measured by affinity HPLC chromatography. Briefly, solution containing antibodies that bind to protein A are applied, e.g., to an Applied Biosystems Poros A/20 column in 200 mM KH.sub.2PO.sub.4, 100 mM sodium citrate, pH 7.4 and eluted with 200 mM NaCl, 100 mM citric acid, pH 2.5 on an Agilent HPLC 1100 system. The eluted antibody is quantified by UV absorbance and integration of peak areas. A purified standard IgG1 antibody served as a standard.

[0421] ELISA Determination:

[0422] Alternatively, the concentration of antibodies and derivatives in solutions is measured by Sandwich-IgG-ELISA. Briefly, StreptaWell High Bind Streptavidin A-96 well microtiter plates (Roche Diagnostics GmbH, Mannheim, Germany) are coated with 100 μL/well biotinylated anti-human IgG capture molecule F(ab′)2<h-FcT>BI (Dianova) at 0.1 μg/mL for 1 hour at room temperature or alternatively overnight at 4° C. and subsequently washed three times with 200 μL/well PBS, 0.05% Tween (PBST, Sigma). Thereafter, 100 L/well of a dilution series in PBS (Sigma) of the respective antibody containing solution is added to the wells and incubated for 1-2 hour on a shaker at room temperature. The wells are washed three times with 200 μL/well PBST and bound antibody is detected with 100 μl F(ab′)2<hFcγ>POD (Dianova) at 0.1 μg/mL as the detection antibody by incubation for 1-2 hours on a shaker at room temperature. Unbound detection antibody is removed by washing three times with 200 μL/well PBST. The bound detection antibody is detected by addition of 100 μL ABTS/well followed by incubation. Determination of absorbance is performed on a Tecan Fluor Spectrometer at a measurement wavelength of 405 nm (reference wavelength 492 nm).

[0423] Preparative Antibody Purification:

[0424] Antibodies were purified from filtered cell culture supernatants referring to standard protocols. In brief, antibodies were applied to a protein A Mab Select SuRe column (GE healthcare) and washed with buffer. Elution of antibodies was achieved at low pH followed by immediate neutralization. Antibody fractions were pooled, frozen and stored at −20° C., −40° C. or −80° C.

[0425] Hydrolytic Activity Determination—Lipase Activity Assay (LEAP):

[0426] The lipase activity was determined by monitoring the conversion of a substrate, such as a nonfluorescent substrate, to a detectable product of the hydrolytic enzyme, such as a fluorescent product. An exemplary method is described, e.g., in WO 2018/035025, which is hereby incorporated by reference in its entirety.

[0427] In more detail, with the LEAP assay hydrolase activity in samples was determined.

[0428] This was done by monitoring the conversion of a fluorogenic substrate ‘4-Methylumbelliferyl Caprylate’ (4-MU-C8, available from Chem Impex Int'l Inc Art. Nr. 01552) by cleavage of the ester bond by hydrolases present in the sample into a fluorescent moiety, i.e. 4-Methylumbelliferyl (4-MU). Cleaved 4-MU-C8, i.e. 4-MU, was excited with a light of wavelength 355 nm. The emitted radiation at a different wavelength of 460 nm was recorded on Tecan Infinite® 200 PRO device as readout. The determination was performed at 37° C. for 2 hours with recording every 10 mins to calculate the rate of substrate hydrolysis.

[0429] The sample to be analyzed was at first buffer exchanged to 150 mM Tris-Cl, pH 8.0, by using Amicon Ultra-0.5 ml centrifugal filter units (10,000 Da cut-off, Merck Millipore, Art. Nr. UFC501096). The assay reaction mixture constituted of 80 μL reaction buffer (150 mM Tris-Cl, pH 8.0, 0.25% (w/v) Triton X-100, 0.125% (w/v) Gum Arabic), 10 μL 4-MU-C8 substrate solution (1 mM in DMSO), and 10 μL protein containing sample. The protein samples' concentration were adjusted to be in the range between 1-30 g/L and 2-3 dilution series were performed for each determination. Each reaction was set up at least in duplicates in 96-well half-area polystyrene plates (black with lid and clear flat bottom, Corning Incorporated Art. Nr. 3882).

[0430] Host Cell Protein (CHOP) Determination:

[0431] The residual CHO HCP content in process samples is determined by an electrochemiluminescence immunoassay (ECLIA) on cobas e 411 immunoassay analyzer (Roche Diagnostics).

[0432] The assay is based on a sandwich principle using polyclonal anti-CHO HCP antibody from sheep.

[0433] First incubation: Chinese hamster ovary host cell protein (CHO HCP) from 15 μL sample (neat and/or diluted) and a biotin conjugated polyclonal CHO HCP specific antibody form a sandwich complex, which becomes bound to streptavidin-coated microparticles via interaction of biotin with streptavidin.

[0434] Second incubation: After addition of polyclonal CHO HCP-specific antibody labeled with ruthenium complex (Tris(2,2′-bipyridyl)ruthenium(II)-complex) a ternary sandwich complex is formed on the microparticles.

[0435] The reaction mixture is aspirated into the measuring cell where the microparticles are magnetically captured onto the surface of the electrode. Unbound substances are then removed in a washing step. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier.

[0436] The concentration of CHO HCP in the test sample is finally calculated from a CHO HCP standard curve of known concentration.

[0437] Host Cell DNA Determination:

[0438] The residual Chinese Hamster Ovary (CHO) deoxyribonucleic acid (DNA) in process samples is determined on FLOW FLEX System (Roche Diagnostics GmbH, Mannheim, Germany).

[0439] The FLOW FLEX System consists of three modules: FLOW PCR SETUP Instrument, MagNA Pure 96 Instrument and LightCycler®480.

[0440] The FLOW PCR SETUP Instrument module is used as PSH (FLOW Primary Sample Handling) for sample transfer from primary tubes into a 96 well processing plate, and as PSU (FLOW PCR SETUP Instrument) for transfer of extracted DNA from the 96 well output plate into the PCR plate.

[0441] The MagNA Pure 96 Instrument module is used for automated isolation of nucleic acids. To release the DNA, the sample material is incubated under denaturing conditions. The released DNA is separated from the other buffer and sample components by binding to magnetic glass particles via a magnet, and the bound DNA is then eluted with buffer. Up to 96 samples can be processed simultaneously.

[0442] The LightCycler®480 module (microplate LightCycler®) is used for quantification of DNA or RNA based on PCR technology. The Residual DNA CHO Kit uses specific PCR of highly conserved regions within the DNA of CHO. The highly specific forward primers and reverse primers bind specifically to the ends of the target sequence of single-stranded DNA. The CHO DNA probe, labeled with a fluorescent reporter dye (FAM) at the 5′ end and a quencher dye at the 3′ end, hybridizes between the primers and the target sequence of single-stranded DNA. As long as the probe is intact, the proximity of the Quencher dye suppresses the fluorescence of the reporter dye. Upon amplification, the Taq polymerase, due to its 5′.fwdarw.3′exonuclease activity, disrupts the probe attached to the target sequence. This releases the reporter dye and the fluorescence increases. The increase in fluorescence is directly proportional to the amount of PCR product. The amount of CHO DNA in the samples is quantified with a standard curve.

[0443] Size Exclusion High Performance Liquid Chromatography (SE-HPLC):

[0444] Size exclusion chromatography (SEC) for the determination of the aggregation and oligomeric state of antibodies was performed by HPLC chromatography. Briefly, protein A purified antibodies were applied to a Tosoh TSK-Gel G3000SWXL (7.8×300 mm; 5 μm (TOSOH Bioscience Nr. 08541)) in 250 mM KCl, 200 mM KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 buffer (pH 7.0) on an Dionex Ultimate® system (Thermo Fischer Scientific). The eluted antibody was quantified by UV absorbance and integration of peak areas. BioRad Gel Filtration Standard 151-1901 served as a standard.

[0445] MCE (Caliper):

[0446] Purity and antibody integrity were analyzed by CE-SDS using microfluidic Labchip technology (PerkinElmer, USA). Therefore, 5 μl of antibody solution was prepared for CE-SDS analysis using the HT Protein Express Reagent Kit according manufacturer's instructions and analyzed on LabChip GXII system using a HT Protein Express Chip. Data were analyzed using LabChip GX Software.

Example 1

[0447] Filtration of a T-Cell Bispecific Anti-GPRC5D Antibody Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with Alkaline Treatment

[0448] Materials: [0449] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0450] 2) Millipore Millistak+® HC Pro X0SP filter with 23 cm.sup.2 filter area; Lot.: CP8MA89804 [0451] 3) equilibration buffer: 150 mM acetic acid/tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0452] 4) alkaline regeneration solution: 1 M NaCl, 0.5 M NaOH, pH 12.6

[0453] Filter Conditioning:

[0454] Following steps were performed sequentially: [0455] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0456] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[0457] Experimental Setup:

[0458] Following steps were performed sequentially: [0459] 1) An intermediate “Affinity Pool pH 5.5” containing the T-cell bispecific antibody was applied to the conditioned 23 cm.sup.2×0SP filter unit. The mass throughput was 600 g/m.sup.2. The corresponding calculated volume throughput was 52.17 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated loading flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0460] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0461] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0462] 4) The flushing flowthrough was collected until the OD280 value dropped below 0.5 AU (1 cm UV cell). Thereafter the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0463] 5) Thereafter, the alkaline regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (200 LMH)). Thus, the contact time was approx. 30 minutes. [0464] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value above 10 for approximately 50 minutes. [0465] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0466] 8) Steps 1) to 7) were repeated for further nine product filtration cycles (steps 1) to 4)) and further eight regeneration cycles (steps 5) to 7)). In total, 6 kg/m.sup.2 (10×0.6 kg/m.sup.2) antibody were applied to the filter. [0467] 9) The separate final filtration pools were kept for analysis at −80° C.

