Continuous manufacturing process for biologics manufacturing by integration of drug substance and drug product processes

Abstract

To provide a fully integrated, continuous, manufacturing process for biologics manufacturing by integration of drug substance and drug product processes.SOLUTION: Provided is a method for producing a recombinant biologic therapeutic, comprising: providing a purified recombinant protein of interest; concentrating or diluting the purified recombinant protein by ultrafiltration; buffer exchanging the purified recombinant protein into a desired formulation by diafiltration; further diluting or concentrating the formulated recombinant protein by ultrafiltration until a target concentration is achieved; adding or combining at least one stability -enhancing excipient once the target concentration is achieved; subjecting the resulting bulk drug substance to filtration to reduce bioburden; subjecting the resulting bulk drug product to sterile filtration; and subjecting the sterile bulk drug product to a fill and finish operation; neither the purified recombinant protein nor the bulk drug substance being subjected to freezing and thawing unit operations.SELECTED DRAWING: Figure 1B

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 797,445, filed Jan. 28, 2019, which is incorporated herein by reference.

[0002] A biopharmaceutical manufacturing process that links drug substance and drug product processes into an integrated, continuous process. [Background technology]

[0003] Leading the drug substance to drug product fill / finish is usually a two-part process, separated by the conversion of the drug substance to a drug product, typically by a freeze / thaw step. The conversion of a purified biopharmaceutical protein of interest to a drug product (DS) and then to a drug product (DP) typically involves concentrating the protein of interest to a desired level in a suitable formulation buffer via an ultrafiltration and diafiltration (UFDF) unit operation. After UFDF, the concentrated, formulated protein is processed through one or more bioburden reduction filters and typically placed into a holding vessel. To enhance protein stability, one or more additional excipients are typically added to the concentrated, formulated protein, whereupon the drug substance is processed once more through one or more bioburden reduction filters and placed into a sterile vessel. The drug substance is typically harvested at this point and certain drug substance attributes are tested against release specifications. The drug substance is then frozen for ease of storage or transfer to another manufacturing facility. If necessary, the drug substance materials are thawed, pooled in a drug product container, mixed, and processed through one or more bioburden reduction filters to produce a filtered bulk drug product. The filtered bulk drug product is then sterile filtered and transferred to a sterile facility for fill / finish operations. During this fill / finish step, additional rounds of attribute and / or release assays, some of which are common to the attribute tests already performed on the drug substance, are performed to evaluate the attributes against release specifications and to ensure that the drug product quality / characteristics have not changed after going through the drug product preparation process.

[0004] This process involves dual operations that contribute to increased manufacturing costs and material waste; multiple holding / storage steps that are not compatible with a continuous manufacturing platform; redundant filtration steps, and freeze and thaw unit operations, all of which can cause drug substance loss and / or instability. Thus, there is a need for a more cost-effective, continuous, integrated conversion of the purified biopharmaceutical protein of interest to drug substance and subsequently to drug product, which would allow for a reduction in the size and amount of equipment and materials required and used; would reduce the footprint of the manufacturing facility or allow the use of manufacturing pods or other compact systems, and may have the advantages of reducing the time and cost of setting up and running the manufacturing facility, as well as the advantages of integrating attribute testing. The invention described herein meets this need by providing a fully integrated, continuous manufacturing process for biopharmaceutical manufacturing by integration of drug substance and drug product processes by removing and / or combining process steps from UFDF to drug substance fill / finish. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides an integrated, continuous method for producing a recombinant biological product, comprising providing a purified recombinant protein of interest; concentrating or diluting the purified recombinant protein by ultrafiltration; buffer exchanging the purified recombinant protein into a desired formulation by diafiltration; further diluting or concentrating the formulated recombinant protein by ultrafiltration until a target concentration is reached; adding or combining at least one stability-enhancing excipient after the target concentration is reached; subjecting the resulting bulk drug substance to filtration to reduce bioburden; subjecting the resulting bulk formulation to sterile filtration; and subjecting the sterile bulk formulation to a fill and finish operation, wherein neither the purified recombinant protein nor the bulk drug substance is subjected to freeze and thaw unit operations. In one embodiment, the stability-enhancing excipient is added to the formulated recombinant protein within the appropriate range. In one embodiment, the stability-enhancing excipient is added directly to the ultrafiltration and diafiltration (UFDF) retentate feed tank. In a related embodiment, the stability-enhancing excipient is added directly to the UFDF retentate feed tank within the appropriate range after the target concentration is reached. In another embodiment, the stability enhancing excipient is a non-ionic detergent or surfactant. In one embodiment, the stability enhancing excipient is a poly-oxy-ethylene (PEO) based surfactant. In one embodiment, the stability enhancing excipient is selected from polysorbate 80 and polysorbate 20. In one embodiment, the concentration of the at least one stability enhancing excipient is 0.001-0.1% (weight / volume). In one embodiment, the bulk formulation is collected in a storage container. In one embodiment, the bulk formulation is sent to a sterile processing facility. In a related embodiment, the sterile processing facility includes at least one filling station. In another related embodiment, the sterile processing facility includes at least one globeless sterile isolator. In another embodiment, the bulk formulation is collected in a storage container and sent directly to a sterile processing facility. In a related embodiment, the storage container is coupled to the sterile processing facility. In another related embodiment, the storage bag containing the bulk product, or the filtrate of a filter processing the bulk product, is connected to a globeless sterile isolator.In another related embodiment, the aseptic processing equipment has a connection to a storage vessel containing the bulk formulation or the filtrate of a filter unit that processes the bulk formulation. In one embodiment, the primary formulation vessel is filled with the sterile bulk formulation. In a related embodiment, the primary formulation vessel is sealed, labeled, and packaged. In one embodiment, there is a continuous flow between one or more steps. In one embodiment, the pool from the UFDF and / or bioburden reduction filtration is collected in a storage vessel. In one embodiment, the formulated recombinant protein is diluted until a target concentration is reached. In one embodiment, the formulated recombinant protein is concentrated by ultrafiltration until a target concentration is reached. In one embodiment, the ultrafiltration is performed using a stabilized cellulose-based hydrophilic membrane, up to 72 g / m of membrane area. 2 In one embodiment, ultrafiltration is performed using stabilized hydrophilic membranes with a target concentration of 3.20 mg / ml or less. In one embodiment, ultrafiltration is performed using stabilized cellulosic hydrophilic membranes with a target excess concentration of 1.1× to 2.5× the starting concentration. In one embodiment, ultrafiltration and diafiltration are performed using stabilized cellulosic hydrophilic membranes with a target excess concentration of 1.1× to 2.5× the starting concentration. In one embodiment, ultrafiltration and diafiltration are performed using a membrane area of ​​up to 170 g / m 2In one embodiment, the ultrafiltration and diafiltration are performed using an alkaline stable membrane of regenerated cellulose loaded up to 9 g / L. In one embodiment, the ultrafiltration and diafiltration are performed using an alkaline stable membrane of regenerated cellulose with a diavolume of up to 13 at an intermediate target excess concentration of 9 g / L or less. In one embodiment, the method described herein further comprises at least one virus filtration operation. In one embodiment, the at least one virus filtration operation follows a UFDF operation. In a related embodiment, the at least one virus filtration operation follows the addition of stability enhancing excipients to the formulated recombinant protein within a suitable range or to the UFDF retentate tank. In one embodiment, a bispecific T cell engager having a formulation concentration of 5 g / L or less is subjected to a virus filtration operation. In one embodiment, the virus filter is selected from a hydrophilized polyvinylidene fluoride (PVDF) hollow fiber filter, a cuprammonium regenerated cellulose hollow fiber filter, or a polyethersulfone (PES) parvovirus retentive filter. In another related embodiment, the at least one virus filtration operation also comprises a prefilter. In another related embodiment, the pre-filter is a depth filter. In one embodiment, one or more additional purified recombinant proteins of interest or drug substances are added prior to sterile filtration. In one embodiment, the purified protein of interest is an antigen binding protein. In one embodiment, the antigen binding protein is a multispecific protein. In one embodiment, the multispecific protein is a bispecific antibody. In one embodiment, the bispecific protein is a bispecific T cell engager. In one embodiment, the bispecific T cell engager is an extended half-life bispecific T cell engager. In a related embodiment, one binding domain of the bispecific T cell engager is specific for a tumor-associated surface antigen on a target cell selected from EGFRvIII, MSLN, CDH19, DLL3, CD19, CD33, CD38, FLT3, CDH3, BCMA, PSMA, MUC17, CLDN18.2, or CD70.In related embodiments, the bispecific T cell engager is selected from blinatumomab, pasotuxizumab, AMG103, AMG330, AMG212, AMG160, AMG420, AMG-110, AMG562, AMG596, AMG427, AMG673, AMG675, or AMG701.

[0006] The invention also provides pharmaceutical compositions comprising the formulations from the methods described herein.

[0007] The present invention also provides a method for producing a recombinant protein of interest comprising: expanding cells expressing the protein of interest to the N-1 stage; seeding and / or feeding a bioreactor with the expanded cells and culturing the cells to express the recombinant protein of interest; recovering the recombinant protein via a harvest unit operation; purifying the harvested recombinant protein via at least one capture chromatography unit operation; purifying the recombinant protein via at least one polish chromatography unit operation; subjecting the purified recombinant protein to ultrafiltration and diafiltration unit operations, including concentrating or diluting the purified recombinant protein by ultrafiltration; and converting the purified recombinant protein into a desired formulation by diafiltration. further diluting or concentrating the formulated, purified recombinant protein by ultrafiltration until a target concentration is reached, adding one or more stability-enhancing excipients directly to the UFDF retentate feed tank containing the formulated, purified recombinant protein to result in a formulated drug substance; subjecting the formulated drug substance to a single unit operation to reduce bioburden to result in a filtered bulk formulation; sterile filtering the bulk formulation; filling a primary formulation container with the sterile bulk formulation; and sealing, labeling, and packaging the primary formulation container, wherein neither the recombinant protein nor the drug substance are subjected to freeze and thaw unit operations.

[0008] The present invention also provides pharmaceutical compositions comprising the recombinant protein preparations of the methods described herein.

[0009] The invention also provides a method for reducing the manufacturing footprint for a drug product production process, comprising subjecting the purified recombinant protein of interest to an ultrafiltration and diafiltration (UFDF) unit operation until a target concentration is reached; adding at least one stability enhancing excipient directly to a UFDF retentate feed tank; subjecting the bulk drug substance to a single unit operation to reduce bioburden and then subjecting it to sterile filtration; subjecting the sterile bulk drug product to a fill and finish unit operation, where neither the recombinant protein nor the drug substance is subject to freeze and thaw unit operations. In one embodiment, a storage vessel containing the bulk drug product is connected to aseptic processing equipment. In one embodiment, the aseptic processing equipment has a connection with a storage vessel containing the bulk drug product or containing the filtrate of a filter that processes the bulk drug product. In one embodiment, there is a continuous flow between one or more steps. In one embodiment, at least one virus filtration unit operation follows the UFDF unit operation.

[0010] The present invention also provides a method for reducing drug product loss and / or destabilization during recombinant therapeutic protein manufacturing, comprising subjecting the purified recombinant protein of interest to a UFDF unit operation; adding at least one stability-enhancing excipient to a UFDF retentate feed tank after a target concentration is reached; and subjecting the UFDF pool to a single filtration to reduce bioburden and result in bulk drug substance, wherein neither the recombinant protein nor the drug substance are subjected to freeze and thaw unit operations.

[0011] The invention also provides a method for reducing viral contamination in a composition comprising a recombinant bispecific T cell engager, comprising providing a sample comprising less than 7.0 g / L of recombinant bispecific T cell engager at a pH of 6.0 or less and having a conductivity of 23-45 mS / cm; subjecting the sample to a virus filtration unit operation comprising a virus filter alone or in combination with a depth filter or a surface modified membrane prefilter; and recovering the eluate of the virus filter comprising the recombinant bispecific T cell engager in a pool or as a stream. In one embodiment, the bispecific T cell engager is an extended half-life bispecific T cell engager. In one embodiment, the sample comprises a chromatography column pool or eluate stream. In one embodiment, the pH of the pool or stream is 4.2-6.

[0012] The present invention also provides purified recombinant extended half-life bispecific T cell engagers produced according to the methods described herein.

[0013] The invention also provides a method for reducing high molecular weight species during the manufacture of a recombinant bispecific T cell engager comprising providing a sample comprising less than 7 g / L of recombinant bispecific T cell engager at a pH of 6.0 or less and having a conductivity of 23-45 mS / cm; subjecting the sample to a virus filtration unit operation comprising a virus filter in combination with a depth filter; and collecting the eluate of the virus filter in a pool or as a stream; wherein the percentage of high molecular weight species in the filter eluate pool is reduced compared to the use of a virus filtration unit operation comprising a virus filter alone or in combination with a surface-modified membrane prefilter. In one embodiment, the bispecific T cell engager is an extended half-life bispecific T cell engager.

[0014] The invention also provides a method for reducing flow decay and reducing high molecular weight species in a virus filtration unit operation during production of a recombinant bispecific T cell engager comprising providing a sample comprising up to 1.75 g / L of recombinant bispecific T cell engager at a pH of 4.2-6.0 and having a conductivity of 23-45 mS / cm; subjecting the purified recombinant bispecific T cell engager to a virus filtration unit operation comprising a virus filter in combination with a depth filter; and recovering the filter eluate in a pool or as a stream; wherein the percentage of high molecular weight species in the filter eluate pool or stream is reduced compared to a virus filtration unit operation comprising a virus filter alone or in combination with a surface modified membrane prefilter. In one embodiment, the bispecific T cell engager is an extended half-life bispecific T cell engager.

