Use of temperature to control asymmetric antibody product quality

Abstract

The present invention relates to the field of biopharmaceutical manufacturing. In particular, the present invention relates to the use of temperature as a means to control product quality in upstream processes.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 180,220, filed April 27, 2021, the entirety of which is incorporated by reference herein for all purposes as if fully set forth herein.

[0002] Sequence Listing The computer readable nucleotide / amino acid sequence listing, which was submitted contemporaneously with this application and is identified as follows, is incorporated by reference in its entirety: "A-2777- US-PSP_SeqListing.txt " 791,966 ASCII (text) files of bytes, Created on April 27, 2021 .

[0003] The present invention relates to the field of biopharmaceutical manufacturing. In particular, the present invention relates to the use of temperature as a means to control product quality in manufacturing processes, especially in upstream cell culture steps. [Background technology]

[0004] Monoclonal antibody biotherapeutics are the largest segment of the biopharmaceutical market. However, because monoclonal antibodies can only bind to a single target, their effectiveness is limited due to the multifactorial nature of many diseases. To overcome these challenges, engineered multispecific antibodies have been developed. These multispecific antibodies can have any number of unique peptide sequences and can be engineered to have multiple target affinities. The unique properties of these proteins provide an improvement over traditional monoclonal antibody therapeutics and have proven to be effective next-generation biotherapeutics that can be utilized in a vast variety of formats to address even more challenging therapeutic indications.

[0005] However, there is little information on the production of multispecific antibodies, especially those that are asymmetric in structure, so platforms and processes developed for monoclonal antibodies are often applied with varying degrees of success. Asymmetric multispecific antibodies are highly engineered, and subjecting such proteins to upstream and downstream manufacturing processes under conditions typical for monoclonal antibodies can affect the product quality of the expressed and / or purified proteins. For example, the generation of product-related impurities during cell culture can complicate downstream purification steps and reduce the product quality of the desired asymmetric multispecific formulation. It would therefore be beneficial to optimize the conditions in the upstream manufacturing steps of asymmetric multispecific antibodies to mitigate any adverse effects on product quality.

[0006] The invention described herein meets this need by using temperature as a tool in the cell line development and cell culture process to improve product quality of recombinant asymmetric multispecific antibody drug substance. Summary of the Invention [Means for solving the problem]

[0007] The present invention provides a method for modulating the product quality of a recombinant asymmetric multispecific antibody expressed by cells during cell culture, comprising the steps of: (a) establishing at least two cell cultures, each inoculated with the same cell line expressing the asymmetric multispecific antibody; (b) culturing at least one cell culture at a first temperature regime consisting of a single temperature and culturing at least one cell culture at a second temperature regime; (c) comparing at least one product-related impurity in the cell cultures at each temperature regime; (d) selecting a temperature regime that reduces the expression of at least one product-related impurity comprising an unpaired or mispaired long heavy chain; (e) culturing the cell line at the selected temperature regime; and (f) recovering the recombinant asymmetric multispecific antibody. In one embodiment, the first temperature regime is selected from 36°C to 37°C. In one embodiment, the second temperature regime comprises a temperature shift from the first temperature to a second temperature higher or lower than the first temperature. In a related embodiment, the second temperature is selected from 28°C to 35°C. In a related embodiment, the second temperature is about 1° C. to about 9° C. lower than the first temperature. In one embodiment, the first temperature regime is a single temperature between 36° C. and 37° C., and the second temperature regime comprises at least one temperature shift from a first temperature between 36° C. and 37° C. to a second temperature between 28° C. and 35° C. In one embodiment, the product-related impurities result from an imbalance in the ratio of long to short heavy chains. In one embodiment, the product-related impurities comprise unpaired or mispaired long heavy chains. In a related embodiment, the product-related impurities comprise unpaired or mispaired long heavy chains, selected from homodimers, half antibodies, protein aggregates, antibody fragments, combinations of antibody fragments, and unpaired antibody fragments. In one embodiment, the cell culture temperature regime may be further selected for modulation of expression levels, productivity, growth, yield, and / or other desired product quality attributes of the asymmetric multispecific antibody. In one embodiment, the asymmetric multispecific antibody comprises a mutein. In one embodiment, the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein linked to one of the two antibody heavy chains of the anti-PD-1 antibody. In a related embodiment, the long heavy chain comprises an anti-PD-1 antibody heavy chain to which is linked an IL-21 mutein.