[0468] Analyses and Results:

[0469] The following analyses were performed with the respective final filtration pools: [0470] protein concentration (using the absorbance of 1.43 at 1 mg/ml as reference) [0471] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0472] LEAP (Lipase Activity Assay; hydrolytic activity) [0473] CHOP values (host cell proteins) by cobas_HCP [0474] DNA values (host cell DNA) by qPRC_DNA [0475] SE-HPLC (size exclusion HPLC) [0476] MCE (Caliper)

[0477] The results are shown in the following Table X-1:

TABLE-US-00002 Final SEC SEC Filtrat Photo- Area- [LMW Vol. Yield Conc. SEC [MainPeak Area- DNA cobas_HCP Filtration [mL] [%] [g/L] [HMW  Area-%] %] [ppb] [ppm] Load not not 11.57 7.6 80.0 12.4 2212.62 15417 X0SP applicable applicable #1 334.35 81.06 3.51 4.4 92.6 3.0 6.05 1111 w/o Reg. #2 331.3 76.57 3.21 4.6 94.6 0.8 0.25 196 #3 329.2 68.28 2.88 4.6 94.3 1.1 2.78 162 #4 298.51 50.92 2.37 3.6 83.0 13.3 3.38 132 #5 323.74 72.87 3.13 2.9 83.9 13.3 9.22 207 #6 330.0 84.21 3.54 3.5 83.7 12.8 7.62 496 #7 331.28 87.27 3.66 3.8 83.3 12.9 7.98 881 #8 331.65 89.17 3.73 3.8 83.3 12.9 10.34 1117 #9 331.51 88.68 3.71 3.8 83.3 12.9 3.42 1352 #10 331.33 89.06 3.73 3.9 83.2 12.9 10.99 1532 LEAP MCE MCE Average LEAP [HMW [MainPeak MCE Converted Rate [hydrolytic Filtration %-CPA] %-CPA] [LMW % CPA] [μM MU/h] activity %] Load 1.0 69.5 29.5 7.9 100.0% X0SP #1 1.1 81.0 17.9 2.3  29.1% w/o Reg. #2 1.1 86.4 12.6 1.2  15.2% #3 1.6 86.8 11.7 1.0  12.7% #4 0.9 72.8 26.2 1.4  17.7% #5 0.5 73.0 26.5 N/A N/A #6 0.6 71.4 28.0 2.2  27.8% #7 0.8 70.3 28.9 2.2  27.8% #8 1.0 70.8 28.3 2.5  31.6% #9 0.9 69.8 29.2 N/A N/A #10 1.0 69.7 29.2 2.4  30.4% Filtration Mass Mainpeak [mg] Yield Mainpeak [%] Load X0SP 1157.30 100.0% #1 1086.67  93.9% w/o Reg. #2 1006.48  87.0% #3 893.62  77.2% #4 587.50  50.8% #5 849.69  73.4% #6 977.28  84.4% #7 1010.42  87.3% #8 1030.79  89.1% #9 1024.26  88.5% #10 1028.16  88.8% N/A = not analyzed indicates data missing or illegible when filed

Example 2

[0478] Filtration of a Bispecific Anti-CD20/TfR Antibody Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with Alkaline Treatment

[0479] Materials: [0480] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0481] 2) Millipore Millistak+® HC Pro X0SP filter with 23 cm.sup.2 filter area; Lot.: CP0BB08624 [0482] 3) equilibration buffer: 40 mM sodium acetate, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0483] 4) alkaline regeneration solution: 1 M NaCl, 0.5 M NaOH, pH 12.6

[0484] Filter Conditioning:

[0485] Following steps were performed sequentially: [0486] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0487] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[0488] Experimental Setup:

[0489] Following steps were performed sequentially: [0490] 1) An intermediate “Affinity Pool pH 5.5” containing the bispecific anti-CD20/TfR antibody was applied to the conditioned 23 cm.sup.2 X0SP filter unit. The mass throughput was 800 g/m.sup.2. The corresponding calculated volume throughput was 100 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated loading flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0491] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell]. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0492] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0493] 4) The flushing flowthrough was collected until OD280 was above 0.5 AU [1 cm UV cell]. When the OD280 value dropped below 0.5 AU (1 cm UV cell), the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0494] 5) Thereafter, the alkaline regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (200 LMH)). Thus, the contact time was approx. 30 minutes. [0495] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value above 10 for approximately 50 minutes. [0496] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0497] 8) Steps 1) to 7) were repeated for further four product filtration cycles (steps 1) to 4)) and further three regeneration cycles (steps 5) to 7)). In total, 4 kg/m.sup.2 (5×0.8 kg/m.sup.2) antibody were applied to the filter. [0498] 9) The separate final filtration pools were kept for analysis at −80° C.

[0499] Analyses and Results:

[0500] The following analyses were done: [0501] protein concentration (using the absorbance of 1.57 at 1 mg/ml as reference) [0502] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0503] LEAP (Lipase Activity Assay; hydrolytic activity) [0504] CHOP values (host cell proteins) by cobas_HCP [0505] DNA values (host cell DNA) by qPRC_DNA [0506] SE-HPLC (size exclusion HPLC)

[0507] The results are shown in the following Table X-2:

TABLE-US-00003 Final Filtrat Photo- SEC SEC SEC Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-% ] Area-% ] Area-%] Load not not 8.07 13.9 81.0 5.11 X0SP applicable applicable #1 295.83 87.69 5.45 6.3 89.2 4.5 w/o Reg. #2 281.21 78.69 5.15 6.6 89.8 3.6 #3 429.12 82.05 3.52 5.0 90.7 4.3 #4 458.05 92.21 3.70 7.0 87.3 5.7 #5 449.38 92.91 3.80 7.4 86.7 6.0 #6 442.34 93.73 3.90 7.5 86.6 5.9 #7 440.08 94.02 3.93 7.6 86.5 5.9 #8 437.41 94.09 3.96 7.5 86.2 6.4 LEAP Average Converted LEAP Rate [μM [hydrolytic Filtration MU/h] activity %] DNA [ppb] cobas_HCP [ppm] Load X0SP 15.6 100.0% 109.54 18388 #1 10.6  67.9% 1.93 849 w/o Reg.   #2 5.9  37.8% 1.55 645 #3 11.1  71.2% 2.27 1384 #4 12.8  82.1% 3.10 3261 #5 13.0  83.3% 4.70 3978 #6 12.4  79.5% 5.59 4417 #7 12.7  81.4% 5.80 4718 #8 12.7  81.4% 5.36 4750 Filtration Mass Mainpeak [mg] Yield Mainpeak [%] Load X0SP 1517.5 100.0% #1 1438.8  94.8% w/o Reg. #2 1300.3  85.7% #3 1369.5  90.2% #4 1480.3  97.5% #5 1480.4  97.6% #6 1494.3  98.5% #7 1495.3  98.5% #8 1493.0  98.4%

Example 3

[0508] Filtration of a Bispecific Anti-CD20/TfR Antibody Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with an Acidic Treatment

[0509] Materials: [0510] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0511] 2) Millipore Millistak+® HC Pro X0SP filter with 23 cm.sup.2 filter area; Lot.: CP0BB08624 [0512] 3) equilibration buffer: 40 mM sodium acetate, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0513] 4) acidic regeneration solution: 167 mM acidic acid, 300 mM phosphoric acid, pH 1.34.

[0514] Filter Conditioning:

[0515] Following steps were performed sequentially: [0516] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0517] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[0518] Experimental Setup:

[0519] Following steps were performed sequentially: [0520] 1) An intermediate “Affinity Pool pH 5.5” containing the bispecific anti-CD20/TfR antibody was applied to the conditioned 23 cm.sup.2 X0SP filter unit. The mass throughput was 814 g/m.sup.2. The corresponding calculated volume throughput was 100 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated loading flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0521] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0522] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0523] 4) The flushing flowthrough was collected until the OD280 value dropped below 0.5 AU (1 cm UV cell). Thereafter the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0524] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (200 LMH)). Thus, the contact time was approx. 30 minutes. [0525] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [0526] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0527] 8) Steps 1) to 7) were repeated for further four product filtration cycles (steps 1) to 4)) and further three regeneration cycles (steps 5) to 7)). In total, 4.07 kg/m.sup.2 (5×0.814 kg/m.sup.2) antibody were applied to the filter. [0528] 9) The separate final filtration pools were kept for analysis at −80° C.

[0529] Analyses and Results:

[0530] The following analyses were performed with the respective final filtration pools: [0531] protein concentration (using the absorbance of 1.57 at 1 mg/ml as reference) [0532] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0533] LEAP (Lipase Activity Assay; hydrolytic activity) [0534] CHOP values (host cell proteins) by cobas_HCP [0535] DNA values (host cell DNA) by qPRC_DNA [0536] SE-HPLC (size exclusion HPLC)

[0537] The results are shown in the following Table X-3:

TABLE-US-00004 Final Filtrat Photo- SEC SEC SEC Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-%] Area-% ] Area-%] Load X0SP not not 8.22 0.67 79.87 4.79 applicable applicable #1 w/o Reg. 310.23 85.5 5.16 0.54 88.794 3.96 #2 314.92 87.36 5.2 0.57 88.491 4.04 #3 307.17 87.07 5.31 0.57 88.235 4.12 #4 306.86 88.12 5.38 0.57 88.11 4.09 #5 310.83 88.19 5.31 0.61 87.838 4.16 LEAP Average Converted LEAP Rate [μM [hydrolytic Filtration MU/h] activity %] DNA [ppb] cobas_HCP [ppm] Load X0SP 15.60 100.0% 276.31 17340 #1 w/o Reg. 10.00  64.1% 1.55 992 #2 10.20  65.4% 1.68 895 #3 10.20  65.4% 2.21 930 #4 10.60  67.9% 2.04 1043 #5 10.70  68.6% 2.27 1115 Filtration Mass Mainpeak [mg] Yield Mainpeak [%] Load X0SP 1497.00  100% #1 w/o Reg. 1421.40 95.0% #2 1449.11 96.8% #3 1439.18 96.1% #4 1454.61 97.2% #5 1449.77 96.8%

Example 4

[0538] Filtration of an IgG Antibody IL-2 Fusion Protein Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with an Acidic Treatment

[0539] Materials: [0540] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0541] 2) Millipore Millistak+® HC Pro X0SP filter with 23 cm.sup.2 filter area; Lot.: CP0BB08624 [0542] 3) equilibration buffer: 150 mM acidic acid/tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0543] 4) acidic regeneration solution: 167 mM acidic acid, 300 mM phosphoric acid, pH 1.34.