[0015] The invention also provides a method for producing a purified, formulated recombinant bispecific T cell engager, the method comprising purifying a harvested recombinant bispecific T cell engager via one or more chromatography unit operations; subjecting the purified recombinant bispecific T cell engager to ultrafiltration and diafiltration unit operations to result in a formulated bispecific T cell engager at a concentration of ≦5 g / L and subjecting the formulated bispecific T cell engager to a viral filtration unit operation; obtaining a purified, formulated recombinant bispecific T cell engager. In one embodiment, the formulated bispecific T cell engager is at a concentration of ≦3.2 g / L. In one embodiment, the formulated bispecific T cell engager is at a concentration of ≦1.79 g / L. In one embodiment, the bispecific T cell engager is an extended half-life bispecific T cell engager. In one embodiment, the ultrafiltration diafiltration unit operations are performed with a stabilized cellulosic hydrophilic membrane or a regenerated cellulose membrane. In one embodiment, the UF diafiltration unit operation is configured to produce up to 71.4 g / m of membrane area with an initial ultrafiltration target concentration of up to 3.20 g / L. 2In one embodiment, the UF diafiltration unit operation is carried out with a stabilized cellulosic hydrophilic membrane loaded to a maximum of 170 g / m per membrane area at an intermediate target excess concentration of up to 9 g / L with a diavolume of up to 13. 2 In one embodiment, the virus filtration unit operation is performed with a hydrophilized polyvinylidene fluoride (PVDF) hollow fiber filter, a cuprammonium regenerated cellulose hollow fiber filter, or a polyethersulfone (PES) parvovirus-retentive filter. In one embodiment, the virus filtration unit operation is performed using a cuprammonium regenerated cellulose hollow fiber filter and a formulated bispecific T cell engager at a concentration of ≦3.2 g / L. In one embodiment, the formulated bispecific T cell engager is at a concentration of ≦1.79 g / L. In one embodiment, the virus filtration unit operation is performed using a hydrophilized polyvinylidene fluoride (PVDF) hollow fiber filter and a formulated bispecific T cell engager at a concentration of ≦1.79 g / L. [Brief description of the drawings]

[0016] [Figure 1A] (A) shows a typical conventional processing step from UFDF operation to DP loading during the DS process. The conventional process can be subdivided into 10 steps or stages. The invention described herein reduces the number of steps or stages to 5, as shown in (B). [Figure 1B] (A) shows a typical conventional processing step from UFDF operation to DP loading during the DS process. The conventional process can be subdivided into 10 steps or stages. The invention described herein reduces the number of steps or stages to 5, as shown in (B). [Diagram 2] NWP % recovery after set of runs 1-3 for the center point of the multiple runs compared to the minimum % recovery. The black bars are the center points of the multiple runs. The grey stippled columns are the minimum % recovery. [Diagram 3]Formulation Buffer Matrix - Cuprammonium Regenerated Cellulose Filter. Flow decay versus throughput for runs in pH 4.2-high concentration [open black circle], pH 4.2-high volume [open black triangle], pH 4.2-extended retention [open black square], pH 4.2-midpoint [grey filled circle], pH 4.2-PVDF filter [black filled circle], and pH 5.0-low concentration [filled diamond pattern]. [Figure 4A] Product quality data Cuprammonium regenerated cellulose filter pH 4.2 midpoint [black bar], pH 4.2 high concentration [gray bar], pH 4.2 extended retention [open white bar], pH 4.2 high volume [solid diamond grid bar], pH 4.2 PVDF filter [patterned circle bar], pH 5.0 low concentration [square grid bar]. A: HMW% B: Clip% C: Basic D% Acidic. [Figure 4B] Product quality data Cuprammonium regenerated cellulose filter pH 4.2 midpoint [black bar], pH 4.2 high concentration [gray bar], pH 4.2 extended retention [open white bar], pH 4.2 high volume [solid diamond grid bar], pH 4.2 PVDF filter [patterned circle bar], pH 5.0 low concentration [square grid bar]. A: HMW% B: Clip% C: Basic D% Acidic. [Figure 4C] Product quality data Cuprammonium regenerated cellulose filter pH 4.2 midpoint [black bar], pH 4.2 high concentration [gray bar], pH 4.2 extended retention [open white bar], pH 4.2 high volume [solid diamond grid bar], pH 4.2 PVDF filter [patterned circle bar], pH 5.0 low concentration [square grid bar]. A: HMW% B: Clip% C: Basic D% Acidic. [Figure 4D]Product quality data Cuprammonium regenerated cellulose filter pH 4.2 midpoint [black bar], pH 4.2 high concentration [gray bar], pH 4.2 extended retention [open white bar], pH 4.2 high volume [solid diamond grid bar], pH 4.2 PVDF filter [patterned circle bar], pH 5.0 low concentration [square grid bar]. A: HMW% B: Clip% C: Basic D% Acidic. [Diagram 5] Flow rate versus load challenge for 0.001-m2 20N filtration cuprammonium regenerated cellulose filters at 1.77 g / L product [open black triangles], 3.15 g / L [gray filled diamonds], and 6.82 g / L [open black circles], [filled black squares]. All load materials were filtered at 19 PSI. [Figure 6A] Product quality data for molecule A in chromatography buffer matrix runs - 1.77 g / L, pH 5, 23 high pressure [black bars], 3.2 g / L, pH 5, 23 [gray bars], 1.77 g / L, pH 5, 28 [white unfilled bars], 1.77 g / L, pH 5, 23 low pressure [dotted circle bars], 6.82 g / L, pH 5.3, 28 [square grid bars], 6.82 g / L, pH 4.5, 28 [light gray bars], 1.77 g / L, pH 5, 23 medium pressure [filled diamond grid bars]. [Figure 6B] Product quality data for molecule A in chromatography buffer matrix runs - 1.77 g / L, pH 5, 23 high pressure [black bars], 3.2 g / L, pH 5, 23 [gray bars], 1.77 g / L, pH 5, 28 [white unfilled bars], 1.77 g / L, pH 5, 23 low pressure [dotted circle bars], 6.82 g / L, pH 5.3, 28 [square grid bars], 6.82 g / L, pH 4.5, 28 [light gray bars], 1.77 g / L, pH 5, 23 medium pressure [filled diamond grid bars]. [Figure 6C]Product quality data for molecule A in chromatography buffer matrix runs - 1.77 g / L, pH 5, 23 high pressure [black bars], 3.2 g / L, pH 5, 23 [gray bars], 1.77 g / L, pH 5, 28 [white unfilled bars], 1.77 g / L, pH 5, 23 low pressure [dotted circle bars], 6.82 g / L, pH 5.3, 28 [square grid bars], 6.82 g / L, pH 4.5, 28 [light gray bars], 1.77 g / L, pH 5, 23 medium pressure [filled diamond grid bars]. [Figure 6D] Product quality data for molecule A in chromatography buffer matrix runs - 1.77 g / L, pH 5, 23 high pressure [black bars], 3.2 g / L, pH 5, 23 [gray bars], 1.77 g / L, pH 5, 28 [white unfilled bars], 1.77 g / L, pH 5, 23 low pressure [dotted circle bars], 6.82 g / L, pH 5.3, 28 [square grid bars], 6.82 g / L, pH 4.5, 28 [light gray bars], 1.77 g / L, pH 5, 23 medium pressure [filled diamond grid bars]. [Figure 7] Hydraulic performance of BiTE® A at mid-pH, low concentration, low conductivity conditions (pH 5.0, 23 mS / cm, 1.75 g / L) for VPro alone [filled black circle], VPro+Shield [filled black triangle], VPro+Shield H [open square], VPro+VPF [filled grey circle], and VPro+X0SP [open black triangle]. [Figure 8] Hydraulic performance of BiTE A® at low pH, high and low concentration and conductivity conditions (pH 4.2, 23 or 28 mS / cm, 1.75 or 7 g / L): VPro [solid black circle], VPro+X0SP low pH [open black triangle], VPro+Shield low pH [solid black triangle], VPro+X0SP high concentration, low pH [solid grey triangle], VPro+Shield H high concentration, low pH [open circle], VPro+Shield high concentration, low pH [open black square]. [Figure 9]Hydraulic performance of BiTE A® at high pH, ​​low and high concentration and conductivity conditions (pH 6.0, 23 or 28 mS / cm, 1.8 or 7 g / L): VPro [closed circle], VPro+Shield H high pH, ​​low concentration [closed triangle], VPro+X0SP [closed grey triangle] high pH, ​​high concentration, VPro+Shield H [open circle] high pH, ​​high concentration, VPro+Shield [open square] high pH, ​​high concentration. [Figure 10A] A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 10B]A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 10C]A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 10D]A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 10E]A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 10F]A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 10G]A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 10H]A: HMW% product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. B: HMW% product quality data for molecule A - 7g / L, pH6 [black bar] and pH4.2 [grey bar]. C: Rce (clip%) product quality data for molecule A - 1.75g / L, pH5 [black bar], pH4.2 [grey bar] and pH6.0 [patterned bar]. D: Rce (clip%) product quality data for molecule A - 7g / L, pH6.0 [black bar] and pH4.2 [grey bar]. E: CEX acidity (%) product quality data for molecule A - 1.75g / L, pH5 [black bar], 4.2 [grey bar] and 6.0 [patterned bar]. F: CEX basicity (%) product quality data for molecule A - 1.75 g / L, pH 5 [black bars], pH 4.2 [grey bars] and pH 6.0 [patterned bars]. G: CEX acidity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. H: CEX basicity (%) product quality data for molecule A - 7 g / L, pH 6 [black bars] and pH 4.2 [grey bars]. [Figure 11] Hydrodynamic performance at midpoint pH and concentration between mAb [filled squares] and 1) BiTE® A X0SP / VPro [grey triangles], 2) BiTE® A VPF / VPro [open black circles]. [Figure 12] Hydrodynamic performance at high pH and concentration between mAb [filled squares] and BiTE®A X0SP / VPro [grey triangles]. [Figure 13] Hydraulic performance of BiTE® B at pH 5.9, 31 mS / cm, 1.8 g / L for VPro alone [filled black circle], VPro+Shield [filled black triangle], VPro+Shield H [open square], and VPro+VPF [filled grey circle], and VPro+X0SP [open black triangle]. [Figure 14]Hydraulic performance of BiTE® B for VPro+Shield H [open black squares], VPro+X0SP [filled grey triangles] at pH 5.9, 45 mS / cm, 1.81 g / L. Hydraulic performance of VPro+Shield H [filled black squares], VPro+X0SP [filled black triangles] at pH 4.2, 31 mS / cm. [Figure 15] BiTE® B product quality HMW% set point pH 5.9 [black bars], low pH 4.2 [grey bars], and high conductivity -45 mS / cm [patterned bars]. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Described herein is a process for biologics manufacturing that is advantageous in that it eliminates or combines steps required in the manufacture of drug substance (DS) and drug product (DP), allowing for a fully integrated, end-to-end continuous manufacturing process for the production of biologics. This process requires only one bioburden reduction filtration step before drug product fill / finish and one sterile filtration step after the UFDF operation. Stabilizing excipients such as polysorbate 80 (PS80), which are usually added after the first bioburden reduction filtration of the UFDF pool, are now combined directly into the UFDF operation, thereby eliminating an entire unit operation dedicated to excipient addition and second bioburden reduction filtration. The filtered bulk drug product is then subjected to sterile filtration and transferred to a filling station used to fill the primary drug product containers, which are then sealed, labeled, and packaged. The transfer of material from the drug product manufacturing station to the drug product processing station and subsequent drug product fill is performed within the holding times and holding temperatures supported by the process operating range. This eliminates the time-consuming freeze and thaw unit operations.

[0018] The present invention reduces the number of steps or stages in a typical manufacturing process by 10-5, as shown in Figure 1. The invention described herein also eliminates the need for drug substance storage from multiple freezing containers, dilution of the formulation, addition of excipients, and similar operations after thawing of the drug substance. It also eliminates the need for a drug substance holding tank prior to sterile filtration. The present invention allows for the use of the same drug substance collection vessel for delivery to and / or connection to the drug substance fill / finish site, or direct transfer, and use when collecting bulk drug substance samples for release assays.

[0019] The invention also allows for the elimination of redundant shipping sampling of the formulated protein and / or drug substance and drug product, and allows assays of attributes common to both to be done only once, such as at the drug product fill / finish stage, which may be combined with other drug product attribute testing.

[0020] The present invention also reduces labor and equipment-related costs by eliminating redundant unit operations, unnecessary collection and / or storage vessels, and the need for freezing and thawing and storage of frozen bulk drug substance. The present invention supports modular, flexible equipment design and the use of miniaturized equipment. Upstream and downstream unit operations can be done on a smaller scale in a continuous or semi-continuous manner. The present invention also allows for just-in-time manufacturing, increasing flexibility for manufacturing campaigns, and is useful in situations where there is low inventory demand for the product or situations that experience seasonal or other demand fluctuations. The present invention allows for minimization of process footprint by eliminating, combining and / or linking various unit operations, reducing the size of the equipment required, eliminating the need for physical separation of unit operations, liberalizing facility design, eliminating the need for separate gowning spaces and air-conditioned manufacturing space before and after virus filtration. The present invention provides a continuous manufacturing process of the product from cell culture to drug substance that can utilize sterile, single-use components. The continuous manufacturing process can be a closed process.

[0021] The present invention provides an integrated, continuous process for producing a recombinant biological product, comprising providing a purified recombinant protein of interest; concentrating or diluting the purified recombinant protein by ultrafiltration; buffer exchanging the purified recombinant protein into a desired formulation by diafiltration; further diluting or concentrating the formulated recombinant protein by ultrafiltration until a target concentration is reached; adding or combining at least one stability-enhancing excipient once the target concentration is reached; subjecting the resulting bulk drug substance to filtration to reduce bioburden; subjecting the resulting bulk formulation to sterile filtration; and subjecting the sterile bulk formulation to fill and finish operations, wherein neither the purified recombinant protein nor the bulk drug substance are subjected to freeze and thaw unit operations.

[0022] The invention provides a method for producing a recombinant protein formulation comprising expanding cells expressing a protein of interest to the N-1 stage; culturing the cells expressing the recombinant protein; recovering the recombinant protein via a harvest unit operation; purifying the harvested recombinant protein via at least one capture chromatography unit operation; purifying the recombinant protein via at least one polish chromatography unit operation; concentrating or diluting the purified recombinant protein by ultrafiltration; buffer exchanging the purified recombinant protein into a desired formulation by diafiltration; further concentrating or diluting the formulated, purified recombinant protein by ultrafiltration to reach a target concentration followed by adding one or more stability-enhancing excipients; subjecting the formulated drug substance to a single unit operation to reduce bioburden and result in a filtered bulk formulation; sterile filtering the bulk formulation; filling a primary formulation container with the sterile bulk formulation; and sealing, labeling, and packaging the primary formulation container, wherein neither the recombinant protein nor the drug substance are subjected to freeze and thaw unit operations.

[0023] As used herein, "drug substance" refers to a purified recombinant protein intended to provide pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease or to affect the structure or any function of any part of the human body. Typically, a drug substance comprises a formulated protein from a UFDF unit operation with the addition of one or more stability-enhancing excipients. "Purified recombinant protein" or "purified protein" are used interchangeably and refer to a recombinant protein that has been purified from undesirable proteins, polypeptides, impurities and / or other contaminants that would interfere with its therapeutic, diagnostic, preventative or other use.

[0024] As used herein, "formulation" refers to a finished dosage form that may contain one or more drug substances in association with one or more pharma- ceutically or physiologically acceptable carriers, diluents, and / or excipients. "Bulk formulation" or filtered bulk formulation are used interchangeably and are used to refer to drug substances after bioburden reduction filtration.

[0025] In one embodiment, the invention provides drug substances and formulations made by the methods described herein.

[0026] The purified recombinant protein is usually subjected to a UFDF unit operation before conversion to a drug substance. Ultrafiltration is usually divided into two parts: a first ultrafiltration step to partially concentrate or dilute the recombinant protein, followed by formulation of the recombinant protein with one or more pharma- ceutically or physiologically acceptable carriers, diluents, and / or excipients by buffer exchange using diafiltration, and a second ultrafiltration step to bring the formulated recombinant protein to the target concentration desired for the final formulation.

[0027] For modalities where recombinant proteins typically need to be concentrated to achieve a desired target concentration desired in the final formulation, such as monoclonal antibodies, the degree of concentration in the first ultrafiltration step depends on the desired target value for the formulation. Typically, the first ultrafiltration step brings the concentration to about half of the desired final target value. The degree of concentration during this first step can be greater or less depending on the circumstances, the desired final target dose, the nature of the recombinant protein, and / or other factors. For the second ultrafiltration step, the target concentration can be anywhere from 20 mg / ml to 40 mg / ml, or it can be higher than the desired final concentration of the formulation taking into account the holdup in the second ultrafiltration system; for example, a higher concentration is set for a larger holdup volume and a lower concentration is set for a smaller holdup volume, or a concentration closer to the desired formulation concentration.

[0028] For highly potent modalities such as bispecific T cell engagers, where the recombinant protein concentration may be higher than the desired final concentration for the formulation, the recombinant protein may be diluted to the desired final concentration during the UFDF unit operation.