[0544] Filter Conditioning:

[0545] Following steps were performed sequentially: [0546] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0547] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0548] Following steps were performed sequentially: [0549] 1) An intermediate “Affinity Pool pH 5.5” containing the IgG-IL2 antibody fusion protein was applied to the conditioned 23 cm.sup.2 X0SP filter unit. The mass throughput was 548 g/m.sup.2. The corresponding calculated volume throughput was 56 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated loading flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0550] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0551] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0552] 4) The flushing flowthrough was collected until the OD280 value dropped below 0.5 AU (1 cm UV cell). Thereafter the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0553] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (200 LMH)). Thus, the contact time was approx. 30 minutes. [0554] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [0555] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0556] 8) Steps 1) to 7) were repeated for further five product filtration cycles (steps 1) to 4)) and further four regeneration cycles (steps 5) to 7)). In total, 2.288 kg/m.sup.2 (6×0.548 kg/m.sup.2) fusion protein was applied to the filter. [0557] 9) The separate final filtration pools were kept for analysis at −80° C.

[0558] Analyses and Results:

[0559] The following analyses were performed with the respective final filtration pools: [0560] protein concentration (using the absorbance of 1.25 at 1 mg/ml as reference) [0561] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0562] LEAP (Lipase Activity Assay; hydrolytic activity) [0563] CHOP values (host cell proteins) by cobas_HCP [0564] DNA values (host cell DNA) by qPRC_DNA [0565] SE-HPLC (size exclusion HPLC)

[0566] The results are shown in the following Table X-4:

TABLE-US-00005 Final Photo- SEC SEC SEC Filtrat Yield Conc. [HMW [MainPeak [LMW Filtration Vol. [mL] [%] [g/L] Area-%] Area-% ] Area-%] Load not not 9.86 6.64% 93.30% 0.05% X0SP applicable applicable #1 w/o Reg. 204.76 81.48% 5.02 2.38% 97.60% 0.02% #2 212.09 86.59% 5.15 2.52% 97.43% 0.05% #3 214.61 87.12% 5.12 2.63% 97.33% 0.04% #4 210.51 87.74% 5.25 2.78% 97.16% 0.06% #5 211.18 87.74% 5.24 2.95% 97.00% 0.04% #6 216.11 88.47% 5.17 2.82% 97.15% 0.03% LEAP Average Converted LEAP Rate [μM [hydrolytic Filtration MU/h] activity %] DNA [ppb] cobas_HCP [ppm] Load X0SP 9.1 100% 1428 7315 #1 w/o Reg. 1.9  21% 1.59 17 #2 2.1  23% 1.55 20 #3 2.3  25% 1.56 23 #4 2.1  23% 1.52 24 #5 2.0  22% 1.53 31 #6 2.3  25% 1.55 33 Filtration Mass Mainpeak [mg] Yield Mainpeak [%] Load X0SP 1,287.91 100% #1 w/o Reg. 1,003.23  78% #2 1,064.19  83% #3 1,069.47  83% #4 1,073.79  83% #5 1,073.39  83% #6 1,085.45  84%

Example 5

[0567] Filtration of an IgG-IL2 Fusion Polypeptide Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment

[0568] Materials: [0569] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0570] 2) Pall PDD1 SUPRAcap 50 (SC050PDD1) filter with 22 cm.sup.2 filter area; Lot.: 103864042 [0571] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0572] 4) acidic regeneration solution: 167 mM acetic acid, 300 mM phosphoric acid, pH 1.34

[0573] Filter Conditioning:

[0574] Following steps were performed sequentially: [0575] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0576] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0577] Following steps were performed sequentially: [0578] 1) An intermediate “Affinity Pool pH 5.5” containing the IgG-IL2 fusion polypeptide was applied to the conditioned 22 cm.sup.2 PDD1 filter unit. The mass throughput was 547 g/m.sup.2. The corresponding calculated volume throughput was 55 L/m.sup.2. The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated loading flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0579] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0580] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0581] 4) The flushing flowthrough was collected until the OD280 value dropped below 0.5 AU (1 cm UV cell). Thereafter the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0582] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (191 LMH)). Thus, the contact time was approx. 30 minutes. [0583] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [0584] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0585] 8) Steps 1) to 7) were repeated for further five product filtration cycles (steps 1) to 4)) and further four regeneration cycles (steps 5) to 7)). In total, 3.282 kg/m.sup.2 (6×0.547 kg/m.sup.2) fusion protein was applied to the filter. [0586] 9) The separate final filtration pools were kept for analysis at −80° C.

[0587] Analyses and Results:

[0588] The following analyses were performed with the respective final filtration pools: [0589] protein concentration (using the absorbance of 1.25 at 1 mg/ml as reference) [0590] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0591] LEAP (Lipase Activity Assay; hydrolytic activity) [0592] CHOP values (host cell proteins) by cobas_HCP [0593] DNA values (host cell DNA) by qPRC_DNA [0594] SE-HPLC (size exclusion HPLC)

[0595] The results are shown in the following Table X-5:

TABLE-US-00006 Final Filtrat Photo- Vol. Yield Conc. Filtration [mL] [%] [g/L] Load not not 9.86 PDD1 applicable applicable #1 w/o 164.28 83.73 6.13 Reg. #2 155.72 90.3 6.97 #3 154.46 92.15 7.17 #4 155.12 91.91 7.12 #5 156 92.28 7.11 #6 154.6 91.85 7.14 LEAP Average Converted LEAP Rate [μM [hydrolytic cobas_HCP Filtration MU/h] activity %] DNA [ppb] [ppm] Load PDD1 9.1 100% 1428 7315 #1 w/o Reg. 4.7  52% 310.00 1299 #2 3.4  37% 58.22 816 #3 3.4  37% 69.97 898 #4 3.7  41% 81.68 962 #5 3.9  43% 94.75 1055 #6 3.9  43% 138.85 1106

Example 6

[0596] Filtration of Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter Unit, with Water and Buffer Application (without Alkaline or Acidic Filter Regeneration)—Comparative Example

[0597] Materials: [0598] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0599] 2) Millipore Millistak+® HC Pro X0SP filter with 23 cm.sup.2 filter area; Lot.: CP0BB08624 [0600] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0601] 4) intermediate solution: water and equilibration buffer

[0602] Filter Conditioning:

[0603] Following steps were performed sequentially: [0604] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0605] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0606] Following steps were performed sequentially: [0607] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 23 cm.sup.2 X0SP filter unit. The mass throughput was 600 g/m.sup.2. The corresponding calculated volume throughput was 41.3 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated loading flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0608] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0609] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0610] 4) The flowthrough of the filter was collected for 70 L/m.sup.2. When the flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool was obtained. [0611] 5) To prepare the filter for the next filtration cycle, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. [0612] 6) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0613] 7) Steps 1) to 6) were repeated for further nine product filtration cycles without harsh regeneration solution between the filtration cycles. In total, 6.0 kg/m.sup.2 (10×0.6 kg/m.sup.2) antibody were applied to the filter. [0614] 8) The separate final filtration pools were kept for analysis at −80° C.

[0615] Analyses and Results:

[0616] The following analyses were performed with the respective final filtration pools: [0617] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0618] total product yield, calculated out ofloaded mass, final filtration pool volume and final filtration pool protein concentration [0619] LEAP (Lipase Activity Assay; hydrolytic activity) [0620] CHOP values (host cell proteins) by cobas_HCP [0621] DNA values (host cell DNA) by qPRC_DNA [0622] SE-HPLC (size exclusion HPLC) [0623] MCE (Caliper)

[0624] The results are shown in the following Table X-6:

TABLE-US-00007 Final Filtration Pool Photo- SEC SEC SEC Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-%] Area-% ] Area-%] Load not not 14.53 5.35 93.66 0.99 X0SP applicable applicable #1 235.13 87.15% 5.12 1.22 98.11 0.67 w/o Reg. #2 237.54 90.23% 5.24 1.45 97.80 0.76 #3 238.78 90.68% 5.24 1.39 97.85 0.75 #4 239.49 93.07% 5.36 1.67 97.58 0.75 #5 240.01 92.98% 5.35 2.27 95.80 0.94 #6 240.01 93.72% 5.39 3.78 95.28 0.94 #7 240.64 94.68% 5.43 4.03 95.01 0.96 #8 240.84 95.10% 5.45 4.20 94.84 0.96 #9 241.44 95.12% 5.44 4.29 94.87 0.84 #10 241.64 95.53% 5.46 4.42 94.61 0.98 LEAP Average Converted LEAP Rate [μM [hydrolytic Filtration MU/h] activity %] DNA [ppb] cobas_HCP [ppm] Load X0SP 20.2 100% 8176.19 12529 #1 w/o Reg. 1.1 5% 0.16 2 #2 1.5 7% 0.15 306 #3 N/A N/A 0.15 1243 #4 3.2 16% 1.49 2913 #5 N/A N/A 12.83 4042 #6 8.2 41% 315.39 4815 #7 10.1 50% 1185.76 4998 #8 10.3 51% 1654.83 5347 #9 11 54% 2070.50 5826 #10 12 59% 2349.28 2006 Filtration Mass Mainpeak [mg] Yield Mainpeak [%] Load X0SP 1292.84 100% #1 w/o Reg. 1180.21  91% #2 1218.14  94% #3 1224.73  95% #4 1253.61  97% #5 1229.57  95% #6 1232.62  95% #7 1241.72  96% #8 1245.01  96% #9 1245.67  96% #10 1247.56  96% N/A = not analyzed

Example 7

[0625] Filtration of Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Derivatized with an Alkaline Pre-Treatment, Regenerated with an Alkaline Treatment

[0626] Materials: [0627] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0628] 2) Millipore Millistak+® HC Pro X0SP filter with 23 cm.sup.2 filter area; Lot.: CP0BB08624 [0629] 3) equilibration buffer: 150 mM acetic acid/tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0630] 4) alkaline pre-treatment and regeneration solution: 1 M NaOH

[0631] Filter Derivatization:

[0632] Following steps were performed sequentially: [0633] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0634] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0635] 3) 100 L/m.sup.2 alkaline pre-treatment/regeneration solution were applied to/flown through the filter. The system flow was paused after 70 L/m.sup.2 flowthrough for four hours to incubate the filter with the alkaline regeneration solution. The regeneration flow was 7.67 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0636] Following steps were performed sequentially: [0637] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the derivatized 23 cm.sup.2 X0SP filter unit. The mass throughput was 600 g/m.sup.2. The corresponding calculated volume throughput was 41.3 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0638] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0639] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0640] 4) The flowthrough of the filter was collected for 70 L/m.sup.2. When the flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool was obtained. [0641] 5) Thereafter, the alkaline regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (200 LMH)). Thus, the contact time was approx. 30 minutes. [0642] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter derivatization: 1)” was carried out. In total the filter was at a pH value above 10 for approximately 50 minutes. [0643] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter derivatization: 2)”. [0644] 8) Steps 1) to 7) were repeated for further nine product filtration cycles (steps 1) to 4)) and further eight regeneration cycles (steps 5) to 7)). In total, 6.0 kg/m.sup.2 (10×0.6 kg/m.sup.2) antibody were applied to the filter. [0645] 9) The separate final filtration pools were kept for analysis at −80° C.

[0646] Analyses and Results:

[0647] The following analyses were performed with the respective final filtration pools: [0648] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0649] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0650] LEAP (Lipase Activity Assay; hydrolytic activity) [0651] CHOP values (host cell proteins) by cobas_HCP [0652] DNA values (host cell DNA) by qPRC_DNA [0653] SE-HPLC (size exclusion HPLC)

[0654] The results are shown in the following Table X-7:

TABLE-US-00008 Final Filtration Photo- SEC SEC SEC Pool Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-% ] Area-% ] Area-%] Load not not 14.53 3.5 94.9 1.6 X0SP applicable applicable #1 233.38 87.36% 5.17 0.3 98.5 1.2 #2 234.8 89.71% 5.27 0.6 98 1.3 #3 236.19 91.27% 5.33 0.7 98 1.4 #4 233.36 92.03% 5.44 0.7 97.9 1.4 #5 230.28 92.36% 5.53 0.7 97.9 1.3 #6 231.01 92.23% 5.51 0.8 97.8 1.4 #7 231.09 92.32% 5.51 0.8 97.9 1.4 #8 230.78 92.60% 5.54 0.8 97.8 1.4 #9 230.61 93.11% 5.57 0.9 97.7 1.4 #10 230.65 92.75% 5.55 0.8 97.8 1.4 LEAP Average Converted LEAP Rate [μM [hydrolytic Filtration MU/h] activity %] DNA [ppb] cobas_HCP [ppm] Load X0SP 13.7 100% 8176.1 12529 #1. 3.3  24% 18.0 848 #2 3.3  24% 398.2 1920 #3 3.7  27% 364.4 2315 #4 N/A N/A 378.4 2460 #5 3.9  28% 379.3 2682 #6 4.4  32% 391.9 2746 #7 4.7  34% 402.6 2744 #8 4.5  33% 436.9 3033 #9 4.4  32% 412.7 3072 #10 4.6  34% 396.3 3064 Filtration Mass Mainpeak [mg] Yield Mainpeak [%] Load X0SP 1309.95 100% #1. 1187.74  91% #2 1213.57  93% #3 1234.71  94% #4 1243.69  95% #5 1247.52  95% #6 1245.09  95% #7 1247.56  95% #8 1250.10  95% #9 1255.74  96% #10 1252.17  96% N/A = not analyzed

Example 8

[0655] Filtration of Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millipore Millistak+® HC Pro X0SP Filter, Regenerated with an Acidic Treatment

[0656] Materials: [0657] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column, Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0658] 2) Millipore Millistak+® HC Pro X0SP filter with 23 cm.sup.2 filter area; Lot.: CP0BB08624 [0659] 3) equilibration buffer: 150 mM acetic acid/tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0660] 4) acid regeneration solution: 0.5 M phosphoric acid

[0661] Filter Conditioning:

[0662] Following steps were performed sequentially: [0663] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0664] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[0665] Experimental Setup:

[0666] Following steps were performed sequentially: [0667] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 23 cm.sup.2 X0SP filter unit. The mass throughput was 600 g/m.sup.2. The corresponding calculated volume throughput was 41.3 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0668] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0669] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0670] 4) The flowthrough of the filter was collected for 70 L/m.sup.2. When the flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool was obtained. [0671] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (200 LMH)). Thus, the contact time was approx. 30 minutes. [0672] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [0673] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0674] 8) Steps 1) to 7) were repeated for further nine product filtration cycles (steps 1) to 4)) and further eight regeneration cycles (steps 5) to 7)). In total, 6.0 kg/m.sup.2 (10×0.6 kg/m.sup.2) antibody were applied to the filter. [0675] 9) The separate final filtration pools were kept for analysis at −80° C.

[0676] Analyses and Results:

[0677] The following analyses were performed with the respective final filtration pools: [0678] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0679] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0680] LEAP (Lipase Activity Assay; hydrolytic activity) [0681] CHOP values (host cell proteins) by cobas_HCP [0682] DNA values (host cell DNA) by qPRC_DNA [0683] SE-HPLC (size exclusion HPLC)

[0684] The results are shown in the following Table X-8:

TABLE-US-00009 Final Filtration Photo- SEC SEC SEC Pool Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-%] Area-%] Area-%] Load X0SP not not 14.53  3.5 94.9 1.6 applicable applicable  #1 w/o Reg. 223.14 84.06% 5.20 0.1 98.9 1.1  #2 224.71 86.12% 5.29 0.2 98.7 1.2  #3 225.46 85.63% 5.24 0.2 98.7 1.2  #4 225.83 85.84% 5.25 0.2 98.6 1.2  #5 225.99 86.24% 5.27 0.2 98.7 1.2  #6 226.25 86.92% 5.30 0.2 98.6 1.2  #7 226.54 86.82% 5.29 0.2 98.6 1.2  #8 226.67 86.64% 5.28 0.2 98.5 1.3  #9 226.84 86.80% 5.28 0.2 98.5 1.2 #10 227.28 87.44% 5.31 0.3 98.5 1.3 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load X0SP 13.7  100% 8176.19 12529 1309.95 100%  #1 w/o Reg. 1.2  9% 0.15 12 1147.56  88%  #2 1.5  11% 0.15 44 1174.28  90%  #3 1.5  11% 0.15 54 1166.59  89%  #4 1.2  9% 0.15 64 1168.36  89%  #5 1.4  10% 1.52 74 1174.90  90%  #6 1.5  11% 1.51 74 1183.03  90%  #7 1.7  12% 1.51 83 1181.69  90%  #8 1.8  13% 1.52 93 1177.95  90%  #9 1.7  12% 1.51 105 1180.11  90% #10 1.4  10% 1.51 101 1188.93  91% N/A = not analyzed

Example 9

[0685] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, with Water and Buffer Application (without Alkaline or Acidic Filter Regeneration)—Comparative Example

[0686] Materials: [0687] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0688] 2) Pall PDD1 SUPRAcap 50 (SC050PDD1) filter with 22 cm.sup.2 filter area; Lot.: 103864042 [0689] 3) equilibration buffer: 150 mM acetic acid/tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0690] 4) intermediate solution: water and equilibration buffer

[0691] Filter Conditioning:

[0692] Following steps were performed sequentially: [0693] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0694] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0695] Following steps were performed sequentially: [0696] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 22 cm.sup.2 PDD1 filter unit. The mass throughput was 599 g/m.sup.2. The corresponding calculated volume throughput was 120 L/m.sup.2.

[0697] The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0698] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0699] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0700] 4) The flowthrough of the filter was collected for 70 L/m.sup.2. When the flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool was obtained. [0701] 5) To prepare the filter for the next filtration cycle, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. [0702] 6) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0703] 7) Steps 1) to 6) were repeated for further nine product filtration cycles without the application of harsh regeneration solution between the filtration cycles. In total, with 5.99 kg/m.sup.2 (100×0.599 kg/m.sup.2) antibody were applied to the filter. [0704] 8) The separate final filtration pools were kept for analysis at −80° C.