[0029] UFDF filters are well known and common in the art and are commercially available from many sources. There are many types of materials available, such as regenerated cellulose Pellicon (MilliporeSigma, Danvers, MA), stabilized cellulose, Sartocon® Slice, Sartocon® ECO Hydrosart® (Sartorius, Goettingen, Germany), polyethersulfone (PES) membrane, Omega (Pall Corporation, Port Washington, NY). Depending on the scale of purification, typical filter sizes range from 0.11 m 2 Areas from less than 1.14m 2 Multiple filters can be used up to the capacity that the holder, skid, or physical mechanics of the UFDF system will allow or that is necessary to achieve the desired end of the production process. For example, in a clinical production setting, 11.4 m2 For commercial production scale, the range is >40m 2 The area may be .

[0030] Bispecific T cell engagers, BiTEs, are highly potent and prone to aggregation during the purification process. BiTEs are prone to aggregation, which can affect their concentration during UFDF procedures. BiTEs with extended half-life were applied to regenerated cellulose membranes with a diavolume of 13 at 170 g / m2 of membrane area. 2 It was found that even loadings as high as 71.4 g / m2 were still within the product profile. 2 Rinsing the stabilized cellulosic membrane with buffer after each cycle of loading with HLE BiTE® was sufficient to clean and did not affect future membrane performance, despite the higher loading and high initial concentration for at least three cycles, allowing optimal reuse of the TFF filters with buffer rinses between cycles without the use of caustic chemical cleaning solutions (sodium hydroxide), allowing for faster processing.

[0031] For bispecific T cell engagers, the stabilized cellulosic membrane may be loaded to an initial target excess concentration that is 2.5x the target concentration. In one embodiment, the target excess concentration is 1.1x to 2.5x. In one embodiment, the target excess concentration is 1.1x to 1.5x. In one embodiment, the target excess concentration is 1.5x to 2.5x.

[0032] Buffer exchange by diafiltration into the desired formulation buffer is usually performed before the second ultrafiltration step. The buffer containing the purified recombinant protein from the first ultrafiltration concentration is exchanged with one containing one or more pharma- ceutically or physiologically acceptable carriers, diluents and / or excipients that are desirable for formulation of the drug product and will act to achieve certain desired results in the final formulation, such as maintaining product quality, stability and / or integrity during subsequent steps, including but not limited to filtration, filling, lyophilization, freezing, packaging, storage, shipping, delivery, thawing and / or administration. Buffers can also be used to adjust attributes such as osmolality, conductivity and / or protein concentration of the final formulation. Components of the formulation may provide protection for the formulation and may be desirable to enhance and / or reduce certain attributes of the formulation, such as protection against degradation pathways; promoting water solubility; reducing toxicity and / or reactivity; providing rapid clearance; reducing immunogenicity; acting as cryoprotectants or lyoprotectants; stabilizing native conformation to maintain efficacy, potency, and safety; protecting against chemical and physical degradation; protein stabilization to reduce surface tension, protein-surface and protein-protein interactions; reducing hydrophobic interactions; optimizing conditions such as pH, ionic strength, and the like; and buffering and stabilization. Excipients are usually prepared in the form of one or more buffer solutions.

[0033] Pharmaceutically or physiologically acceptable carriers, diluents and / or excipients may include, but are not limited to, one or more of the following: a sterile diluent such as water for injection; saline solutions such as saline solution, neutral buffered saline, phosphate buffered saline, physiological saline, Ringer's solution, isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides which may serve as solvents or suspending media; polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid or glutathione; sugars such as glucose, mannose, sucrose or dextran, mannitol; proteins; non-ionic surfactants; detergents; emulsifiers; amino acids such as polypeptides or glycine; acetates, citrates or phosphates, agents for adjusting osmotic pressure, e.g., sodium chloride or dextrose; adjuvants (e.g., aluminum hydroxide); and preservatives. Excipients that are sensitive to concentration or filtration or for any other reason may require special handling or consideration and may be added during the second ultrafiltration step or after the UFDF unit operation.

[0034] Typically, after the UFDF unit operation, the UFDF pool is filtered to reduce bioburden, then recovered in an external holding tank, and a unit operation for the addition of stability-enhancing excipients to the UFDF pool is followed by another filtration to reduce bioburden of the formulated drug substance.

[0035] As described herein, the present invention eliminates the need for a separate unit operation to add a stability-enhancing excipient, such as polysorbate 80, to an external holding tank containing a reduced bioburden UFDF pool and filter again. The present invention provides for adding or combining such stability-enhancing excipients directly into the UFDF retentate tank. When an excipient is added to the UFDF retentate tank, it does not need to be passed through a UFDF filter. Access to the filter may be closed so that the excipient does not interact with the UFDF filter. In one embodiment, one or more stability-enhancing excipients are added to or combined with the formulated recombinant protein. In one embodiment, one or more stability-enhancing excipients are added to the formulated recombinant protein within the appropriate range. In a related embodiment, one or more stability-enhancing excipients are added directly to the ultrafiltration and diafiltration (UFDF) retentate tank. In one embodiment, one or more excipients are added to or combined with the formulated recombinant protein after reaching a target concentration. In one embodiment, one or more excipients are added to the formulated recombinant protein within the appropriate range after the target concentration is reached. The excipients can also be added directly to the holding vessel within the appropriate range along with the UFDF pool flow. In one embodiment, the stability enhancing excipients and the UFDF pool are added separately to the storage vessel.

[0036] Stability enhancing excipients include, but are not limited to, non-ionic surfactants, detergents, and / or emulsifiers. Non-ionic surfactants include, but are not limited to, poly-oxy-ethylene (PEO) surfactants, polyethylene oxide-polypropylene oxide block copolymers; polyoxyethylene (20) sorbitan monooleate; polysorbate 20 and 80, Tween® 20 and Tween® 80; polyethylene glycol (PEG), Pluronics; poloxamers such as poloxamer 188 and poloxamer 407.

[0037] In one embodiment, the stability-enhancing excipient is a non-ionic detergent or surfactant. In one embodiment, the stability-enhancing excipient is a poly-oxy-ethylene (PEO) surfactant. In one embodiment, the stability-enhancing excipient is selected from polysorbate 80 or polysorbate 20.

[0038] The amount of stability enhancing excipient depends on the desired final formulation of the drug product. For example, a typical range for polysorbate 80 is 0.001-0.1% (weight / volume). In one embodiment, the concentration of polysorbate 80 is 0.01% (weight / volume) in the drug substance formulation buffer. For excipients such as polysorbate 80, whose solutions may be viscous, dilution to 0.01% in the formulation buffer reduces the viscosity and simplifies flushing of lines and bioburden reduction filters.

[0039] In one embodiment of the invention, one or more additional formulated recombinant proteins and / or drug substances may be added prior to bioburden reduction filtration and / or sterile filtration to ultimately form the combined formulation.

[0040] After the UFDF unit operation and addition of any stability enhancing excipients, the drug substance is filtered to reduce bioburden and the pool is collected in a holding vessel such as a sterilized single-use storage bag. Prior to addition of the drug substance to the bioburden reduction filter, the lines connecting the UFDF unit to the bioburden reduction unit may be flushed with a formulation buffer containing the stabilizing excipients at the target concentration, and then the bioburden reduction filter may be saturated with the same buffer. This helps achieve the correct concentration of the stabilizing excipients in the formulated recombinant protein. As used herein, bioburden reduction refers to the removal of unwanted microorganisms from the formulation in the final formulation. Suitable filters are known and are widely used for bioburden reduction, such as SHC and PVDF filters and common 0.2 micron filters, and are commercially available from a number of sources.

[0041] In a typical biopharmaceutical manufacturing process, the drug substance would be frozen for ease of storage or transport to a drug processing facility. The present invention eliminates the freeze and thaw unit operations and the conversion of the drug substance to the drug product is instantaneous and continuous. This is useful for integrated end-to-end continuous therapeutic biopharmaceutical manufacturing platforms, platforms that are automated, platforms that operate with minimal or no operator intervention, just-in-time manufacturing platforms, production platforms where the demand for drug product is variable or limited, or where it is undesirable or impossible to maintain an inventory of frozen drug substance. It also reduces the amount and timing of attribute testing since attributes that are common between the drug substance and the drug product can be performed only once at the fill / finish stage of the drug product. Also, any additional processing of the drug substance after freeze / thaw that is required to convert to a bulk drug product is eliminated.

[0042] After the UFDF unit operation, one or more additional unit operations, such as viral filtration, may be performed. Multispecific modalities, due in part to their very specific design and function, can achieve the desired therapeutic potency at low concentrations, unlike monoclonal antibodies, which require much higher concentrations to achieve the desired potency. In particular, some bispecific antibodies, such as bispecific T cell engagers, can achieve the desired potency at very low concentrations and thus have drug substance formulation concentrations of <10 g / L, while for most therapeutic monoclonal antibodies, the drug substance formulation concentrations are much higher, 70 g / L or more. At such high concentrations, the formulated antibody solution can quickly clog a viral filter.

[0043] Because the pore size of the virus filter is so small, highly concentrated formulations, such as those containing monoclonal antibodies, will clog the filter at a much smaller volume. For highly concentrated antibody formulations >10 g / L, the filter or membrane area required to process such solutions would make their use in manufacturing impractical. In a typical sequence of operations for monoclonal antibody processing, virus filtration usually follows a polishing step at some point in the manufacturing process when the antibody pool is at its most dilute state. A subsequent UFDF operation concentrates the antibody formulation. For high potency bispecific T cell engagers that are at low concentrations both before and after UFDF, it has been found that the filter or membrane area required to process the formulated bispecific T cell engager is reasonable for virus filtration, regardless of the sequence of virus filter and UFDF operations, and virus filtration of formulated BiTE® is possible as described herein.

[0044] The invention also provides a method for reducing viral contamination in a composition comprising a recombinant bispecific T cell engager comprising providing a sample comprising less than 7.0 g / L of recombinant bispecific T cell engager at a pH of 6.0 or less and having a conductivity of 23-45 mS / cm; subjecting the sample to a virus filtration unit operation comprising a virus filter alone or in combination with a depth filter or a surface modified membrane prefilter; and collecting the eluate of the virus filter comprising the recombinant bispecific T cell engager in a pool or as a stream.

[0045] The invention also provides a method for reducing high molecular weight species during production of recombinant bispecific T cell engagers comprising: providing a sample comprising less than 7 g / L of recombinant bispecific T cell engager at a pH of 6.0 or less and having a conductivity of 23-45 mS / cm; subjecting the sample to a virus filtration unit operation comprising a virus filter in combination with a depth filter; and collecting the eluate of the virus filter in a pool or as a stream; wherein the percentage of high molecular weight species in the filter eluate pool is reduced compared to use of a virus filtration unit operation comprising a virus filter alone or in combination with a surface-modified membrane prefilter.

[0046] The invention also provides a method for producing a purified, formulated recombinant bispecific T cell engager comprising purifying a harvested recombinant bispecific T cell engager via one or more chromatography unit operations; subjecting the purified recombinant bispecific T cell engager to ultrafiltration and diafiltration unit operations to result in formulated bispecific T cell engager at a concentration of < 5 g / L and subjecting the formulated bispecific T cell engager to a viral filtration unit operation; obtaining a purified, formulated recombinant bispecific T cell engager.

[0047] As described herein, low concentration drug substance formulations containing bispecific T cell engager drug substance have been successfully processed through viral filtration procedures. Formulated bispecific T cell engagers having a concentration of <10 g / L, preferably ≦5 g / L, are within the scope of the present invention. Preferably, the formulated bispecific T cell engager has a concentration of ≦0.10 g / L, ≦0.5 g / L, ≦1 g / L, ≦2 g / L, ≦3 g / L, ≦4 g / L. In one embodiment, the concentration is ≦3.5 g / L. In one embodiment, the concentration is ≦1.79 g / L. In one embodiment, the concentration is between 1.59 g / L and 3.16 g / L. In one embodiment, the formulated bispecific T cell engager has a concentration of between 1.59 g / L and 1.79 g / L. In one embodiment, the formulated bispecific T cell engager has a concentration of between 1.79 g / L and 3.16 g / L. In one embodiment, the concentration of the formulated bispecific T cell engager is 1.59 g / L. In one embodiment, the concentration of the formulated bispecific T cell engager is 1.79 g / L. In one embodiment, the concentration of the formulated bispecific T cell engager is 3.2 g / L.

[0048] In one embodiment, the invention provides subjecting a formulated multispecific protein, a formulated multispecific protein with a stability enhancing agent, a bulk drug substance comprising a multispecific protein, and / or a bulk drug product comprising a multispecific protein to viral filtration. In one embodiment, the multispecific protein is a bispecific antibody. The viral filtration step may be followed by bioburden reduction and / or sterile filtration. A stability enhancing agent may be added to the viral filtration pool. Optionally, the viral filtration pool may be stored at 2-8°C for short term or at -70°C for long term.

[0049] The unit operations may be linked continuously or semi-continuously through the viral filtration step, the bioburden reduction filtration or sterile filtration step, or the fill / finish operation. The viral filtration and post viral filtration steps may be performed in the same space as the viral filtration step.

[0050] Non-enveloped viruses are difficult to inactivate without risk to the protein therapeutic being manufactured, but such viruses can be removed by size-based filtration methods that use filters with small pore sizes to remove viral particles. Virus filtration may be performed using micro- or nano-filters such as those available from Plavona® (Asahi Kasei, Chicago, IL), Virosart® (Sartorius, Goettingen, Germany), Viresolve® Pro (MilliporeSigma, Burlington, MA), Pegasus™ Prime (Pall Biotech, Port Washington, NY), CUNO Zeta Plus VR, (3M, St. Paul, Mn), and may occur at one or more steps in downstream operations of the biomanufacturing process. Typically, virus filtration precedes the UFDF operation, but may also occur after UFDF.

[0051] Bispecific T cell engagers such as HLE BiTE® are very potent and prone to aggregation during the purification process. Bispecific T cell engagers are sensitive to purification conditions and prone to aggregation, which can result in reduced throughput and increased flow rate decay during virus filtration operations. Prefilters may be used in combination with virus filters to help remove certain contaminants in the product pool or eluate stream before the pool or eluate is applied to the virus filter, maintaining a continuous flow during virus filtration operations and extending the life of the filter. Prefilters are commercially available and include surface-modified polyethersulfone membrane filters such as Viresolve® Pro Shield, Viresolve® Pro Shield H, and depth filters such as Viresolve® prefilters and Millistak+® HC Pro X0SP, all from MilliporeSigma (Burlington, Mass). As described herein, depth filter prefilters have been found to be particularly effective in virus filtration operations for bispecific T cell engagers.

[0052] There is little information on downstream processing of bispecific antibodies, so platforms developed for monoclonal antibodies are often applied (Shulka and Norman, Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates, in Process Scale Purification of Antibodies Second Edition, Uwe Gottswchalk editor, p559-594, John Wiley & Sons, 2017). However, these processes do not necessarily work the same way for bispecific proteins such as recombinant bispecific T cell engagers as for monoclonal antibodies. As described herein, the addition of a prefilter to a virus filter did not uniformly improve performance when processing recombinant bispecific T cell engager proteins with extended half-life, especially the bispecific T cell engagers with extended half-life. It has been found that limiting the concentration of extended half-life bispecific T cell engager proteins to less than 7.0 g / L at a pH of 6.0 or less with a conductivity of 23-45 mS / cm improves performance when the protein is subjected to a virus filtration unit operation comprising a virus filter alone or in combination with a depth filter prefilter or a surface modified membrane prefilter. In particular, the use of a depth filter prefilter in combination with a virus filter reduced flow attenuation and / or reduced % HMW compared to the use of a virus filter alone or in combination with a surface modified membrane prefilter.

[0053] The invention also provides a method for reducing flow decay and reducing high molecular weight species in a virus filtration unit operation during production of recombinant bispecific T cell engagers comprising: providing a sample comprising no more than 1.75 g / L of recombinant bispecific T cell engager at a pH of 4.2-6.0 and having a conductivity of 23-45 mS / cm; subjecting the purified recombinant bispecific T cell engager to a virus filtration unit operation comprising a virus filter in combination with a depth filter; and collecting the filter eluate in a pool or as a stream; wherein the percentage of high molecular weight species in the filter eluate pool or stream is reduced compared to a virus filtration unit operation comprising a virus filter alone or in combination with a surface modified membrane prefilter.

[0054] In one embodiment, the pH of the pool or stream is 4.0-6.0. In one embodiment, the pH of the pool or stream is 4.2-6.0. In a related embodiment, the pH of the pool or stream is 4.2-5.9. In a related embodiment, the pH of the pool or stream is 4.2-5.0. In one embodiment, the pH of the pool or stream is 5.0-6.0. In one embodiment, the pH of the pool or stream is 5.0-5.9. In one embodiment, the conductivity of the pool or stream is 23-45. In one embodiment, the conductivity of the pool or stream is 23-32. In one embodiment, the conductivity of the pool or stream is 23-28. In one embodiment, the concentration of the extended half-life bispecific T cell engager is 1.75-7.0 g / L. In one embodiment, the concentration of the extended half-life bispecific T cell engager is 7.0 g / L. In one embodiment, the concentration of the extended half-life bispecific T cell engager is 1.75 g / L. In a related embodiment, the concentration of the extended half-life bispecific T cell engager is 1.75-1.18 g / L.

[0055] In one embodiment, the pH is 5.0, the concentration of the extended half-life bispecific T cell engager is 1.75 g / L. In a related embodiment, the pH is 6.0, the concentration of the extended half-life bispecific T cell engager is 7.0 g / L, and the conductivity is 28 mS / cm. In one embodiment, the pH is 5.9, the concentration of the extended half-life bispecific T cell engager is 1.81 g / L, and the conductivity is 31.36-45 mS / cm. In one embodiment, the pH is 4.2-5.9, the concentration of the extended half-life bispecific T cell engager is 1.75-1.81 g / L, and the conductivity is 23-45 mS / cm. In one embodiment, the pH is 4.2-5.0, the concentration of the extended half-life bispecific T cell engager is 1.75 g / L, and the conductivity is 23 mS / cm. In one embodiment, the pH is 5.9, the concentration of the extended half-life bispecific T cell engager is 1.81 g / L and the conductivity is 31.36-45 mS / cm. In one embodiment, the purified recombinant extended half-life bispecific T cell engager is 7.0 g / L or less, has a pH of 6.0 or less and a conductivity of 23-45 mS / cm.

[0056] In one embodiment, the virus filtration unit operation comprises a virus filter in combination with a depth filter prefilter. In a related embodiment, the depth filter prefilter is an absorbent depth filter or a synthetic depth filter. In one embodiment, the virus filtration unit operation comprises a virus filter in combination with a surface-modified membrane prefilter. In a related embodiment, the virus filtration unit operation comprises a virus filter in combination with a surface-modified polyethersulfone membrane prefilter. In one embodiment, the virus filtration unit operation comprises only a virus filter.

[0057] The filtered bulk drug product may also be subjected to bioburden reduction filtration and / or sterile filtration to ensure that it is free of viable microorganisms, and then introduced into the aseptic processing equipment used to fill the primary drug product containers, which are then sealed, labeled, and packaged.

[0058] A sterile processing facility is a facility that is maintained with minimized sources of contaminants that may affect the sterility of the drug product. Such a facility may be divided into a clean room with one or more filling stations for drug product fill / finish, each of which includes one or more automated fillers with multiple needles for filling multiple drug product containers at one time. The sterile processing facility may also be a freestanding globeless aseptic isolator station. Such a station may be located in an open ballroom manufacturing facility, in particular such a station may be located in or near the drug substance preparation area. Such modular globeless aseptic isolators for liquid and lyophilized drug products include, but are not limited to, Vanrx (Barnaby, British Columbia, Canada). Such a system allows for the development of a continuous system that does not require operator intervention. Small modular workstations with robots to perform material handling, filling and closing activities within a completely enclosed isolator may allow for a reduction in the size of the manufacturing plant, allowing for flexible space for modular and reconfigurable use, but may require the intervention of several operators. The present invention is fully robotic, involves aseptic filling in a globeless isolator, and can leverage existing single-use assemblies to create new flow paths with low capital and a smaller footprint than traditional built-in-place equipment.

[0059] The present invention provides a method for reducing the manufacturing footprint for a drug product production process that includes subjecting a purified recombinant protein of interest to a UFDF unit operation until a target concentration is reached; adding at least one stability enhancing excipient directly to a UFDF retentate tank; subjecting the bulk drug substance to a single unit operation to reduce bioburden and then subjecting it to sterile filtration; and subjecting the bulk drug product to a fill and finish unit operation, where neither the recombinant protein nor the drug substance is subjected to freeze and thaw unit operations. A viral filtration unit operation may precede or follow the UFDF operation.

[0060] In one embodiment of the present invention, the bulk formulation is sent to a sterile processing facility and sterile filtered before filling / finishing. In one embodiment, the sterile processing facility includes at least one filling station. In one embodiment, the filtered bulk formulation is in a storage container that can be delivered to the sterile processing facility. In another embodiment, the storage container can be directly connected to the sterile processing facility. In one embodiment, the formulation is filtered into a storage bag that is sent and / or connected directly to the sterile processing facility. In one embodiment, the bulk formulation can be sent directly from the bioburden reduction filtration to the sterile processing facility via piping or other connection.

[0061] In one embodiment, the bulk drug formulation is delivered to a sterile processing facility, which may be, for example, a robotic unit such as a gloveless sterile isolator. In one embodiment, the robotic unit has a connection to a storage vessel or filter that contains or processes the bulk drug formulation. The ability to directly couple drug substance processing to drug formulation processing, particularly by directly coupling to a robotic filter, offers the potential to reduce the footprint of the process. Compressing the process and eliminating redundant or unnecessary equipment or process steps, and elimination of freeze / thaw of the drug substance allows for the design and execution of a process with a footprint of 3,000 square feet or less.

[0062] In one embodiment of the invention, the primary product container is filled with the sterile bulk product. In another embodiment, the primary product container is sealed, labeled, and packaged. In one embodiment, the primary product container is a vial, an ampoule, a cartridge, a syringe or a syringe-containing device, or other suitable storage or delivery device, apparatus, or system.

[0063] The present invention provides a method for reducing drug product loss and / or destabilization during recombinant therapeutic protein manufacturing that includes subjecting a purified recombinant protein of interest to a UFDF unit operation; adding at least one stability-enhancing excipient to a UFDF retentate tank once a target concentration has been reached; and subjecting the UFDF pool to a single filtration to reduce bioburden and result in bulk drug substance, where neither the recombinant protein nor the drug substance is subjected to a freeze and thaw unit operation. A viral filtration unit operation may precede or follow the UFDF operation.

[0064] The proteins that make up drug substances are the result of a delicate balance between various interactions, including covalent bonds, hydrophobic interactions, electrostatic interactions, hydrogen bonds, and van der Waals forces, that form and maintain their folded three-dimensional structure. The folded state of a protein is only slightly more stable than the unfolded state, and changes in the protein's environment can induce degradation or inactivation, which directly impacts product quality.

[0065] The present invention reduces the number of bioburden reduction filtration steps, which is advantageous in reducing product losses due to volume holdup during filtration and avoiding any impact on product quality and protein structure due to shear-induced PQ changes that may result from multiple filtrations. It also streamlines the manufacturing process and increases compatibility with continuous manufacturing platforms; it reduces the footprint of drug substance manufacturing facilities and is advantageous in that it shortens manufacturing turnaround times and potentially allows for earlier availability of packaged drug product. There is also cost savings and waste reduction compared to typical biologics manufacturing platforms, where three or more bioburden reduction filters and associated holding tanks or collection vessels can be used from the UFDF unit operation to the fill / finish of the drug product.

[0066] The method of the present invention eliminates the "freeze and thaw unit operation" which is freezing, freezing storage, thawing, mixing and storage of the thawed drug substance. Freezing and thawing of bulk drug substance during manufacturing can be detrimental to protein stability and affect product quality. Ice-liquid interface, freeze concentration (concentration of protein as liquid freezing can result in changes in protein structure), excipient crystallization, pH shift (due to selective precipitation of buffer components, destabilizing the protein), increased protein concentration can result in aggregation or precipitation, cold denaturation (spontaneous unfolding at low temperature), container surface interactions, leachables and extractables from the container. (Rathore and Rajan, Biotechnol. Prog. 24:504-514, 2008).

[0067] The terms "polynucleotide" or "nucleic acid molecule" are used interchangeably throughout and include both single-stranded and double-stranded nucleic acids, and include genomic DNA, RNA, mRNA, cDNA, or synthetic origin or some combination thereof that is not associated with a sequence normally found in nature. The term "isolated polynucleotide" or "isolated nucleic acid molecule" specifically refers to a sequence of synthetic origin or one that is not normally found in nature. An isolated nucleic acid molecule that includes a specified sequence may, in addition to a sequence that expresses a protein of interest, include coding sequences for up to 10 or even up to 20 other proteins or portions thereof, may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequence, and / or may include vector sequences. The nucleotides that include the nucleic acid molecule may be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. Modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and modifications of internucleotide linkages such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoroaniladates and phosphoroamidates.

[0068] As used herein, the term "isolated" means (i) free from at least some other proteins or polynucleotides with which it is normally found; (ii) substantially free from other proteins or polynucleotides from the same source, e.g., the same species; (iii) separated from at least about 50 percent of the polypeptides, polynucleotides, lipids, carbohydrates or other materials with which it is naturally associated; (iv) operably associated (by covalent or noncovalent interactions) with polypeptides or polynucleotides with which it is not naturally associated; or (v) not naturally occurring.

[0069] The terms "polypeptide" or "protein" are used interchangeably throughout and refer to molecules that contain two or more amino acid residues linked together by peptide bonds. Polypeptides and proteins also include polymers that have one or more deletions, insertions, and / or substitutions from the amino acid residues of the native sequence, i.e., polypeptides or proteins produced by naturally occurring non-recombinant cells, or molecules produced by genetically engineered or recombinant cells that have one or more deletions, insertions, and / or substitutions from the amino acid residues of the native protein's amino acid sequence. Polypeptides and proteins also include amino acid polymers in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. Polypeptides and proteins also include modifications, including, but not limited to, glycosylation, lipid conjugation, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. The terms "isolated protein," "isolated recombinant protein," or "purified recombinant protein," may be used interchangeably, and refer to a polypeptide or protein of interest that has been purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research, or other use. In particular, drug substances and pharmaceutical preparations made from recombinant proteins of interest processed using the invention as described herein may be referred to as "recombinant protein preparations," "recombinant biologic therapeutics."

[0070] Polypeptides and proteins, including protein therapeutics, are of scientific or commercial interest. Proteins of interest include, inter alia, secreted proteins, non-secreted proteins, intracellular proteins, or membrane-bound proteins. Proteins of interest may be produced by recombinant animal cell lines using the methods described herein and may be referred to as "recombinant proteins" or "recombinant protein therapeutics." The expressed protein may be produced intracellularly or secreted into the culture medium where it may be recovered and / or collected. Proteins of interest may include, for example, proteins that exert a therapeutic effect by binding to a target, particularly those listed below, targets derived therefrom, targets related thereto, and modifications thereof.

[0071] The protein of interest may include an "antigen-binding protein." An antigen-binding protein refers to a protein or polypeptide that contains an antigen-binding region or portion that has a strong affinity for another molecule (antigen) to which it binds. Antigen-binding proteins include antibodies, peptibodies, antibody fragments, antibody derivatives, antibody analogs, fusion proteins (including single-chain variable fragments (scFv) and two-chain (bivalent) scFv), DARPins®, muteins, multispecific proteins, bispecific proteins, xMAbs, as well as chimeric antigen receptors (CAR or CAR-T) and T cell receptors (TCR).

[0072] "Multispecific," "multispecific protein," and "multispecific antibody" are used herein to refer to proteins that have been engineered to simultaneously bind and neutralize at least two different antigens or at least two different epitopes on the same antigen. For example, multispecific proteins can be engineered to target immune effectors to tumors or infectious agents in combination with targeted cytotoxic agents. These multispecific proteins have been found to be useful in a variety of applications, such as redirecting immune effector cells to tumor cells, blocking signaling pathways to alter cell signaling, targeting tumor angiogenesis, in cancer immunotherapy by blocking cytokines, and as pretargeting delivery vehicles for drugs such as chemotherapeutic agents, radiolabeling (to improve detection sensitivity) and nanoparticles (to be directed to specific cells / tissues such as cancer cells).

[0073] The most common and most diverse group of multispecific proteins are those that bind two antigens and are referred to herein as "bispecifics," "bispecific proteins," and "bispecific antibodies." Bispecific proteins can be divided into two broad categories: immunoglobulin G (IgG)-like molecules and non-IgG-like molecules. IgG-like molecules retain Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP), and the Fc region helps improve solubility and stability and facilitates some purification procedures. Non-IgG-like molecules are smaller and have enhanced tissue penetration (see Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Fan et al., J Hematol & Oncology 8:130-143, 2015; Spiess et al., Mol Immunol 67, 95-106, 2015; Williams et al., Chapter 41 Process Design for Bispecific Antibodies in Biopharmaceutical Processing Development, Design and Implementation of Manufacturing Processes, Jagschies et al., eds., 2018, pages 837-855). Bispecific proteins may have binding specificities for different antigens or several epitopes and be used as a framework for additional components that increase the binding specificity of the molecule.

[0074] Formats for bispecific proteins, including bispecific antibodies, are constantly evolving and include quadromas, knobs-in-holes, cross-Mab, dual variable domain IgG (DVD-IgG), IgG-single chain Fv (scFv), scFv-CH3 KIH, dual acting Fab (DAF), half molecule exchange, κλ-body, tandem scFv, scFv-Fc, diabody, single chain diabody (sc diabody), sc diabody-CH3, triple body, mini antibody, minibody, TriBi minibody, tandem diabody, sc diabody-HAS, tandem scFv-toxin, dual affinity retargeting molecule (DART), nanobody, nanobody-HSA, dock -and-lock (DNL), strand-exchange engineered domain SEED body, Triomab, Leucine zipper (LUZ-Y), XmAb®; Fab-arm exchange, DutaMab, DT-IgG, charge pair, Fcab, orthogonal Fab, IgG(H)-scFv, scFV-(H)IgG, IgG(L)-scFV, IgG(L1H1)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-VV(L)-IgG, KIH IgG-scFab, 2scFV-IgG, IgG-2scFv, scFv4-Ig, Zybody, DVI-Ig4 (quadrature function), Fab-scFv, scFv-CH-CL-scFV, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, intrabody, ImmTAC, HSABody, IgG-IgG, Cov-X-Body, scFv1-PEG-scFv2, bispecific T cell engager (BiTE®) and half-life extended bispecific T cell engager (HLE BiTE) (Fan supra; Spiess supra; Sedykh supra; Seimetz et al.,Cancer Treat Rev 36(6)458-67,2010;Shulka and Norman,Chapter 26 Downstream Processing of Fc Fusion Proteins, Bispecific Antibodies, and Antibody-Drug Conjugates,in Process Scale Purification of Antibodies Second Edition,Uwe Gottswchalk editor,p559-594,John Wiley & Sons,2017;Moore et al.,MAbs 3:6,546-557,2011).