[0705] Analyses and Results:

[0706] The following analyses were performed with the respective final filtration pools: [0707] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0708] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0709] LEAP (Lipase Activity Assay; hydrolytic activity) [0710] CHOP values (host cell proteins) by cobas_HCP [0711] DNA values (host cell DNA) by qPRC_DNA [0712] SE-HPLC (size exclusion HPLC)

[0713] The results are shown in the following Table X-9:

TABLE-US-00010 Final Filtration Photo- SEC SEC SEC Pool Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-%] Area-%] Area-%] Load PDD1 not not 4.97 3.5 94.9 1.6 applicable applicable  #1 w/o Reg. 397.73 88.08% 2.92 0.1 98.9 1.0  #2 408.22 92.89% 3.00 0.4 98.3 1.3  #3 408.18 94.64% 3.05 1.1 97.5 1.4  #4 409.48 94.98% 3.06 1.4 97.2 1.4  #5 409.49 96.60% 3.11 1.8 96.7 1.4  #6 409.78 96.76% 3.11 2.5 95.9 1.5  #7 410.01 96.78% 3.11 2.6 95.9 1.5  #8 410.05 97.23% 3.12 2.7 95.7 1.6  #9 410.26 97.65% 3.13 2.5 96.0 1.5 #10 410.39 97.74% 3.14 2.2 96.3 1.5 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load PDD1 16.2 100% 5955.73 12114 1249.96 100%  #1 w/o Reg. 1.0  6% N/A 27 1147.42  92%  #2 1.5  9% 17.3 511 1202.64  96%  #3 5.1  31% 753.1 2586 1215.42  97%  #4 8.9  55% 2396.1 4036 1215.93  97%  #5 10.9  67% 2825.9 4592 1230.30  98%  #6 11.8  73% 3299.0 5495 1222.16  98%  #7 11.6  72% 3859.8 5760 1222.46  98%  #8 12.4  77% 4585.3 5961 1225.52  98%  #9 13.5  83% 4357.3 6494 1230.78  98% #10 12.5  77% 4564.9 6487 1239.76  99% N/A = not analyzed

Example 10

[0714] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Derivatized with an Alkaline Pre-Treatment, Regenerated with an Alkaline Treatment

[0715] Materials: [0716] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0717] 2) Pall PDD1 SUPRAcap 50 (SC050PDD1) filter with 22 cm.sup.2 filter area; Lot.: 103864042 [0718] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0719] 4) alkaline regeneration solution: 1 M NaOH

[0720] Filter Derivatization:

[0721] Following steps were performed sequentially: [0722] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0723] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0724] 3) 100 L/m.sup.2 alkaline pre-treatment/regeneration solution were applied to/flown through the filter. The system flow was paused after 70 L/m.sup.2 flowthrough for four hours to incubate the filter with the alkaline regeneration solution. The regeneration flow was 7.67 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0725] Following steps were performed sequentially: [0726] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the derivatized 22 cm.sup.2 PDD1 filter unit. The mass throughput was 600 g/m.sup.2. The corresponding calculated volume throughput was 41.3 L/m.sup.2. The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0727] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0728] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0729] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0730] 5) Thereafter, the alkaline regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (191 LMH)). Thus, the contact time was approx. 30 minutes. [0731] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter derivatization: 1)” was carried out. In total the filter was at a pH value above 10 for approximately 50 minutes. [0732] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter derivatization: 2)”. [0733] 8) Steps 1) to 7) were repeated for further nine product filtration cycles (steps 1) to 4)) and further eight regeneration cycles (steps 5) to 7)). In total, 6.0 kg/m.sup.2 (10×0.6 kg/m.sup.2) antibody were applied to the filter. [0734] 9) The separate final filtration pools were kept for analysis at −80° C.

[0735] Analyses and Results:

[0736] The following analyses were performed with the respective final filtration pools: [0737] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0738] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0739] LEAP (Lipase Activity Assay; hydrolytic activity) [0740] CHOP values (host cell proteins) by cobas_HCP [0741] DNA values (host cell DNA) by qPRC_DNA [0742] SE-HPLC (size exclusion HPLC)

[0743] The results are shown in the following Table X-10:

TABLE-US-00011 Final Photo- SEC SEC SEC Filtrat Yield Conc. [HMW [MainPeak [LMW Filtration Vol. [mL] [%] [g/L] Area-%] Area-%] Area-%] LoadP DD1 not not 13.76 5.33 93.69 0.97 applicable applicable  #1 228.18 82.43% 4.772 1.3  98.07 0.63  #2 227.38 81.16% 4.715 1.25 98.16 0.59  #3 227.1 80.51% 4.683 1.26 98.14 0.59  #4 226.94 80.18% 4.667 1.27 98.13 0.59  #5 226.32 79.39% 4.634 1.27 98.14 0.59  #6 225.79 79.41% 4.646 1.27 98.13 0.6   #7 225.4 80.20% 4.7 1.29 98.12 0.6   #8 227.46 79.85% 4.637 1.42 97.97 0.61  #9 240.44 80.33% 4.413 1.25 98.19 0.57 #10 244.99 80.16% 4.3222 1.36 98.06 0.58 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load PDD1 18.5 100% 5595.93 10237 1237.61 100%  #1 1  5% 0.17 23 1067.86  86%  #2 1.1  6% 0.17 21 1052.37  85%  #3 1  5% 0.17 23 1043.73  84%  #4 0.9  5% 0.17 26 1039.32  84%  #5 0.9  5% 1.73 25 1029.26  83%  #6 0.8  4% 10.68 38 1029.40  83%  #7 1.1  6% 122.13 204 1039.46  84%  #8 1.7  9% 417.13 740 1033.32  83%  #9 2.6  14% 802.18 1411 1041.86  84% #10 3.6  19% 1267.87 2265 1038.35  84%

Example 11

[0744] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment

[0745] Materials: [0746] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0747] 2) Pall PDD1 SUPRAcap 50 (SC050PDD1) filter with 22 cm.sup.2 filter area; Lot.: 103864042 [0748] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Millipore Super Q) [0749] 4) acid regeneration solution: 0.5 M phosphoric acid

[0750] Filter Conditioning:

[0751] Following steps were performed sequentially: [0752] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0753] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[0754] Experimental Setup:

[0755] Following steps were performed sequentially: [0756] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 22 cm.sup.2 PDD1 filter unit. The mass throughput was 600 g/m.sup.2. The corresponding calculated volume throughput was 41.3 L/m.sup.2. The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0757] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0758] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0759] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0760] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (191 LMH)). Thus, the contact time was approx. 30 minutes. [0761] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total the filter was at a pH value below 2 for approximately 50 minutes. [0762] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0763] 8) Steps 1) to 7) were repeated for further nine product filtration cycles (steps 1) to 4)) and further eight regeneration cycles (steps 5) to 7)). In total, 6.0 kg/m.sup.2 (10×0.6 kg/m.sup.2) antibody were applied to the filter. [0764] 9) The separate final filtration pools were kept for analysis at −80° C.

[0765] Analyses and Results:

[0766] The following analyses were performed with the respective final filtration pools: [0767] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0768] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0769] LEAP (Lipase Activity Assay; hydrolytic activity) [0770] CHOP values (host cell proteins) by cobas_HCP [0771] DNA values (host cell DNA) by aPRC_DNA [0772] SE-HPLC (size exclusion HPLC)

[0773] The results are shown in the following Table X-11:

TABLE-US-00012 Final Photo- SEC SEC SEC Filtrat Yield Conc. [HMW [MainPeak [LMW Filtration Vol. [mL] [%] [g/L] Area-%] Area-%] Area-%] Load PDD1 not not 13.76 4.2 94 1.8 applicable applicable  #1 w/o Reg. 229.84 87.17% 4.96 0.1 98.8 1.1  #2 233.03 90.84% 5.10 0.2 98.5 1.3  #3 233.22 91.06% 5.10 0.2 98.4 1.3  #4 233.56 90.98% 5.09 0.3 98.4 1.3  #5 233.82 91.32% 5.11 0.2 98.5 1.3  #6 234.01 90.80% 5.07 0.3 98.3 1.3  #7 234.1  90.84% 5.07 0.3 98.4 1.3  #8 234.12 90.83% 5.07 0.4 98.3 1.3  #9 234.16 90.77% 5.07 0.4 98.3 1.3 #10 234.23 91.29% 5.09 0.4 98.2 1.4 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load PDD1 18.5 100% 5988.37 10742 1228.77 100%  #1 w/o Reg. 1  5% <0.16 43 1126.33  92%  #2 1.3  7% <0.16 348 1170.63  95%  #3 1.5  8% <0.16 420 1170.39  95%  #4 N/A N/A <0.16 463 1169.80  95%  #5 1.6  9% <1.57 533 1176.90  96%  #6 N/A N/A <0.16 568 1166.26  95%  #7 1.6  9% <0.16 597 1167.90  95%  #8 1.7  9% <0.16 612 1166.81  95%  #9 1.6  9% <0.16 669 1167.01  95% #10 1.6  9% <0.16 694 1170.77  95%

Example 12

[0774] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millistak+® HC Pro Synthetic Depth Filter X0SP (Reference Example)

[0775] Materials: [0776] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0777] 2) Millistak+® HC Pro Synthetic Depth Filter X0SP with 23 cm.sup.2 filter area [0778] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS).

[0779] Filter Conditioning:

[0780] Following steps were performed before the first sample application: [0781] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0782] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[0783] Experimental Setup:

[0784] Following steps were performed sequentially: [0785] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 23 cm.sup.2 X0SP filter unit. The mass throughput was 660 g/m.sup.2. The corresponding calculated volume throughput was 49.6 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0786] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0787] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0788] 4) The flowthrough of the filter was collected for 70 L/m.sup.2. When the flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0789] 5) Steps 1) to 4) were repeated for further five product filtration cycles without the application of a solution between the filtration cycles. In total, 3.96 kg/m.sup.2 (6×0.66 kg/m.sup.2) antibody were applied to the filter. [0790] 6) The procedure has resulted in six fractions. For further analysis the respective fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0791] 7) The final pools were kept for analysis at −80° C.