[0075] Some embodiments include bispecific T cell engager (BiTE®) antibody constructs, which are recombinant protein constructs made from two flexibly linked antibody-derived binding domains (see WO 99 / 54440 and WO 2005 / 040220). One binding domain of the construct is specific for a selected tumor-associated surface antigen on target cells, such as EGFRvIII, MSLN, CDH19, DLL3, CD19, CD33, CD38, FLT3, CDH3, BCMA, PSMA, MUC17, CLDN18.2, or CD70; the second binding domain is specific for CD3, a subunit of the T cell receptor complex on T cells. BiTE® constructs may also include the ability to bind a context-independent epitope at the N-terminus of the CD3s chain (WO 2008 / 119567) to more specifically activate T cells. BiTE® antibody constructs with extended half-life are those that include fusions of small bispecific antibody constructs, preferably with larger proteins that do not interfere with the therapeutic effect of the BiTE® antibody construct. Examples include bispecific T cell engagers that include bispecific Fc molecules, as described, for example, in US2014 / 0302037, US2014 / 0308285, WO2014 / 151910, and WO2015 / 048272. Another strategy is the use of human serum albumin (HAS) fused to the bispecific molecule, or simply fusion of a human albumin binding peptide (see, for example, WO2013 / 128027, WO2014 / 140358). Another HLE BiTE® strategy involves fusing a first domain that binds to a target cell surface antigen, a second domain that binds to an extracellular epitope of the human and / or rhesus CD3e chain, and a third domain that is a specific Fc modality (WO 2017 / 134140).

[0076] In some embodiments, the bispecific protein is selected from the group consisting of blinatumomab, catumaxomab, ertumasomab, solitomab, targomiR, rutikizumab (ABT981), vanucizumab (RG7221), lemtolumab (ABT122), ozoralizumab (ATN103), flotetuzumab (MGD006), pasotuxizumab (AMG112, MT112), lymphomun (FBTA05), (ATN-103), AMG103 (anti-CD19xanti-CD3 BiTE® antibody), AMG211 (MT111, Medi-1565) (anti-carcinoembryonic antigen x anti-CD3 antibody), AMG330 (anti-CD33xanti-CD3 BiTE® antibody), AMG212 (anti-PSMAxanti-CD3 BiTE® antibody), AMG160 (anti-PSMAx anti-CD3 BiTE® antibody), AMG420 (B1836909), (anti-BCMAx anti-CD3 BiTE® antibody), AMG-110 (MT110), AMG562 (anti-CD19x anti-CD3 BiTE® antibody), AMG596 (anti-EGFRvIIIx anti-CD3 BiTE® antibody), AMG427 (extended half-life anti-FLT3x anti-CD3 BiTE® antibody), AMG673 (extended half-life anti-CD33x anti-CD3 BiTE® antibody), AMG675 (extended half-life anti-DLL3x anti-CD3 BiTE® antibody), AMG701 (extended half-life anti-BCMAx anti-CD3 BiTE® antibody), AMG424 (anti-CD38 anti-CD3Xmab), MDX-447, TF2, rM28, HER2Bi-aATC, GD2Bi-aATC, MGD006, MGD007, MGD009, MGD010, MGD011 (JNJ64052781), IMCgp100, Indium-labeled IMP-205, xm734, LY3164530, OMP-305BB3, REGN1979, COV322, ABT112, ABT165, RG-6013 (ACE910), RG7597 (MEDH7945A), RG7802, RG7813 (RO6895882), RG7386, BITS7201A (RG7990), RG7716, BFKF8488A (RG7992), MCLA-128, MM-111, MM141, MOR209 / ES414, MSB0010841, ALX-0061, ALX0761, ALX0141; BII034020, AFM13, AFM11, SAR156597, FBTA05, PF06671008, GSK2434735, MEDI3902, MEDI0700, MEDI7352, and molecules or variants or analogs thereof and biosimilars of any of the foregoing.

[0077] Multispecific proteins also include triabodies capable of binding to multiple targets, tetravalent bispecific antibodies, multispecific proteins without an antibody component such as diabodies, triabodies or tetrabodies, minibodies, and single chain proteins. Coloma, MJ, et. al., Nature Biotech. 15 (1997) 159-163.

[0078] An scFv is a single chain antibody fragment that has the variable regions of the heavy and light chains of an antibody linked together. See U.S. Patent Nos. 7,741,465 and 6,319,494 and Eshhar, et al., Cancer Immunol. Immunotherapy (1997) 45:131-136. An scFv retains the ability of the parent antibody to specifically interact with a target antigen.

[0079] The term "antibody" includes reference to both glycosylated and nonglycosylated immunoglobulins of any isotype or subclass, or antigen-binding regions thereof that compete with the intact antibody for specific binding. Unless otherwise specified, antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, hetero-IgG, bispecific, and oligomers or antigen-binding fragments thereof. Antibodies include IgG1, IgG2, IgG3, or IgG4 types. Also included are Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single chain antibody molecules, single domain V H Also included are proteins having antigen-binding fragments or regions, such as H, complementarity determining region (CDR) fragments, scFvs, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a target polypeptide.

[0080] Also included are human, humanized, and other antigen binding proteins, such as human and humanized antibodies, that do not produce a significant adverse immune response when administered to humans.

[0081] Also included are modified proteins, such as proteins that are chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. Also included are proteins that further include one or more post-translational modifications, which may be made by cell engineering systems, or modifications introduced ex vivo by enzymatic and / or chemical methods, or otherwise introduced.

[0082] Proteins of interest may also include recombinant fusion proteins that contain multimerization domains, such as, for example, leucine zippers, coiled coils, the Fc portion of an immunoglobulin, etc. Also included are proteins that contain all or part of the amino acid sequence of differentiation antigens (referred to as CD proteins) or their ligands, or proteins substantially similar to any of these.

[0083] In some embodiments, the protein may include a colony stimulating factor, such as granulocyte colony stimulating factor (G-CSF). Such G-CSF agents include, but are not limited to, Neupogen® (filgrastim) and Neulasta® (pegfilgrastim). Also included are Epogen® (epoetin alfa), Aranesp® (darbepoetin alfa), Dynepo® (epoetin delta), Mircera® (methoxypolyethylene glycol-epoetin beta), Hematide®, MRK-2578, INS-22, Retacrit® (epoetin zeta), Neorecormon® (epoetin beta), Silapo® (epoetin zeta), Binocrit® (epoetin alpha), epoetin alfa, and the like. Also included are erythropoiesis stimulating agents (ESAs) such as αHexal, Abseamed® (epoetin alfa), Ratioepo® (epoetin theta), Eporatio® (epoetin theta), Biopoin® (epoetin theta), epoetin alfa, epoetin beta, epoetin zeta, epoetin theta, and epoetin delta, epoetin omega, epoetin iota, tissue plasminogen activator, GLP-1 receptor agonists, and molecules or variants or analogs thereof and biosimilars of any of the foregoing.

[0084] In some embodiments, the proteins may include proteins that specifically bind to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, osteoinductive factors, insulin and insulin-related proteins, coagulation and coagulation-related proteins, colony stimulating factors (CSFs), other blood and serum proteins, blood group antigens; receptors, receptor-related proteins, growth hormones, growth hormone receptors, T cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0085] In some embodiments, the protein binds to one or more of the following, alone or in any combination: CD proteins, including but not limited to CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD30, CD33, CD34, CD38, CD40, CD70, CD123, CD133, CD138, CD171, and CD174; HER receptor family proteins, including HER2, HER3, HER4; EGF receptor EGFRvIII; cell adhesion molecules, such as LFA-1, Mol, p15. 0, 95, VLA-4, ICAM-1, VCAM, and alpha v / beta 3 integrin, growth factors including, but not limited to, vascular endothelial growth factor ("VEGF"), VEGFR2, growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian inhibitory substance, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), nerve growth factors such as NGF-beta, platelet derived growth factors (PDGF), including fibroblast growth factors such as aFGF and bFGF. Insulin and insulin-related proteins including, but not limited to, cell growth factor, epidermal growth factor (EGF), Cripto, transforming growth factors (TGFs) including TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, insulin-like growth factors-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins. Other blood and serum proteins, including, but not limited to, coagulation and coagulation-related proteins such as factor VIII, tissue factor, von Willebrand factor, plasminogen activators such as protein C, alpha-1-antitrypsin, urokinase, and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin, and thrombopoietin receptors, colony stimulating factors (CSFs), including M-CSF, GM-CSF, and G-CSF, among others, albumin, IgE, and blood group antigens, e.g.,(x) receptors and receptor-associated proteins, including flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor, and T cell receptor; (x) neurotrophic factors, including, but not limited to, bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); (xi) relaxin A chain, relaxin B chain, and prorelaxin, interferons, including, for example, interferon-alpha, -beta, and -gamma, interleukins (IL), for example, IL-1 through IL-10, IL-12, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49 ...9, IL-40, IL-41, IL-42, IL-43, IL-44 (xiv) viral antigens including, but not limited to, AIDS envelope virus antigens, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (Regulated Antigens of Antibodies), and the like. on activation normal T-cell expressed and secreted), mouse gonadotropin-related peptide, Dnase, FR-alpha, inhibin, and activin, integrin, protein A or D, rheumatoid factor, immunotoxin, bone morphogenetic protein (BMP), superoxide dismutase, surface membrane protein, decay accelerating factor (DAF), AIDS envelope, transport protein, homing receptor, MIC (MIC-a, MIC-B), ULBP 1-6, EPCAM, addressin, regulatory protein, immunoadhesin, antigen-binding protein, somatropin, CTGF, CTLA4, eotaxin-1, MUC1, CEA, c-MET, claudin-18, GPC-3, EPHA2, FPA, LMP1, MG7, NY-ESO-1, PSCA, ganglioside GD2, ganglioside GM2, BAFF, ICOS, OPGL (RANKL), myostatin, Dickkopf-1 (DKK-1), Ang2, NGF, IGF-1 receptor, hepatocyte growth factor (HGF), TRAIL-R2, c-Kit,B7RP-1, PSMA, NKG2D-1, programmed cell death protein 1 and ligand, PD1 and PDL1, mannose receptor / hCGβ, Hepatitis C virus, mesothelin dsFv [PE38 conjugate, Legionella pneumophila pneumophila) (lly), IFN gamma, interferon gamma-inducible protein 10 (IP10), IFNAR, TALL-1, thymic stromal lymphopoietin (TSLP), proprotein convertase subtilisin / kexin type 9 (PCSK9), stem cell factor, Flt-3, calcitonin gene-related peptide (CGRP), OX40L, α4β7, platelet specific (platelet glycoprotein Iib / IIIb (PAC-1), transforming growth factor beta (TFGβ), zona pellucida sperm-binding protein 3 (ZP-3), TWEAK, TSLP, platelet-derived growth factor receptor alpha (PDGFRα), sclerostin, Jagged-1, and biologically active fragments or variants of any of the foregoing.

[0086] AMG506 (FAPx4-1BB targeting DARPin®), AMG592 (IL2 mutein Fc fusion), AMG890 (interfering RNA Lp(a)), AMG119 (DLL3 CART).

[0087] In another embodiment, the protein is selected from the group consisting of abciximab, adalimumab, adecatumumab, aflibercept, alemtuzumab, alirocumab, anakinra, atacicept, basiliximab, belimumab, bevacizumab, biosuzumab, blinatumomab, brentuximab vedotin, brodalumab, cantuzumab mertansine, canakinumab, cetuximab, certolizumab pegol, conatumumab, daclizumab, denosumab, eculizumab, edrecolomab, efalizumab, epratuzumab, erenumab, etanercept, etelcalcetide, evolocumab, galiximab, ganitumab, gemtuzumab, golimumab, ibritumomab tiuxetan, infliximab, ipilimumab, , ixekizumab, lerdelimumab, lumiliximab, mapatuzumab, motesanib diphosphate, muromonab-CD3, natalizumab, nesiritide, nimotuzumab, nivolumab, ocrelizumab, ofatumumab, omalizumab, oprelvekin, palivizumab, panitumumab, pembrolizumab, pertuzumab, pexelizumab, ranibizumab These include, but are not limited to, rilotumumab, rituximab, romiplostim, romosozumab, sargramostim, tezepelumab, tocilizumab, tositumomab, trastuzumab, tratuzumap, ustekinumab, vedolizumab, visilizumab, volociximab, zanolimumab, zalutumumab, and biosimilars of any of the foregoing.

[0088] Proteins according to the invention encompass all of the above and further include antibodies comprising one, two, three, four, five or six of the complementarity determining regions (CDRs) of any of the above antibodies. Also included are variants comprising regions that are 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, particularly 97% or more, more particularly 98% or more, even more particularly 99% or more amino acid sequence identical to the reference amino acid sequence of the protein of interest. Identity in this regard can be determined using a variety of well-known and readily available amino acid sequence analysis software. Preferred software includes those that implement the Smith-Waterman algorithm, which is considered a satisfactory solution to the problem of searching and aligning sequences. Other algorithms may also be employed, especially where speed is an important consideration. Commonly used programs for DNA, RNA, and polypeptide alignment and homology matching that can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter of which is an implementation of the Smith-Waterman algorithm for running on massively parallel processors created by MasPar.

[0089] Also provided herein are expression systems and constructs in the form of plasmids, expression vectors, transcription cassettes or expression cassettes comprising at least one of the above nucleic acid molecules, and host cells comprising such expression systems or constructs. As used herein, "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, transposon, cosmid, chromosome, virus, viral capsid, virion, naked DNA, complexed DNA, etc.) suitable for use in transferring and / or transporting information-encoding proteins to a host cell and / or a specific location and / or compartment within a host cell. Vectors may include viral and non-viral vectors, non-episomal mammalian vectors. Vectors are often referred to as expression vectors, e.g., recombinant expression vectors and cloning vectors. A vector may be introduced into a host cell to allow the vector to replicate itself, thereby amplifying copies of the polynucleotides contained therein. Cloning vectors may contain sequence components that generally include, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These factors can be selected as needed by those skilled in the art.

[0090] A "cell" or "cells" includes any prokaryotic or eukaryotic cell. Cells may exist either ex vivo, in vitro or in vivo, either separately or as part of a higher order structure such as a tissue or organ. Cells include "host cells", also referred to as "cell lines", that have been genetically engineered to express a polypeptide of commercial or chemical interest. Host cells are usually derived from a lineage arising from a primary culture that can be maintained in culture for an indefinite period of time. Genetic engineering of host cells includes transfecting, transforming or transducing the cells with recombinant polynucleotide molecules and / or otherwise modifying them (e.g., by homologous recombination and gene activation, or fusion of recombinant cells with non-recombinant cells) to cause the host cells to express a desired recombinant polypeptide. Methods and vectors for genetically engineering cells and / or cell lines to express a polypeptide of interest are well known to those of skill in the art; for example, various techniques are described in Current Protocols in Molecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1990, and updated quarterly); Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989); Kaufman, RJ, Large Scale Mammalian Cell Culture, 1990, pp. 15-69.

[0091] A host cell can be any prokaryotic cell (e.g., E. coli) or eukaryotic cell (e.g., yeast cells, insect cells, or animal cells (e.g., CHO cells). Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or gene transfer techniques.