[0792] Analyses and Results:

[0793] The following analyses were performed with the respective final filtration pools: [0794] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0795] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0796] LEAP (Lipase Activity Assay; hydrolytic activity) [0797] CHOP values (host cell proteins) by cobas_HCP [0798] DNA values (host cell DNA) by qPRC_DNA [0799] SE-HPLC (size exclusion HPLC)

[0800] The results are shown in the following Table X-12:

TABLE-US-00013 Final Filtration Photo- SEC SEC SEC Pool Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] (theo.) [%] [g/L] Area-%] Area-%] Area-%] Load X0SP not not 13.37 6.62 92.22 1.17 applicable applicable Fraction #1 270.30 93.15 5.08 1.67 97.42 0.91 w/o Reg. Pool #2 270.30 95.16 5.19 2.13 96.90 0.96 Pool #3 270.30 97.73 5.33 2.6 96.40 0.99 Pool #4 270.30 96.81 5.28 2.9 96.02 0.10 Pool #5 270.30 97.91 5.34 3.33 95.64 1.02 Pool #6 270.30 98.46 5.37 3.15 95.89 0.95 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic DNA cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] [ppb] [ppm] [mg] [%] (*) Load X0SP 18.1 100.00 5703.59 8432.00 1359.98   100% Fraction #1 1.4 7.73 N/A N/A 1337.70  98.4% w/o Reg. Pool #2 N/A N/A 147.98 428.00 1359.37 100.0% Pool #3 3.3 18.23 457.79 834.00 1388.83 102.1% Pool #4 4.6 25.41 799.24 1218.00 1370.38 100.8% Pool #5 5.3 29.28 1063.67 1545.00 1380.47 101.5% Pool #6 6.3 34.81 1299.81 1665.00 1391.85 102.3% N/A = not analyzed (*) caluclation based on the theoretical volume and not the actual sample volume

Example 13

[0801] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millistak+® HC Pro Synthetic Depth Filter X0SP, Regenerated with an Acidic Treatment.

[0802] Materials: [0803] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0804] 2) Millistak+® HC Pro Synthetic Depth Filter X0SP, with 23 cm.sup.2 filter area. [0805] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS). [0806] 4) acidic regeneration solution: 167 mM acetic acid, 300 mM phosphoric acid, pH 1.34

[0807] Filter Conditioning:

[0808] Following steps were performed sequentially: [0809] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0810] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0811] Following steps were performed sequentially: [0812] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 23 cm.sup.2 X0SP filter unit. The mass throughput was 660 g/m.sup.2. The corresponding calculated volume throughput was 49.6 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0813] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0814] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0815] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0816] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (200 LMH)). Thus, the contact time was approx. 30 minutes. [0817] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [0818] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0819] 8) Steps 1) to 7) were repeated for further five product filtration cycles (steps 1) to 4)) and further four regeneration cycles (steps 5) to 7)). In total, 3.96 kg/m.sup.2 (6×0.66 kg/m.sup.2) antibody were applied to the filter. [0820] 9) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0821] 10) The separate final filtration pools were kept for analysis at −80° C.

[0822] Analyses and Results:

[0823] The following analyses were performed with the respective final filtration pools: [0824] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0825] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0826] LEAP (Lipase Activity Assay; hydrolytic activity) [0827] CHOP values (host cell proteins) by cobas_HCP [0828] DNA values (host cell DNA) by qPRC_DNA [0829] SE-HPLC (size exclusion HPLC)

[0830] The results are shown in the following Table X-13:

TABLE-US-00014 Final Photo- SEC SEC SEC Filtrat Yield Conc. [HMW [MainPeak [LMW Filtration Vol. [mL] [%] [g/L] Area-%] Area-%] Area-%] Load X0SP not not 13.17 7.04 91.89 1.06 applicable applicable #1 w/o Reg. 291.2 98.29 5.83 1.76 97.38 0.86 Pool #2 291.2 98.59 5.85 1.74 97.38 0.87 Pool #3 291.2 99.09 5.88 1.9 97.22 0.89 Pool #4 291.2 99.26 5.89 1.89 97.19 0.92 Pool #5 291.2 99.26 5.89 1.87 97.24 0.9 Pool #6 291.2 100.27 5.95 1.89 97.21 0.9 LEAP Average Converted LEAP Rate [μM [hydrolytic cobas_HCP Filtration MU/h] activity %] DNA [ppb] [ppm] Load X0SP 18.1 100.00 6124.62 8804.00 #1 w/o N/A N/A <0.14  18.00 Pool #2 N/A N/A <0.14  39.00 Pool #3  1.3  7.18 <0.14  50.00 Pool #4 N/A N/A <0.15  68.00 Pool #5 N/A N/A <0.15  80.00 Pool #6  1.5  8.29 <0.15  80.00 Mass Yield Mainpeak Mainpeak Filtration [mg] [%] (*) Load X0SP 1587.77 100% #1 w/o Reg. 1653.22 104% Pool #2 1658.89 104% Pool #3 1656.16 104% Pool #4 1511.31  95% Pool #5 1509.26  95% Pool #6 1542.76  97% (*) caluclation based on the theoretical volume and not the actual sample volume

Example 14

[0831] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Millistak+® HC Pro Synthetic Depth Filter X0SP, with Water and Buffer Regeneration (without Alkaline or Acidic Filter Regeneration), with a Pre-Incubation with 1 M NaOH for Four Hours Prior to First Use

[0832] Materials: [0833] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0834] 2) Millistak+® HC Pro Synthetic Depth Filter X0SP with 23 cm.sup.2 filter area. [0835] 3) equilibration buffer: 150 mM acetic acid/tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS). [0836] 4) regeneration solution: water and equilibration buffer

[0837] Filter Derivatization:

[0838] Following steps were performed sequentially: [0839] 1) The X0SP filter was flown through with 1 M NaOH, deaerated and the flow was stopped until a final incubation time with 1 M NaOH of 4 hours was reached. The flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0840] 2) 100 L/m.sup.2 water were applied to/flown through the filter. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0841] 3) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0842] Following steps were performed sequentially: [0843] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the derivatized 23 cm.sup.2 X0SP filter unit. The mass throughput was 660 g/m.sup.2. The corresponding calculated volume throughput was 50.65 L/m.sup.2. The feed flow was adjusted to 200 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0844] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0845] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0846] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0847] 5) To prepare the filter for the next filtration cycle, water and equilibration buffer were applied with same conditions as described in “filter derivatization: 2) and 3)”. [0848] 6) Steps 1) to 4) were repeated for further five product filtration cycles and step 5) for further four regeneration cycles. In total, 3.96 kg/m.sup.2 (6×0.66 kg/m.sup.2) antibody were applied to the filter. [0849] 7) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0850] 8) The separate final filtration pools were kept for analysis at −80° C.

[0851] Analyses and Results:

[0852] The following analyses were performed with the respective final filtration pools: [0853] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0854] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0855] LEAP (Lipase Activity Assay; hydrolytic activity) [0856] CHOP values (host cell proteins) by cobas_HCP [0857] DNA values (host cell DNA) by qPRC_DNA [0858] SE-HPLC (size exclusion HPLC)

[0859] The results are shown in the following Table X-14:

TABLE-US-00015 Final Filtration Photo- SEC SEC SEC Pool Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-%] Area-%] Area-%] Load X0SP not not 13.03 5.13 94.05 0.82 applicable applicable Fraction #1 250.20 86.86 5.27 1.69 97.5  0.81 Pool #2 256.80 90.51 5.42 1.82 97.44 0.74 Pool #3 257.50 91.16 5.43 2.4 96.67 0.92 Pool #4 257.70 91.17 5.45 1.86 97.42 0.73 Pool #5 257.80 92.59 5.49 1.88 97.35 0.76 Pool #6 258.50 93.07 5.51 1.3 97.97 0.72 Pool #7 258.90 93.71 5.54 2.06 97.22 0.72 Pool #8 259.30 93.66 5.53 1.99 97.28 0.73 Pool #9 259.40 93.92 5.54 2.29 97.00 0.72 LEAP Average Converted LEAP Mass Yield Rate [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration [μM MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load X0SP 15.2 100 4328.47 9015.00 1427.67 100.0% Fraction #1 2.7 17.76 <0.15 684.00 1285.59  90.0% Pool #2 3.1 20.39 <0.15 858.00 1356.22  95.0% Pool #3 3.2 21.05 <0.15 1145.00 1351.66  94.7% Pool #4 3.7 24.34 11.27 1184.00 1368.23  95.8% Pool #5 3.74 24.61 76.5 1428.00 1377.82  96.5% Pool #6 4.2 27.63 195.64 1904.00 1395.42  97.7% Pool #7 5.4 35.53 351.99 3131.00 1394.43  97.7% Pool #8 6 39.47 470.16 2809.00 1394.93  97.7% Pool #9 6.2 40.79 667.87 3185.00 1393.96  97.6%

Example 15

[0860] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter (Reference Example)

[0861] Materials: [0862] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0863] 2) Pall PDD1 SUPRAcap 50 (SC050PDD1) filter with 22 cm.sup.2 filter area. [0864] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS)

[0865] Filter Conditioning:

[0866] Following steps were performed before the first sample application: [0867] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0868] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0869] Following steps were performed sequentially: [0870] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 22 cm.sup.2 PDD1 filter unit. The mass throughput was 690 g/m.sup.2. The corresponding calculated volume throughput was 51.9 L/m.sup.2. The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0871] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0872] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0873] 4) The flowthrough of the filter was collected for 70 L/m.sup.2. When the flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0874] 5) Steps 1) to 4) were repeated for further five product filtration cycles without the application of a solution between the filtration cycles. In total, 4.14 kg/m.sup.2 (6×0.69 kg/m.sup.2) antibody were applied to the filter. [0875] 6) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0876] 7) The final pools were kept for analysis at −80° C.

[0877] Analyses and Results:

[0878] The following analyses were performed with the respective final filtration pools: [0879] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0880] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0881] LEAP (Lipase Activity Assay; hydrolytic activity) [0882] CHOP values (host cell proteins) by cobas_HCP [0883] DNA values (host cell DNA) by qPRC_DNA [0884] SE-HPLC (size exclusion HPLC)

[0885] The results are shown in the following Table X-15:

TABLE-US-00016 Final Filtration Pool Vol. Photo- SEC SEC SEC [mL] Yield Conc. [HMW [MainPeak [LMW Filtration (theo.) [%] [g/L] Area-%] Area-%] Area-%] Load PDD1 not not 13.29 6.23 92.56 1.12 applicable applicable Fraction #1 274.2 97.92 5.42 1.28 97.71 1.00 Pool #2 274.2 99.37 5.5 1.67 97.38 0.95 Pool #3 274.2 101.53 5.62 2.58 96.34 1.08 Pool #4 274.2 100.99 5.59 3.05 95.98 0.97 Pool #5 274.2 104.06 5.76 3.52 95.38 1.11 Pool #6 274.2 102.44 5.67 3.76 95.15 1.10 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] (*) Load PDD1 18.1 100 5658.39 8817 1404.80 100% Fraction #1 1.1 6.08 <1.48 52 1452.13 103% Pool #2 1.7 9.39 65.45 241 1468.59 105% Pool #3 3.3 18.23 537.37 1033 1484.60 106% Pool #4 4.4 24.31 991.06 1436 1471.16 105% Pool #5 4.6 25.41 1427.08 1653 1506.42 107% Pool #6 5.8 32.04 1696.65 1782 1479.31 105% (*) caluclation based on the theoretical volume and not the actual sample volume

Example 16

[0886] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment

[0887] Materials: [0888] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0889] 2) Pall PDD1 SUPRAcap 50 (SC050PDD1) filter with 22 cm.sup.2 filter area. [0890] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS). [0891] 4) acidic regeneration solution: 167 mM acetic acid, 300 mM phosphoric acid, pH 1.34

[0892] Filter Conditioning:

[0893] Following steps were performed sequentially: [0894] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0895] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0896] Following steps were performed sequentially: [0897] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 22 cm.sup.2 PDD1 filter unit. The mass throughput was 690 g/m.sup.2. The corresponding calculated volume throughput was 52.39 L/m.sup.2. The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0898] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0899] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0900] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0901] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (191 LMH)). Thus, the contact time was approx. 30 minutes. [0902] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [0903] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [0904] 8) Steps 1) to 7) were repeated for further five product filtration cycles (steps 1) to 4)) and further four regeneration cycles (steps 5) to 7)). In total, 4.14 kg/m.sup.2 (6×0.69 kg/m.sup.2) antibody were applied to the filter. [0905] 9) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0906] 10) The separate final filtration pools were kept for analysis at −80° C.

[0907] Analyses and Results:

[0908] The following analyses were performed with the respective final filtration pools: [0909] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0910] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0911] LEAP (Lipase Activity Assay; hydrolytic activity) [0912] CHOP values (host cell proteins) by cobas_HCP [0913] DNA values (host cell DNA) by qPRC_DNA [0914] SE-HPLC (size exclusion HPLC)

[0915] The results are shown in the following Table X-16:

TABLE-US-00017 Final Photo- SEC SEC SEC Filtrat Yield Conc. [HMW [MainPeak [LMW Filtration Vol. [mL] [%] [g/L] Area-%] Area-%] Area-%] Load PDD1 not not 13.17 6.56 92.38 1.07 applicable applicable Fraction #1 275.26  99.01 5.46 1.29 97.77 0.94 w/o Reg. Pool #2 275.26 101.73 5.61 1.28 97.85 0.86 Pool #3 275.26 100.64 5.55 1.60 97.39 1.00 Pool #4 275.26 100.82 5.56 1.66 97.32 1.01 Pool #5 275.26 101.55 5.6 1.71 97.27 1.03 Pool #6 275.26 101.00 5.57 1.75 97.24 1.01 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load PDD1 18.1 100 6590.74 8277 1402.30 100% Fraction #1 1.1 6.08 <1.47 52 1469.40 105% w/o Reg. Pool #2 N/A N/A <1.43 176 1511.01 108% Pool #3 1.3 7.18 <1.44 178 1487.82 106% Pool #4 N/A N/A <1.44 281 1489.43 106% Pool #5 N/A N/A <1.43 313 1499.37 107% Pool #6 1.3 7.18 <1.44 354 1490.88 106% N/A = not analyzed

Example 17

[0916] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Silica-Containing Pall PDD1 SUPRAcap 50 Filter, Regenerated with an Acidic Treatment, without Intermediate Water Flush for Reducing Process Time

[0917] Materials: [0918] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0919] 2) Pall PDD1 SUPRAcap 50 (SC050PDD1) filter with 22 cm.sup.2 filter area; Lot.: 104072586. [0920] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS). [0921] 4) acidic regeneration solution: 167 mM acetic acid, 300 mM phosphoric acid, pH 1.5

[0922] Filter Conditioning:

[0923] Following step was performed: [0924] 1) 200 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[0925] Experimental Setup:

[0926] Following steps were performed sequentially: [0927] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 22 cm.sup.2 PDD1 filter unit. The mass throughput was 627 g/m.sup.2. The corresponding calculated volume throughput was 48 L/m.sup.2. The feed flow was adjusted to 191 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0928] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0929] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0930] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0931] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (191 LMH)). Thus, the contact time was approx. 30 minutes. [0932] 6) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [0933] 7) Steps 1) to 6) were repeated for further five product filtration cycles (steps 1) to 4)) and further four regeneration cycles (steps 5) and 6)). In total, 3.762 kg/m.sup.2 (6×0.627 kg/m.sup.2) antibody were applied to the filter. [0934] 8) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0935] 9) The separate final filtration pools were kept for analysis at −80° C.

[0936] Analyses and Results:

[0937] The following analyses were performed with the respective final filtration pools: [0938] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0939] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0940] LEAP (Lipase Activity Assay; hydrolytic activity) [0941] CHOP values (host cell proteins) by cobas_HCP [0942] DNA values (host cell DNA) by qPRC_DNA [0943] SE-HPLC (size exclusion HPLC)

[0944] The results are shown in the following Table X-17:

TABLE-US-00018 Final Photo- SEC SEC SEC Filtrat Yield Conc. [HMW [MainPeak [LMW Filtration Vol. [mL] [%] [g/L] Area-%] Area-%] Area-%] Load PDD1 not not 13.07 5.19 93.98 0.84 applicable applicable Fraction #1 245.2 90.25 5.08 1.26 98.00 0.73 w/o Reg. Pool #2 248.6 92.13 5.15 1.47 97.65 0.88 Pool #3 249.0 92.39 5.15 1.55 97.64 0.80 Pool #4 249.5 92.39 5.14 1.39 97.85 0.76 Pool #5 249.5 92.85 5.16 1.39 97.83 0.78 Pool #6 249.9 93.13 5.17 1.41 97.83 0.76 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load PDD1 13.5 100 5524.1 9359 1297.10 100% Fraction #1 1 7.41 <0.16 35 1220.70  94% w/o Reg. Pool #2 1.2 8.89 <0.16 95 1250.20  96% Pool #3 1.1 8.15 <1.55 180 1252.09  97% Pool #4 1.4 10.37 <1.56 222 1254.86  97% Pool #5 1.3 9.63 <1.55 253 1259.48  97% Pool #6 1.2 8.89 <1.55 359 1263.95  97%

Example 18

[0945] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Zeta Plus™ Biocap VR02 without Intermediate Flush (Reference Example)

[0946] Materials: [0947] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0948] 2) Zeta Plus™ Biocap VR02 filter with 25 cm.sup.2 filter area; (Lot.: 3923451). [0949] 3) equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS).

[0950] Filter Conditioning:

[0951] Following steps were performed before the first sample application: [0952] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0953] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0954] Following steps were performed sequentially: [0955] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 25 cm.sup.2 VR02 filter unit. The mass throughput was 659 g/m.sup.2. The corresponding calculated volume throughput was 54.8 L/m.sup.2. The feed flow was adjusted to 184 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0956] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0957] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0958] 4) The flowthrough of the filter was collected for 70 L/m.sup.2. When the flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0959] 5) Steps 1) to 4) were repeated for further five product filtration cycles without the application of a solution between the filtration cycles. In total, 3.95 kg/m.sup.2 (6×0.659 kg/m.sup.2) antibody were applied to the filter. [0960] 6) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0961] 7) The final pools were kept for analysis at −80° C.

[0962] Analyses and Results:

[0963] The following analyses were performed with the respective final filtration pools: [0964] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0965] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0966] LEAP (Lipase Activity Assay; hydrolytic activity) [0967] CHOP values (host cell proteins) by cobas_HCP [0968] DNA values (host cell DNA) by qPRC_DNA SE-HPLC (size exclusion HPLC)

[0969] The results are shown in the following Table X-18:

TABLE-US-00019 Final Filtration Photo- SEC SEC SEC Pool Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] (theo.) [%] [g/L] Area-%] Area-%] Area-%] Load VR02 not not 13.07 4.64 94.54 0.83 applicable applicable Fraction #1 274.9 95.63 5.74 1.97 97.23 0.8  Pool #2 277.0 97.84 5.85 3.71 95.33 0.95 Pool #3 277.3 100.18 5.98 4.1  94.94 0.96 Pool #4 277.5 98.77 5.89 4.36 94.66 0.98 Pool #5 277.6 99.33 5.92 4.51 94.49 1.00 Pool #6 277.5 99.21 5.91 4.67 94.37 0.97 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load VR02 15.5 100% 4697.78 9430.00 1557.40 100% Fraction #1 4.5  29% 616.72 2253.00 1534.22  99% Pool #2 8.4  54% 2037.61 3836.00 1544.77  99% Pool #3 10.6  68% 2729.1 4244.00 1574.35 101% Pool #4 11.2  72% 2964.35 4929.00 1547.19  99% Pool #5 12.9  83% 3378.38 5013.00 1552.84 100% Pool #6 13.1  85% 3722.5 5170.00 1547.69  99%

Example 19

[0970] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Zeta Plus™ Biocap VR02 Filter, with Water and Buffer Application (without Alkaline or Acidic Filter Regeneration)—Comparative Example

[0971] Materials: [0972] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [0973] 2) Zeta Plus™ Biocap VR02 filter with 25 cm.sup.2 filter area; (Lot.: 3923451). [0974] 3) Equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS). [0975] 4) intermediate solution: water and equilibration buffer

[0976] Filter Conditioning:

[0977] Following steps were performed sequentially: [0978] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [0979] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

Experimental Setup

[0980] Following steps were performed sequentially: [0981] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the equilibrated 25 cm.sup.2 VR02 filter unit. The mass throughput was 659 g/m.sup.2. The corresponding calculated volume throughput was 54.8 L/m.sup.2. The feed flow was adjusted to 184 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [0982] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [0983] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [0984] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [0985] 5) To prepare the filter for the next filtration cycle, water and equilibration buffer were applied with same conditions as described in “filter conditioning: 1) and 2)”. [0986] 6) Steps 1) to 4) were repeated for further five product filtration cycles and step 5) for further four regeneration cycles. In total, 3.95 kg/m.sup.2 (6×0.659 kg/m.sup.2) antibody were applied to the filter. [0987] 7) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 resulted in a fraction “Pool #3”). [0988] 8) The separate final filtration pools were kept for analysis at −80° C.