[0092] In one embodiment, the cell is a host cell. When the host cell is cultured under appropriate conditions, it expresses the protein of interest, which can then be recovered from the culture medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if it is not secreted). The selection of an appropriate host cell will depend on various factors, such as the desired expression level, the modification of the polypeptide that is desired or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule.

[0093] "Culture" or "culturing" refers to the growth and propagation of cells outside of a multicellular organism or tissue. Culture conditions suitable for mammalian cells are known in the art. Cell culture medium and tissue culture medium are used interchangeably to refer to a medium suitable for the growth of host cells in in vitro cell culture. Typically, cell culture media contain buffers, salts, an energy source, amino acids, vitamins and trace essential elements. Any medium capable of supporting the growth of suitable host cells in culture may be used. Cell culture media, which may be further supplemented with other components that maximize cell growth, cell viability, and / or recombinant protein production in a particular cultured host cell, are commercially available and include, among others, serum-free media such as RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and EX-CELL™ 300 series, which may be obtained from American Type Culture Collection or SAFC Biosciences, and other vendors. Cell culture media may be serum-free, protein-free, growth factor-free, and / or peptone-free media. Cell cultures may also be enriched by the addition of nutrients and used at higher than their usual recommended concentrations.

[0094] Various media formulations may be used during the culture period, for example, to facilitate the transition from one stage (e.g., growth stage or phase) to another stage (e.g., production stage or phase) and / or optimize the conditions in the cell culture (e.g., concentrated media provided during perfusion culture). Growth media formulations may be used to promote cell growth and minimize protein expression. Production media formulations may be used to promote production of the protein of interest and maintenance of the cells with minimal new cell growth. Feed media consumed during the production phase of the cell culture, usually media containing more concentrated components such as nutrients and amino acids, may be used to supplement and maintain the active culture, especially cultures operated in fed-batch, semi-perfusion, or perfusion modes. Such concentrated feed media may contain most of the components of cell culture media, for example, at about 5x, 6x, 7x, 8x, 9x, 10x, 12x, 14x, 16x, 20x, 30x, 50x, 100x, 200x, 400x, 600x, 800x, or about 1000x of their normal amounts.

[0095] The growth phase may occur at a higher temperature than the production phase. For example, the growth phase may occur at a first temperature of about 35°C to about 38°C, and the production phase may occur at a second temperature of about 29°C to about 37°C, optionally about 30°C to about 36°C, or about 30°C to about 34°C. In addition, chemical inducers of protein production, such as, for example, caffeine, butyrate, and hexamethylene bisacetamide (HMBA), may be added simultaneously with, before, and / or after the temperature shift. If inducers are added after the temperature shift, they may be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift.

[0096] Host cells can be cultured in suspension or in attached form attached to a solid culture medium. Cell cultures can be established in fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors with or without microcarriers.

[0097] Cell cultures may be operated in batch, fed-batch, continuous, semi-continuous, or perfusion modes. Mammalian cells, such as CHO cells, may be cultured in bioreactors on a small scale of less than 100 ml to less than 1000 ml. Alternatively, large scale bioreactors containing 1000 ml to more than 20,000 liters of medium may be used. Large scale cell cultures, such as those for clinical and / or commercial scale biomanufacturing of protein therapeutics, may be maintained for weeks and months while the cells produce the desired protein.

[0098] The resulting recombinant protein can then be collected from the cell culture medium. Methods for collecting proteins from suspended cells are known in the art and include accelerated sedimentation such as acid precipitation, flocculation, gravity separation, centrifugation, sonication, membrane filtration, including but not limited to filtration using ultrafiltration membranes, microfilters, tangential flow filters, alternative tangential flow, depth, and sedimentation filters. Recombinant proteins expressed by prokaryotes are collected in inclusion bodies in the cytoplasm by redox folding processes known in the art.

[0099] The harvested protein can then be purified or partially purified from any impurities, such as residual cell culture medium, cell extracts, undesired components, host cell proteins, improperly expressed proteins, etc., using one or more unit operations. The term "unit operation" is a term of art and refers to a functional step that can be performed in the process of purifying a recombinant protein from a liquid culture medium. For example, units of operation can include filtration (e.g., removal of contaminating bacteria, yeast, viruses or microorganisms, and / or particulate matter from a liquid containing the recombinant protein), capture, removal of epitope tags, purification, retention or storage, polishing, viral inactivation, adjustment of the ionic concentration and / or pH of the liquid containing the recombinant protein, and removal of undesired salts.

[0100] For example, a unit operation may include steps such as, but not limited to, capture, purification, polishing, viral inactivation, viral filtration, and / or adjustment of the concentration and formulation containing the recombinant protein of interest. A unit operation may also include holding or storage steps between processing steps. A single unit operation may be designed to accomplish multiple objectives in the same operation, such as capture and viral inactivation.

[0101] Capture unit operations include capture chromatography, such as affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), etc., that utilizes resins and / or membranes containing agents that will bind to the recombinant protein of interest. Such materials are known in the art and are commercially available. Affinity chromatography can include substrate-bound capture mechanisms, antibody or antibody fragment-bound capture mechanisms, aptamer-bound capture mechanisms, and cofactor-bound capture mechanisms, such as Protein A, Protein G, Protein A / G, Protein L-bound capture mechanisms, etc. In particular, a continuous upstream manufacturing process for bispecific T cell engagers using Protein L is described in WO2019118426. The recombinant protein of interest may be tagged with a polyhistidine tag and subsequently purified from IMAC using imidazole, or by using an epitope such as FLAG® followed by the use of a specific antibody directed to such an epitope.

[0102] One or more capture unit operations include viral inactivation and / or viral filtration. To ensure patient safety, viral inactivation and viral filtration are necessary components of the purification process when producing protein therapeutics. The liquid to be subjected to viral inactivation and viral filtration can be obtained from the effluent stream, eluate, pool, holding or storage vessel.

[0103] Enveloped viruses are susceptible to inactivation methods such as heat inactivation / pasteurization, pH inactivation, UV and gamma irradiation, use of high intensity broad spectrum white light, addition of chemical inactivators, surfactants, and solvent / detergent treatments, so that they can no longer infect cells, replicate, and / or spread. One method to achieve virus inactivation is incubation at low pH or other solution conditions to achieve virus inactivation. Low pH virus inactivation can be followed by a neutralization unit operation that re-adjusts the virus-inactivated solution to a pH more compatible with the requirements of the next unit operation. It can also be followed by filtration, such as depth filtration, to remove the resulting turbidity or precipitate.

[0104] The term "polishing" is used herein to refer to one or more chromatographic steps performed to remove residual contaminants and impurities such as DNA, host cell proteins, product specific impurities, mutant products and aggregates, and viral adsorption from a liquid containing recombinant protein close to the final desired purity. For example, polishing can be performed in a bind and elute mode by passing the liquid containing recombinant protein through a chromatographic column or membrane absorber that selectively binds either the recombinant protein or contaminants or impurities present in the liquid containing recombinant protein. In such an example, the eluate / filtrate of the chromatographic column or membrane absorber contains the recombinant protein.

[0105] Polish chromatography unit operations utilize resins and / or membranes containing agents that can be used in either a flow-through mode (where the protein of interest is contained in the eluate and contaminants and impurities are bound to the chromatography medium) or a bind-and-elute mode (where the protein of interest is bound to the chromatography medium and eluted after contaminants and impurities have passed through or been washed out of the chromatography medium). Examples of such chromatography methods include ion exchange chromatography (IEX), such as anion exchange chromatography (AEX) and cation exchange chromatography (CEX); hydrophobic interaction chromatography (HIC); mixed-mode or multimodal chromatography (MM), hydroxyapatite chromatography (HA); reversed-phase chromatography, and gel filtration.

[0106] Key attributes and performance parameters can be measured to better inform decisions regarding the performance of each step during production. These key attributes and parameters can be monitored in real time, near real time, and / or after the fact. Key parameters such as consumed media components (such as glucose), metabolic levels of accumulating by-products (such as lactate and ammonia), and those related to cell maintenance and survival such as dissolved oxygen content can be measured. Key attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, yield, and titer can be monitored during and after the process. Monitoring and measurements can be done using known techniques and commercially available equipment.

[0107] The present invention eliminates the need for redundant release sampling of concentrated, formulated drug substance and drug product, and allows assays of attributes common to both to be done only once, such as at the drug product fill / finish stage, which may be combined with other drug product attribute tests.

[0108] The terminology used in this application is standard within the art, but certain definitions are provided herein to ensure clarity and clarity to the meaning of the claims. Units, prefixes, and symbols may be denoted in their SI accepted form. Numerical ranges recited herein are inclusive of the numbers defining the range, and include and support each integer within the defined range. Unless otherwise stated, the methods and techniques described herein are generally performed according to conventional methods well known in the art, and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990). All documents or portions of documents cited in this application, including but not limited to patents, patent applications, articles, books, and journal articles, are expressly incorporated herein by reference. Anything described in an embodiment of the invention may be combined with other embodiments of the invention.

[0109] The present invention is not limited in scope by the specific embodiments described herein, which are intended as single descriptions of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. EXAMPLES

[0110] Example 1 Concatenated DS-DP Operation A 50 L bioreactor run was performed to produce recombinant monoclonal antibody and was processed forward through a series of purification unit operations until a viral filter pool was obtained. An Akta flux 6 skid (GE Healthcare, Piscataway, NJ) was used for UFDF. Millipore Pellicon cassette holders were used for a total of 1.14 m 2 A 30 kD UFDF membrane was used to house the resulting mixture (Millipore Sigma, Burlington, MA). An Opticap XL 600 sterile large volume filter (Millipore Sigma, Burlington, MA) was used as the bioburden reduction filter. The final drug substance was collected in a Mobius single-use mixing bag (Millipore Sigma, Burlington, MA).

[0111] The virus filter pool was used as the starting material for the UFDF run, polysorbate 80 (PS80) addition, and final 0.2 micron filtration. The skid and other product contact parts were held overnight in 0.2 N sodium hydroxide before starting the run. The formulation buffer used for diafiltration and final protein formulation had the composition 272 mM proline, 10 mM acetate at pH 4.1. A 1% PS80 stock solution was prepared in the formulation buffer and added to the final UFDF flush buffer and the final protein pool in the retentate tank to give 0.01% weight / volume PS80 in the final DS.

[0112] Prior to starting the UFDF run, a DIW flush was utilized and the pH of the permeate side liquid was checked to ensure that hydroxides had been washed out. After the DIW flush, normalized water permeability (NWP) was measured to ensure the filter met the acceptance criteria. The virus filter pool was loaded onto the UFDF skid and a concentration of 70 mg / mL was targeted for the Ultrafiltration 1 (UF1) run. Diafiltration (DF) was performed at 70 mg / mL with formulation buffer equivalent to a diavolume (DV) of 10. After DF, the diafiltered pool was recirculated for approximately 10 minutes and a sample was tested for protein concentration from the retentate tank using a Solo VPE. This protein concentration and amount was used to calculate the volume reduction required to concentrate the protein pool to 175 mg / mL in the UF2 run. During concentration, the feed pressure, TMP and flow rate were controlled to maintain the TMP at approximately 15 psi. After the UF2 target value of 175 mg / mL was obtained, the target volume to be achieved for a final DS concentration of 145 mg / mL was calculated. The required amount of flush buffer was added to reach a target final concentration of 140 mg / mL.

[0113] The amount of PS80 stock solution needed to achieve 0.01% w / v PS80 in the retentate tank was calculated and added directly to the protein solution in the retentate tank. A 1% PS80 stock solution prepared in formulation buffer was used for this purpose.

[0114] A 0.01% weight / volume stock solution of PS80 in formulation buffer was prepared and a bioburden reduction filter (Opticap XL 600) was filled with approximately 80 L / m2 of PS80 to saturate the loading sites on the filter. 2The filter outlet was connected to a Y such that one arm of the Y was connected to a flush buffer bag to collect the flush buffer and the other arm was connected to a Mobius bag used to collect the final DS. Traces of buffer remaining in the SHC filter housing were removed by pumping air through the filter. After the remaining buffer was removed from the filter, the flush buffer collection bag was clamped and DS filtration in the product collection bag was started. Prior to filtration, a sample of the final DS concentration was taken directly from the retentate tank and three concentration measurements were obtained.

[0115] The two vial filter pools (sublots) were combined to make one UFDF / DS / DP lot. Common product quality assays between DS and DP were tested once. Target product quality profiles for drug substance and formulation process were compared and all were within specifications.

[0116] The UFDF / DS / DP lots were then filtered using a 0.2μ bioburden reduction filter and collected into a storage bag. The bag was connected to a Vanrx SA25 unit (Burnaby, British Columbia, Canada) and the bulk formulation was sterile filtered prior to formulation fill / finish.

[0117] Example 2 UFDF membrane circulation after buffer washing This experiment evaluated UF-DF performance using a scaled down single-use stabilized cellulosic hydrophilic membrane, reusing the membrane after an equilibration buffer wash representative of a single-use UFDF skid in production, to determine the impact of circulation and feed conditions on the UFDF process and product quality performance using an extended half-life bispecific T cell engager feedstream.

[0118] Frozen eluate pool material from a multimodal anionic (MMA) column containing the extended half-life bispecific T cell engager, HLE BiTE®, was thawed prior to processing. The eluate pool material was then loaded onto three columns (A, B, C) of equilibrated stabilized cellulosic hydrophilic membranes, Sartocon Slice 200 ECO (10 kD MWCO cutoff) (Sartorius, Goettingen, Germany), with a membrane area of ​​0.018 m. 2 and the feed pressure ranged from 20 to 36 psi, with 71.4 g / m 2 The TFF pool was loaded at 1000 µL / ml. Samples were concentrated (UF1) to an intermediate target value ranging from 0.5 g / L to 4 g / L (see Table 1 for initial concentration target values) and the samples were diafiltered with 10 volumes of formulation buffer (10 mM acetate, 180 mM NaCl, pH 5.0). Samples were collected in a pool vessel and then pooled to a volume sufficient for mixing and collection in a collection vessel by system tracing. The TFF pool was then diluted as needed with formulation buffer to a target concentration of 1-2 g / L.

[0119] After the first cycle, the membranes were flushed with equilibration buffer (100 mM acetate, 180 mM NaCl, pH 5.0) and a standardized water permeability [NWP] test was performed on each membrane to determine membrane consistency. Standardized water permeability (NWP) is a measurement of membrane cleanliness after cleaning. The passage of clean water through the membrane was measured under standard pressure and temperature conditions. The rate of clean water flux through each membrane was measured in liters per membrane area per hour (L / m 2 The water flow rate divided by the transmembrane pressure is called the normalized water permeability or NWP (L / m 2 NWP values ​​can be compared to initial (pre-treatment) levels and analyzed for trends over time.

[0120] The UFDF eluate was collected as a bulk pool for all runs and analyzed for product quality. Product quality attributes were assessed in the viral filtrate: high molecular weight (HMW) impurities were measured using size-exclusion ultra-high performance liquid chromatography (SE-UHPLC), clips were measured using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS or r-CE) analysis under reducing conditions, and charge profile, acidic and basic variants were measured using cation exchange high performance liquid chromatography (CEX-HPLC).

[0121] Membranes B and C were then subjected to two additional cycles as outlined in Table 1.

[0122] All membranes are loaded at high loadings, typically 55 g / m 2 Instead of 71.4 g / m 2 Run 1 was one complete cycle performed on one Sartocon membrane (A) without any additional cycles on that membrane. Runs 2-4 were performed on one Sartocon membrane (B) with no caustic chemical cleaning and only one buffer flush in between cycles, each run was performed on consecutive days over three days with one cycle per day (10-12 h pause in between runs). Runs 5-7 were performed on one Sartocon membrane (C) with no caustic chemical cleaning and only one buffer flush in between cycles, runs were performed with rapid transitions without any pauses in between cycles. All experiments were performed using an AKTA crossflow UF / DF skid (GE Healthcare, Chicago, Ill).