[0989] Analyses and Results:

[0990] The following analyses were performed with the respective final filtration pools: [0991] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [0992] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [0993] LEAP (Lipase Activity Assay; hydrolytic activity) [0994] CHOP values (host cell proteins) by cobas_HCP [0995] DNA values (host cell DNA) by qPRC_DNA [0996] SE-HPLC (size exclusion HPLC)

[0997] The results are shown in the following Table X-19:

TABLE-US-00020 Final Filtrat Photo- SEC SEC SEC Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-%] Area-%] Area-%] Load VR02 not not 13.07 5.5 93.56 0.95 applicable applicable Fraction #1 275.8 97.79 5.85 2.09 97.17 0.74 Pool #2 277.4 100.08 5.97 3.89 95.14 0.96 Pool #3 277.4 100.36 5.98 4.27 94.75 0.98 Pool #4 277.3 97.87 5.83 4.5 94.53 0.97 Pool #5 277.4 99.41 5.92 4.63 94.41 0.97 Pool #6 277.6 98.6 5.87 4.76 94.27 0.98 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load VR02 15.5 100% 4483.55 9715.00 1541.25 100% Fraction #1 4.7  30% 762.39 2510.00 1567.77 102% Pool #2 7.0  45% 1805.7 3923.00 1575.59 102% Pool #3 10.2  66% 2588.63 4259.00 1571.76 102% Pool #4 11.5  74% 3138.94 4675.00 1528.23  99% Pool #5 11.4  74% 2949.32 4930.00 1550.41 101% Pool #6 12.3  79% 8926.75 5265.00 1536.14 100%

Example 20

[0998] Filtration of a Crovalimab (Anti-C5 Antibody) Solution with a Zeta Plus™ Biocap VR02 Filter, Regenerated with an Acidic Treatment

[0999] Materials: [1000] 1) Experiments were performed with an Äkta Avant 150 (Cytiva, Uppsala, Sweden) chromatographic skid. The filter was installed to the column valve instead of a column. Pressure, pH value, conductivity, OD280 were monitored. The applied volume was regulated by a sample pump. [1001] 2) Zeta Plus™ Biocap VR02 filter with 25 cm.sup.2 filter area; Lot.: 3923451. [1002] 3) Equilibration buffer: 150 mM acetic acid/Tris, adjusted to pH 5.5 in purified water type II (Advantec CCS-020-D1DS). [1003] 4) Acidic regeneration solution: 167 mM acetic acid, 300 mM phosphoric acid, pH 1.34

[1004] Filter Conditioning:

[1005] Following steps were performed sequentially: [1006] 1) 100 L/m.sup.2 water were applied to/flown through the filter. Thereafter the filter was deaerated. The washing flow was 10.0 mL/min at a max. feed pressure of 5.0 bar. [1007] 2) 100 L/m.sup.2 equilibration buffer were applied to/flown through the filter (equilibration of the system and the filter). The equilibration flow was 10.0 mL/min at a max. feed pressure of 5.0 bar.

[1008] Experimental Setup:

[1009] Following steps were performed sequentially: [1010] 1) An intermediate “Affinity Pool pH 5.5” containing crovalimab was applied to the conditioned 25 cm.sup.2 VR02 filter unit. The mass throughput was 659 g/m.sup.2. The corresponding calculated volume throughput was 54.8 L/m.sup.2. The feed flow was adjusted to 184 LMH (liter per square meter per hour; L*m.sup.−2*h.sup.−1). This resulted in a calculated feed flow of 7.67 mL/min. The maximum feed pressure was 5.0 bar. [1011] 2) The filter flowthrough was not collected until the OD280 (280 nm) exceeded 0.5 AU determined using a 1 cm light path length UV-cell. After exceeding the threshold signal level, the flowthrough was collected in a tared and sterile bottle (Nalgene) resulting in a filtration pool. [1012] 3) When the intended loading volume had been applied, the filter was flushed with equilibration buffer (washing out of remaining protein solution). [1013] 4) The flushing flowthrough was collected for 70 L/m.sup.2. When the flushing flowthrough of 70 L/m.sup.2 was reached, the collecting was stopped. Thereby a final filtration pool (comprising the filter flowthrough and the flushing flowthrough) was obtained. [1014] 5) Thereafter, the acidic regeneration solution was applied to the filter with the same flow rate as the intermediated “Affinity Pool pH 5.5” (7.67 mL/min (184 LMH)). Thus, the contact time was approx. 30 minutes. [1015] 6) To remove the regeneration solution from the filter, a water wash with same conditions as described in “filter conditioning: 1)” was carried out. In total, the filter was at a pH value below 2 for approximately 50 minutes. [1016] 7) To equilibrate the filter for the next filtration cycle, equilibration buffer was applied with same conditions as described in “filter conditioning: 2)”. [1017] 8) Steps 1) to 7) were repeated for further five product filtration cycles (steps 1) to 4)) and further four regeneration cycles (steps 5) to 7)). In total, 3.95 kg/m.sup.2 (6×0.659 kg/m.sup.2) antibody were applied to the filter. [1018] 9) The procedure has resulted in six fractions. For further analysis the fractions were pooled in a final volume of 12 mL (e.g. 4 mL fraction 1, 4 mL fraction 2 and 4 mL fraction 3 is “Pool #3”). [1019] 10) The separate final filtration pools were kept for analysis at −80° C.

[1020] Analyses and Results:

[1021] The following analyses were performed with the respective final filtration pools: [1022] protein concentration (using the absorbance of 1.44 at 1 mg/ml as reference) [1023] total product yield, calculated out of loaded mass, final filtration pool volume and final filtration pool protein concentration [1024] LEAP (Lipase Activity Assay; hydrolytic activity) [1025] CHOP values (host cell proteins) by cobas_HCP [1026] DNA values (host cell DNA) by qPRC_DNA [1027] SE-HPLC (size exclusion HPLC)

[1028] The results are shown in the following Table X-20:

TABLE-US-00021 Final Filtrat Photo- SEC SEC SEC Vol. Yield Conc. [HMW [MainPeak [LMW Filtration [mL] [%] [g/L] Area-%] Area-%] Area-%] Load VR02 not not 13.07 5.64 93.42 0.94 applicable applicable Fraction #1 272.8 95.90 5.8 2.08 97.15 0.77 w/o Reg. Pool #2 274.0 96.44 5.82 2.11 97.12 0.76 Pool #3 274.4 97.22 5.86 2.15 97.05 0.8 Pool #4 274.8 98.64 5.94 2.19 97.01 0.79 Pool #5 275.1 97.23 5.85 2.23 97.02 0.75 Pool #6 275.5 99.13 5.96 3.24 95.81 0.94 LEAP Average Converted LEAP Mass Yield Rate [μM [hydrolytic cobas_HCP Mainpeak Mainpeak Filtration MU/h] activity %] DNA [ppb] [ppm] [mg] [%] Load VR02 15.5 100% 1371.08 8962.00 1538.95 100% Fraction #1 4.3  28% 817.24 2233.00 1537.15 100% w/o Reg. Pool #2 4.0  26% 934.71 2396.00 1548.75 101% Pool #3 4.5  29% 1020.48 2436.00 1560.55 101% Pool #4 5.3  34% 1033.67 2490.00 1583.51 103% Pool #5 5.2  34% 1001.71 2782.00 1561.38 101% Pool #6 5.1  33% 946.66 2677.00 1573.18 102%

Example 21

[1029] Hydrolytic Activity Determination—Lipase Activity Assay (LEAP):

[1030] The lipase activity was determined by monitoring the conversion of a substrate, such as a nonfluorescent substrate, to a detectable product of the hydrolytic enzyme, such as a fluorescent product.

[1031] In more detail, with the LEAP assay hydrolase activity in samples was determined. This was done by monitoring the conversion of a fluorogenic substrate ‘4-Methylumbelliferyl Caprylate’ (4-MU-C8, available from Chem Impex Int'l Inc Art. Nr. 01552) by cleavage of the ester bond by hydrolases present in the sample into a fluorescent moiety, i.e. 4-Methylumbelliferyl (4-MU). Cleaved 4-MU-C8, i.e. 4-MU, was excited with a light of wavelength 355 nm. The emitted radiation at a different wavelength of 460 nm was recorded on Tecan Infinite® 200 PRO device as readout. The determination was performed at 37° C. for 2 hours with recording every 10 mins to calculate the rate of substrate hydrolysis.

[1032] The sample to be analyzed was at first buffer exchanged to 150 mM Tris-Cl, pH 8.0, by using Amicon Ultra-0.5 ml centrifugal filter units (10,000 Da cut-off, Merck Millipore, Art. Nr. UFC501096). The assay reaction mixture constituted of 80 μL reaction buffer (150 mM Tris-Cl, pH 8.0, 0.25% (w/v) Triton X-100, 0.125% (w/v) Gum Arabic), 10 μL 4-MU-C8 substrate solution (1 mM in DMSO), and 10 μL protein containing sample. The protein samples' concentration were adjusted to be in the range between 1-30 g/L and 2-3 dilution series were performed for each determination. Each reaction was set up at least in duplicates in 96-well half-area polystyrene plates (black with lid and clear flat bottom, Corning Incorporated Art. Nr. 3882).