[0123] [Table 1]

[0124] Figure 2 shows the NWP recovery values ​​for the Sartocon membrane after each run starting from midpoint 1 of the runs [Run 5]. Also shown is the minimum NWP recovery % observed after 20 cycles performed on similar membranes during process characterization. The NWP recovery % after midpoint 3 of the runs [Run 7] was high compared to the minimum recovery %. This observation indicates that after 3 cycles with only buffer washes in the middle of the run was good enough to maintain the NWP recovery % well above the minimum observed for 20 cycles. This further provided data that without any type of caustic chemical wash [e.g., no sodium hydroxide CIP, etc.], the membrane did not lose permeability and was good enough to be used for reprocessing after only buffer washes for at least 3 cycles.

[0125] Table 2 summarizes the loading and final pool product quality values ​​(HMW%, clip%, acid%, basic%) for all runs performed. Despite the high loading and high initial concentrations, the HMW% of the final pool for all runs was comparable. Overall, the product quality performance for the stabilized cellulose-based hydrophilic membranes as designed in these experiments met the clinical and commercial process requirements and specifications. From a process performance perspective, rinsing the membrane with equilibration buffer also reduced the loading and final pool product quality values ​​up to 71.4 g / m 2 It was sufficient to perform additional cycle loading up to

[0126] [Table 2]

[0127] Example 3 High membrane loading and increased diavolume for UFDF This experiment used a feedstream of extended half-life bispecific T cell engager molecules to evaluate the performance of UF-DF using specifically loaded regenerated cellulose membranes with high membrane loading and increasing diavolume numbers on product quality performance.

[0128] Frozen pooled eluate material from a multimodal anionic (MMA) column containing extended half-life bispecific T cell engagers was thawed prior to processing. The pooled eluate material was then transferred to a 0.0088 m membrane area centrifuge. 2 The eluate was loaded onto a regenerated cellulose membrane (Pelicon 3 (10 kD MWCO cutoff) (EDM Millipore, Danvers, MA)) having a pH of 10. The experimental conditions are summarized in Table 3. All experiments were performed using an AKTA Crossflow UF / DF skid (GE Healthcare, Chicago, Ill.).

[0129] The filters were equilibrated with 100 mM acetate, 180 mM NaCl, pH 5.0. After membrane equilibration, the eluate pool material was concentrated to the desired initial concentration (see Table 3) and the feed flow was ≥ 10 L / m 2 After concentration, the pool material was diafiltered with 10 or 13 diavolumes of formulation buffer (10 mM glutamate, 9% sucrose, pH 4.2) (see Table 3). The product was collected in a pool vessel, and the system was tracked to collect the pool to sufficient volume and sample in the collection vessel. The TFF pool was then diluted to the target concentration with formulation buffer.

[0130] [Table 3]

[0131] Run 4 had the highest HMW% compared to the other runs, but was below the acceptable quality target of <5% (Table 4).

[0132] [Table 4]

[0133] Example 4 Viral Filtration of Formulated Bispecific T Cell Engagers This experiment demonstrated viral filtration of extended half-life bispecific T cell engagers (HLE BiTE®) in formulation buffer.

[0134] Extended half-life bispecific T cell engagers at various concentrations in formulation buffer (9% sucrose, 10 mM glutamate, pH 4.2) were filtered in a filter train at constant pressure through hydrophilized polyvinylidene fluoride (PVDF) hollow fiber filters (Plavona™ BioEx) and cuprammonium-regenerated cellulose hollow fiber filters (Planova 20N), 0.001 m 2 A viral removal filter (Asahi Kasei Bioprocesses, Glenville, Ill.) was used to evaluate process and product quality performance. The filter train consisted of a pressure regulator connected to a pressure reservoir with a valve connected to the viral removal filter. The viral removal filter opened directly to a collection vessel attached to a balance. The filter train was connected to a computer for data collection and a compressed air supply for pressure regulation.

[0135] Volumetric throughput was determined by measuring the amount of filtrate [mL or L] collected at a given time interval and then dividing by the effective filtration surface area of ​​the filter being used. Flow decay was determined based on dividing the instantaneous flow rate by the initial flow rate and then subtracting that value from 1 (Flow decay = 1 - Flow reduction = 1 - [J / J0]). Since the initial flow rate, -J0, was the buffer permeability, flow decay was normalized with respect to -J0 and expressed as a percentage. Viral filtrate was collected as a bulk pool for all runs and analyzed for product quality. Specific product quality attributes were evaluated in the viral filtrate: high molecular weight (HMW) impurities were measured using size-exclusion ultra-high performance liquid chromatography (SE-UHPLC), clips were measured using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS or r-CE) analysis under reducing conditions, and charge profile, acidic and basic variants were measured using cation exchange high performance liquid chromatography (CEX-HPLC).

[0136] Experiments were performed using aliquots of feed material (thawed or fresh) and runs under the conditions for each of runs 1-6 as provided in Table 5. The feed material was a purified eluate pool containing bispecific T cell engagers formulated in 10 mM glutamate, 9% sucrose, pH 4.2.

[0137] [Table 5]

[0138] Table 6 shows the hydraulic performance of each run separated by feed conditions. The results are shown in terms of volumetric throughput (L / m 2 ) are shown in terms of normalized flux decay (relative to buffer permeability).

[0139] [Table 6]

[0140] Figure 3 shows the effect of different feed conditions on volumetric throughput. For all runs, there was no effect of feed conditions (fresh, extended hold, and high volume) on flow rate decay, except for runs at high concentration and high pH.

[0141] product quality There is an increase in HMW at high concentration (Run 1) and increasing pH (Run 6), resulting in a flow rate reduction of 73% and 78%, respectively. 2 The filterability of the PVDF filter at was lower than the cuprammonium-regenerated cellulose filter with higher flow attenuation (see Figures 4A HMW%, 4B Clip%, 4C Basic%, and 4D Acidic%).

[0142] In a second experiment, the extended half-life bispecific T cell engager was formulated in chromatography pool buffer (100 mM acetate, 180 mM sodium chloride, pH 5.0) and evaluated for process and product quality performance. Cuprammonium-regenerated cellulose hollow fiber viral removal filters (Plavona™ 20N), 0.001 m 2 (Asahi, Glenville, Ill.) was used in the filter train. Experiments were conducted using runs of aliquots of feed under the conditions for each of runs 7-13 as provided in Table 7.

[0143] [Table 7]

[0144] Figure 5 shows the effect of concentration, pH and conductivity in the chromatography buffer matrix. At pH 5.0, both concentrations (1.77 g / L and 3.15 g / L) were within 13% flux attenuation (Table 8). At pH 5.3 with a concentration of 6.82 g / L, flux attenuation was minimal (3%), while at pH 4.5, flux attenuation was significant (32%). This is believed to be due to increased aggregates (>20% at low pH, Figure 6A). Conductivity had no effect on virus filtration for the conditions tested. Regardless of pressure (14, 17 or 19 psi), flux attenuation was minimal (Table 8).

[0145] [Table 8]

[0146] Runs in the formulation buffer matrix had lower flow rates compared to runs in the chromatography buffer, but they were still within acceptable limits for use.

[0147] Example 5. Process and product quality performance of bispecific T cell engagers during viral filtration Virus filtration is usually operated under one of two modes, the first being the constant pressure mode, where the inlet pressure is kept constant by using a pressure regulator. In this mode, there is a flow rate [L / m] that decreases with the progression of time and with increasing volumetric throughput. 2 ], flow rate decay vs. volumetric throughput [L / m 2 In the second, constant flow regime, the flow rate is kept constant by using a pump to push the feed load at a constant flow rate. In this regime, the flow rate increases as the volumetric throughput increases [L / m 2 ], pressure increases over time. This is usually measured as resistance [the inverse of permeability] versus volumetric throughput [L / m 2 ] is plotted as

[0148] This experiment will evaluate viral filtration of an extended half-life bispecific T cell engager (HLE BiTE® A) feed stream at normal flow rate under constant pressure mode and will extend to constant flow rate mode and the effect of feed conditions [pH, conductivity and concentration] on the hydraulic performance and product quality attributes of the viral filter with and without various prefilters.

[0149] Viresolve® Pro (VPro), polyethersulfone (PES) (3.1 cm 2 Parvovirus-retentive filter) The virus filter was used alone and in a set of four filters: two surface-modified prefilters, Viresolve® Pro Shield (Shield) (3.1 cm 2 ) and Viresolve® Pro Shield H (Shield H) (3.1 cm 2 ), a surface-modified polyethersulfone membrane filter; and two depth filters, Viresolve® Prefilters, (VPF) (5 cm 2 ), an adsorptive depth filter, and Millistak+® HC Pro X0SP (X0SP) (5 cm 2 / 3.1cm 2), in combination with a synthetic depth filter consisting of a double-layer silica filter supported by polyacrylic acid fibers (all from MilliporeSigma, Burlington, Mass.).

[0150] A feedstream of the extended half-life bispecific T cell engager, BiTE® A, was evaluated for process and product quality performance of these filter combinations.

[0151] Experiments were performed using a compressed air supply connected through a pressure regulator to a pressurized supply vessel with a valve connected to either the surface modified membrane prefilter or depth filter, and further connected to the virus filter. As a control, the supply vessel valve was connected directly to the virus filter device only. The virus filter was opened directly to a collection vessel attached to a balance. The filter train was connected to a computer for data collection and a compressed air supply for pressure regulation.

[0152] The feed side pressure for the virus filter was set at a constant 30 psi and the filtrate volume was measured at predefined time intervals. During filter installation, the virus filter device and the surface modified membrane prefilter or depth filter device were separately flushed with water at 30 psi. The virus filter device and the prefilter or depth filter device were then connected and a buffer flush was performed at 30 psi. The average water and buffer flow rates and permeabilities for each virus filter and prefilter or depth filter device were recorded and were within recommended limits.

[0153] Volumetric throughput was determined by measuring the amount of filtrate [mL or L] collected at a given time interval and then dividing by the effective filtration surface area of ​​the filter being used (3.1 cm for the VPro device). 2). The flow decay was determined based on dividing the instantaneous flow rate by the initial flow rate and then subtracting that value from 1 (flow decay=1-flow reduction=1-[J / J0]). Because the initial flow rate, -J0, was the buffer permeability, the flow decay was normalized with respect to -J0 and expressed as a percentage. The virus filtrate was collected as a bulk pool for all runs and analyzed for product quality. Specific product quality attributes were evaluated in the virus filtrate: high molecular weight (HMW) impurities were measured using size-exclusion ultra-high performance liquid chromatography (SE-UHPLC), clips were measured using capillary electrophoresis-sodium dodecyl sulfate (CE-SDS or r-CE) analysis under reducing conditions, and charge profile, acidic and basic variants were measured using cation exchange high performance liquid chromatography (CEX-HPLC).

[0154] The purified eluate pool containing the feed material, BiTE® A, was thawed prior to processing. After thawing, aliquots of the feed material were adjusted to the target conditions (pH, conductivity, concentration) as described in Table 9. The conditions for each of Runs 1-16 are provided in Table 10.

[0155] [Table 9]

[0156] [Table 10]

[0157] Figures 7-9 show the hydraulic performance of each run separated by feed conditions. The results are shown in terms of volumetric throughput (L / m 2 ) are shown in terms of normalized flux decay (relative to buffer permeability) as a function of pH. Table 11 and Figures 7-9 provide a summary of the filtration results. Table 12 provides a summary of the product quality attributes HMW% (SEC), Clip% (rCE) and charge profile, acidity and baseness (CEX).

[0158] [Table 11]

[0159] Product Quality Results

[0160] [Table 12]

[0161] result 1) Mid-point pH, low concentration, and low conductivity for BiTE® A (pH 5, conductivity 23 mS / cm, 1.75 g / L) hydraulic performance Virus filters alone and in combination with surface modified membrane prefilters and depth filters, Shield, Shield H, VPF, and X0SP, were tested with BiTE® A at midpoint pH, low concentration, and low conductivity conditions (pH 5, conductivity 23 mS / cm, 1.75 g / L), Runs 1-3, 6, and 9, Table 10. Virus filters in combination with depth filters (VPF or X0SP) reached steady state with approximately 10% flow decay. Virus filters in combination with surface modified membrane filters (Shield or Shield H) showed more initial fouling, but also reached steady state with approximately 25-30% flow decay. Virus filters alone without surface modified membrane prefilter or depth filter had a flow rate of 250 L / m 2 A 40% flow decay was observed, see Table 11, Runs 1-3, 6 and 9, FIG. 7.

[0162] product quality Of the combinations tested, the viral filter in combination with the depth filter (X0SP) had the greatest impact on product quality, reducing aggregate (HMW%) levels to 0.9%. See Table 12, Runs 1-3, 6 and 9, Figures 10A, 10C, 10E, 10G, Load Quality, Table 12, row (A).

[0163] 2) Low pH, low concentration, low conductivity conditions (pH4.2, 23mS / cm, 1.75g / L) hydraulic performance Virus filters alone or in combination with a depth filter (X0SP) and a surface modified prefilter (Shield) were tested at low pH, low concentration, and low conductivity conditions (pH 4.2, 23 mS / cm, 1.75 g / L) (Runs 4, 5, and 10, Table 10). Low pH conditions had a small effect on the virus and depth filter combination, with a 300 L / m 2 The virus filter alone and in combination with the surface modified prefilter underwent 80% flow attenuation and showed significant fouling at low pH conditions. See Table 11, runs 4, 5, and 10, Figure 8.

[0164] product quality Due to the better aggregate removal ability, the flow rate attenuation for the combination of the viral filter and depth filter was only 15% compared to the viral filter alone or the viral filter combined with the surface modified prefilter, which each had a flow rate attenuation of 80%. Clip and charge profiles were similar across the various combinations (see Runs 4, 5, and 10 in Table 12, Figures 10A, 10C, 10E, and 10G).

[0165] 3) High pH, ​​low concentration, low conductivity (1.75g / L, pH6, 23mS / cm) hydraulic performance The virus filter alone or in combination with a surface modified prefilter (Shield H) was tested at low concentration, high pH, ​​low conductivity conditions (1.75 g / L, pH 6, 23 mS / cm) (Table 10, Runs 7 and 8). Both the virus filter alone or the virus filter in combination with the surface modified prefilter had a flow rate attenuation of approximately 20%.

[0166] product quality The combination of the viral filter and the surface modified prefilter was not significantly different from the viral filter alone in removing aggregates. The charge profile and clip were similar across both combinations. See Table 12, runs 7 and 8, Figures 10A, 10C, 10E, 10G.

[0167] 4) Low pH, high conductivity, high concentration (7g / L, pH4.2, 23mS / cm) hydraulic performance A virus filter in combination with a depth filter (X0SP) and two surface modified prefilters (Shield and Shield H) were tested at low pH and high concentration conditions (7 g / L, pH 4.2, 23 mS / cm) (see Runs 11, 12, and 14 in Table 10). All three combinations experienced greater than 80% flux decay (see Runs 11, 12, and 14 in Table 11, FIG. 8).

[0168] product quality No combination was more effective at removing the increased aggregates produced under these conditions, and of the three, the viral filter and prefilter combination was optimal at removing aggregates (see Runs 11, 12, and 14 in Table 12). Charge profiles and clips were similar across the three prefilter conditions (see Runs 11, 12, and 14 in Table 12, Figures 10B, 10D, and 10F, rows (B) and (D) in Table 12).

[0169] 5) High pH, ​​high conductivity, high concentration (7g / L, pH6, 28mS / cm) hydraulic performance A virus filter in combination with a synthetic depth filter (X0SP) and two surface modified prefilters (Shield and Shield H) were tested at high pH, ​​high conductivity and concentration conditions (7 g / L, pH 6, 28 mS / cm) (see Table 10, runs 13, 15 and 16). All three combinations experienced greater than 90% flux decay (see Table 11, runs 13, 15 and 16 in Figure 9).

[0170] product quality The combination of the viral filter and the synthetic depth filter reduced aggregate levels to a significantly low level of 0.07%, and reduced aggregate levels to a very low level of 0.07% (see runs 13, 15 and 16 in Table 12, Figures 10B, 10D, 10E, 10H, row (C) in Table 12).

[0171] When the 7 g / L concentrated feed at the midpoint pH of 5.0 and low conductivity (pH 5, 23 mS / cm, aggregate level 3.8%) was titrated to pH 6.0 and high conductivity (28 mS / cm), less aggregate was observed compared to titration to low pH 4.2 and high conductivity (28 mS / cm). At pH 6.0, the loaded aggregate level was 2.92% compared to 4.4% at the low pH (pH 4.2) condition. There may be a maximum threshold aggregate level that the virus filter in combination with the depth filter (X0SP) can reduce, which may be why the filter was still able to reduce aggregate levels even though the flow rate was still high at high pH. However, at low pH, the theoretical maximum level is exceeded.

[0172] Example 6 Viral Filtration with Bispecific T Cell Engagers and Monoclonal Antibodies An extended half-life bispecific T cell engager, BiTE® A, and a monoclonal antibody, Mab A, were compared for process and product quality performance using a combination of viral and depth filters. For BiTE® A and Mab A, the viral filters were Viresolve® Pro (VPro), polyethersulfone (PES) (3.1 cm 2 ) Parvovirus-retentive filter, an absorbent depth filter, Viresolve® Prefilter (VPF) (5 cm 2 ) (both from MilliporeSigma, Burlington, Mass.) were tested in combination.

[0173] BiTE® A also contains the VPro viral filter and the synthetic depth filter, Millistak+® HC Pro X0SP (X0SP) (5 cm 2 / 3.1cm 2 ) (both from MilliporeSigma, Burlington, Mass.) were used in combination.

[0174] The BiTE® A loading concentration was low, 1.75 g / L, with a midpoint pH of 5.0, and a midpoint conductivity of 23 mS / cm (see Example 5, Runs 6 and 9). For Mab A, the eluate pool containing Mab A was adjusted to a midpoint pH of 6.7, a conductivity of 20 mS / cm, and a loading concentration of 12.4 g / L.

[0175] hydraulic performance For Mab A, the combination of viral filter and absorbent depth filter achieved >1000 L / m with a flow rate attenuation of approximately 40%. 2 (4 h treatment time), while BiTE® A with either virus filter / prefilter combination was able to reach >250 L / m 2 , but with a flow rate decay of approximately 10%. Due to feed limitations, no further data could be obtained for BiTE® A. Figure 11 shows the volumetric throughput (L / m) versus its pH and feed stream concentration. 2 ) are shown. At low concentrations and mid-high pH [above 5], with BiTE A (and also BiTE B in Example 7), the volumetric throughput obtained is similar to that of antibody when using VPF or synthetic filters.

[0176] product quality For Mab A, the load pool and viral filtrate pool samples showed virtually no difference in HMW%. For BiTE® A, the combination of the viral filter and the synthetic depth filter reduced aggregate levels (HMW%) in the viral filter pool (see Table 12, run 9 and row (A)).

[0177] BiTE® A was also tested at higher loading concentrations, pH and conductivity, 7 g / L, pH 6.0, and 28 mS / cm, using a combination of a VPro viral filter and a synthetic depth filter (XOSP) as described in Example 5, Run 16. Figure 11 shows the volumetric throughput (L / m) for both high pH and high feed stream concentrations. 2 ) are shown. For Mab A, the load pool and viral filtrate pool showed virtually no difference in HMW%. For higher concentrations and pH of BiTE® A, the combination of the viral filter and the synthetic depth filter was able to significantly reduce aggregate levels in the viral filtrate pool (see run 16 and row (D) of Table 12, FIG. 10A).

[0178] Filtering a feed stream containing Mab A (12.7 g / L) at midpoint pH (6.7) and conductivity (20 mS / cm) was easier to obtain a relatively high volumetric throughput with minimal flow rate decay compared to high concentration BiTE® A (7 g / L) at high pH (6.0) and conductivity (28 mS / cm), which had a lower volumetric throughput with significant flow rate decay (see Table 11, runs 13, 15, and 16). The aggregate content of BiTE® A may be different at higher concentrations compared to Mab A, where there was no change in the aggregate (% HMW) content between the pre-filtrate and post-filtrate pools. For BiTE® A, the prefilter removed some of the aggregates, but some higher order forms of aggregates may remain, which may be the cause of the poor filterability.

[0179] The filterability of BiTE® A and Mab A at low feed concentrations was similar (see FIG. 11). However, even at low feed concentrations, the prefilter was able to remove some of the aggregates associated with BiTE® A, and presumably the remaining aggregate content was reduced due to the relatively high filterability of BiTE® A, which allowed for a high volumetric throughput [>250 L / m] with less flow rate decay (10%). 2 ] is achieved (see FIG. 10A and Table 12, run 9). For BiTE®, the synthetic depth filter was very sensitive to removing aggregates from the load, going from 2.96% (see Table 12, row (A)) to 0.92% (see Table 12, run 9) in the filtrate pool, while the absorptive depth filter reduced it to 2.37% under the same conditions (see Table 12, run 6). The overall difference between the absorptive and synthetic depth filters is that one is synthetic, and the synthetic filter may be better suited to removing the types of aggregates formed by BiTE® under these conditions.

[0180] Example 7 Viral Filtration Performance with BiTE® B This experiment evaluated the impact of feed conditions [pH, conductivity and concentration] on the hydraulic performance and product quality attributes of the virus filter in a constant pressure regime and normal flow rate filtration for a half-life extended bispecific T cell engager (HLE BiTE® B) molecule feed stream with and without various pre-filters.

[0181] Viresolve® Pro (VPro), polyethersulfone (PES) (3.2 cm 2 Parvovirus-retentive virus filters include, but are not limited to, surface-modified polyethersulfone, membrane prefilters, Viresolve® Pro Shield H (Shield H) (3.2 cm 2); and two depth filters, an adsorptive depth filter Viresolve® prefilter, (VPF) (5 cm 2 ), and a synthetic depth filter Millistak+® HC Pro X0SP (X0SP, composed of a double layer of silica fiber supported by polyacrylic acid fiber) (5 cm 2 ) (all from MilliporeSigma, Burlington, Mass.). After viral filtration, BiTE® B was evaluated for product quality and performance. The feed conditions used in the experiments are shown in Table 13 and the run conditions based on the feed conditions are provided in Table 14.

[0182] [Table 13]

[0183] [Table 14]

[0184] Product quality profiles for BiTE® B were obtained only for high molecular weight aggregates in the viral filtrate pool (Table 16). For midpoint pH and low conductivity conditions (pH 5.9, 1.81 g / L, 31.36 mS / cm) (Runs 17-20 in Table 14), the combination of the viral filter and the synthetic depth filter (X0SP) removed a higher percentage of aggregates compared to the combination of the viral filter with an absorbent depth filter (VPF) or a surface-modified prefilter (Shield H) (Runs 17-20 in Table 15). The combination of the viral filter with either prefilter did not result in significant flux attenuation (see FIG. 13, Runs 17-20 in Table 15).

[0185] For low pH, low conductivity conditions (1.81 g / L, pH 4.2, 31.36 mS / cm) (Runs 23, 24), the combination of the virus filter and the synthetic depth filter had only a 20% flow rate attenuation compared to an 80% flow rate attenuation for the combination of the virus filter and the surface modified prefilter (see FIG. 14, Runs 23-24 in Table 15). From a product quality perspective, the virus filter combined with the synthetic depth filter was significantly better at removing high molecular weight aggregates (1.6%) compared to the virus filter combined with the surface modified prefilter (2.1%) (Runs 23-24 in Table 16) (see FIG. 15, Run 24 in Table 16).

[0186] For the high pH, ​​high conductivity conditions (1.81 g / L, pH 5.9, 45 mS / cm) (Runs 21-22 in Table 14), the virus filter combined with either the synthetic depth filter or the surface modified prefilter had very low flow rate attenuation, 3.7% and 5.2% (see FIG. 14, Runs 21-22 in Table 15), but from a product quality perspective, the virus filter and synthetic prefilter combination performed very well in removing high molecular weight aggregates to a final value of 0.3%, compared to 1.8% for the virus filter and surface modified prefilter combination, which was not very effective (Runs 21-22 in Table 16, FIG. 15). Higher conductivity did not appear to significantly change hydraulic and aggregate removal performance (midpoint pH, low conductivity conditions) (Run 20 compared to Run 22 in Table 16).

[0187] [Table 15]

[0188] [Table 16]

[0189] For BiTE B, at midpoint pH conditions, the virus filter alone was able to provide good volumetric throughput with relatively low flow decay, and the addition of a prefilter reduced the flow decay. However, the synthetic prefilter significantly reduced aggregates. At midpoint pH and high conductivity, both the surface modified and synthetic depth prefilters performed well, achieving volumetric throughput with low flow decay, but only the synthetic depth filter was able to significantly remove aggregates.

[0190] At low pH and low conductivity, only the synthetic depth filter provided high volumetric throughput with minimal flow rate decay, while the surface modified prefilter underwent significant flow rate decay and achieved relatively low throughput compared to the synthetic depth prefilter. At this condition, none of the prefilters tested were able to significantly remove aggregates.

Claims

1. A method of preparing a sample of recombinant bispecific T cell engager comprising: providing a sample of recombinant bispecific T cell engager comprising less than 7.0 g / L at a pH of 6.0 or less and having a conductivity of 23-45 mS / cm; subjecting the sample to a virus filtration unit operation comprising a virus filter alone or in combination with a depth filter or a surface-modified membrane prefilter; collecting the eluate of the virus filter containing the recombinant bispecific T cell engager in a pool or as a stream. The method includes:

2. 2. The method of claim 1, wherein the bispecific T cell engager is an extended half-life bispecific T cell engager.

3. 10. The method of claim 1, wherein the sample comprises a chromatography column pool or an eluate stream.

4. 2. The method of claim 1, wherein the pH of the pool or stream is between 4.2 and 6.

5. The method of claim 1, comprising subjecting the sample to a virus filtration unit operation comprising a virus filter in combination with a depth filter or a surface-modified membrane prefilter.

6. The method of claim 1, comprising subjecting the sample to a virus filtration unit operation comprising a virus filter in combination with a depth filter.

7. The method of claim 1, wherein the sample comprises 3.5 g / L or less of the recombinant bispecific T cell engager.

8. The method of claim 1, wherein the sample comprises less than 1.79 g / L of recombinant bispecific T cell engager.

9. The method of claim 1, wherein the step of providing a sample of recombinant bispecific T cell engager comprises subjecting the recombinant bispecific T cell engager to ultrafiltration and diafiltration operations resulting in less than 7.0 g / L of recombinant bispecific T cell engager.

10. A method of preparing a sample of recombinant bispecific T cell engager comprising: providing a sample of recombinant bispecific T cell engager comprising: less than 7 g / L of recombinant bispecific T cell engager at a pH of 6.0 or less; and having a conductivity of 23-45 mS / cm; subjecting the sample to a virus filtration unit operation comprising a virus filter in combination with a depth filter; collecting the eluate of the virus filter in a pool or as a stream. Including, A method in which the percentage of high molecular weight species in the eluate pool of the filter is reduced compared to use of a virus filtration unit operation comprising a virus filter alone or a virus filter in combination with a surface-modified membrane prefilter.

11. 11. The method of claim 10, wherein the bispecific T cell engager is an extended half-life bispecific T cell engager.

12. The method of claim 10, wherein the pH of the pool or stream is between 4.2 and 6.

13. The method of claim 10, wherein the sample comprises 3.5 g / L or less of the recombinant bispecific T cell engager.

14. The method of claim 10, wherein the sample comprises less than 1.79 g / L of recombinant bispecific T cell engager.

15. The method of claim 1, comprising the steps of: providing a sample comprising 1.75 g / L or less of a recombinant bispecific T cell engager at a pH of 4.2 to 6.0 and having a conductivity of 23 to 45 mS / cm; subjecting the purified recombinant bispecific T cell engager to a virus filtration unit operation comprising a virus filter in combination with a depth filter; collecting the effluent of the filter in a pool or as a stream. Including, A method in which the percentage of high molecular weight species in the eluate pool or stream of the filter is reduced compared to a virus filtration unit operation comprising a virus filter alone or a virus filter in combination with a surface-modified membrane prefilter.

16. 16. The method of claim 15, wherein the bispecific T cell engager is an extended half-life bispecific T cell engager.

17. 1. A method for producing a purified, formulated recombinant bispecific T cell engager, comprising: purifying the harvested recombinant bispecific T cell engagers via one or more chromatographic unit operations; subjecting the purified recombinant bispecific T cell engager to ultrafiltration and diafiltration unit operations to result in a formulated bispecific T cell engager at a concentration of < 5 g / L; subjecting the formulated bispecific T cell engager to a virus filtration unit operation; Obtaining a purified and formulated recombinant bispecific T cell engager. The method includes:

18. 18. The method of claim 17, wherein the formulated bispecific T cell engager is at a concentration of < 3.2 g / L.

19. 18. The method of claim 17, wherein the formulated bispecific T cell engager is at a concentration of < 1.79 g / L.

20. 18. The method of claim 17, wherein the bispecific T cell engager is an extended half-life bispecific T cell engager.

21. 20. The method of claim 17, wherein the ultrafiltration diafiltration unit operation is carried out with a stabilized cellulosic hydrophilic membrane or a regenerated cellulose membrane.

22. The ultrafiltration diafiltration unit operation can achieve up to 71.4 g / m per membrane area with an initial ultrafiltration target concentration of up to 3.20 g / L. ２ 20. The method of claim 17, carried out on a stabilized cellulosic hydrophilic membrane loaded to .

23. The ultrafiltration diafiltration unit operation is capable of delivering up to 170 g / m2 of membrane area at an intermediate target excess concentration of up to 9 g / L with a diavolume of up to 13. ２ 20. The method of claim 17, carried out on a regenerated cellulose membrane loaded to .

24. 20. The method of claim 17, wherein the virus filtration unit operation is performed with a hydrophilized polyvinylidene fluoride (PVDF) hollow fiber filter, a cuprammonium regenerated cellulose hollow fiber filter, or a polyethersulfone (PES) parvovirus-retentive filter.

25. 20. The method of claim 17, wherein the viral filtration unit operation is performed using a cuprammonium regenerated cellulose hollow fiber filter and a formulated bispecific T cell engager at a concentration of ≦3.2 g / L.

26. 20. The method of claim 17, wherein the virus filtration unit operation is performed using a hydrophilized polyvinylidene fluoride (PVDF) hollow fiber filter and a formulated bispecific T cell engager at a concentration of ≦1.79 g / L.

27. The method of claim 17, comprising subjecting the sample to a virus filtration unit operation comprising a virus filter in combination with a depth filter.

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