[0008] The present invention provides a method for regulating the product quality of a recombinant asymmetric multispecific antibody comprising a mutein expressed by cells during cell culture, comprising the steps of: (a) establishing at least two cell cultures, each inoculated with the same cell line expressing the asymmetric multispecific antibody; (b) culturing at least one cell culture at a first temperature regime consisting of a single temperature and culturing at least one cell culture at a second temperature regime; (c) comparing at least one product-related impurity comprising unpaired or mispaired long heavy chains produced by the cell cultures at each temperature regime; and (d) selecting a temperature regime that reduces the expression of at least one product-related impurity comprising unpaired or mispaired long heavy chains. In one embodiment, the mutein is an IL-21 mutein. In a related embodiment, the IL-21 mutein comprises an amino acid substitution at any two of positions 5, 9, 73, and 76 of SEQ ID NO:1, wherein the amino acid substitutions are selected from A, E, or Q at position 5, E or A at position 9, A or Q at position 73, and A, D, or E at position 76. In a related embodiment, the IL-21 mutein comprises the amino acid sequence of any of SEQ ID NOs:233-245. In one embodiment, the asymmetric multispecific antibody comprises a mutein attached to the C-terminus of one of the two antibody heavy chains of an anti-PD-1 antibody. In a related embodiment, the anti-PD-1 antibody comprises two light chains comprising LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequences of SEQ ID NOs:385, 386, and 387, respectively, and two heavy chains comprising HC CDR1, HC CDR2, and HC CDR3 comprising the amino acid sequences of SEQ ID NOs:382, 383, and 384, respectively. In a related embodiment, the anti-PD-1 antibody comprises two light chains comprising the amino acid sequence of SEQ ID NO: 389 and two heavy chains comprising the amino acid sequence of SEQ ID NO: 388. In a related embodiment, the anti-PD-1 antibody comprises two light chains comprising LC CDR1, LC CDR2, and LC CDR3 comprising the amino acid sequences of SEQ ID NOs: 365, 366, and 367, respectively, and two heavy chains comprising HC CDR1, HC CDR2, and HC CDR3 comprising the amino acid sequences of SEQ ID NOs: 362, 363, and 364, respectively.In a related embodiment, the anti-PD-1 antibody comprises two light chains comprising the amino acid sequence of SEQ ID NO: 369 and two heavy chains comprising the amino acid sequence of SEQ ID NO: 368. In a related embodiment, the anti-PD-1 antibody comprises (i) two light chains each comprising the amino acid sequence of SEQ ID NO: 391, a heavy chain bound to an IL-21 mutein comprising the amino acid sequence of any one of SEQ ID NOs: 501-506, and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 556-558, or (ii) two light chains each comprising the amino acid sequence of SEQ ID NO: 371, a heavy chain bound to a single IL-21 mutein comprising the amino acid sequence of any one of SEQ ID NOs: 513-518, and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 559-561. In a related embodiment, the anti-PD-1 antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 391, one heavy chain comprising the amino acid sequence of SEQ ID NO: 556, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 501. In a related embodiment, the anti-PD-1 antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, one heavy chain comprising the amino acid sequence of SEQ ID NO: 559, and one heavy chain linked to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 513. In one embodiment, the asymmetric multispecific antibody comprises an IL-21 mutein linked to the C-terminus of one of the two antibody heavy chains of the anti-PD-1 antibody. In one embodiment, the long heavy chain comprises an anti-PD-1 antibody heavy chain linked to an IL-21 mutein.

[0009] The present invention provides a method for modulating the product quality of a recombinant asymmetric multispecific antibody expressed by cells during cell culture. The present invention provides a method for modulating the product quality of a recombinant asymmetric multispecific antibody comprising a mutein expressed by cells during cell culture. In particular, the present invention provides a method for modulating the product quality of a conjugate of an IL-21 mutein linked to the C-terminus of one of the two antibody heavy chains of an anti-PD-1 antibody expressed by cells during cell culture, comprising the steps of (a) establishing a cell culture inoculated with a cell line expressing the antibody, (b) culturing the cells at 36±1° C. for a culture period, and (c) recovering the antibody, wherein at a certain point during the culture, the amount of at least one product-related impurity resulting from an imbalance in the ratio of long heavy chains to short heavy chains in the harvested cell culture is reduced compared to the amount of the same product-related impurity in the harvest from a cell culture exposed to a temperature of 32° C.-34° C.

[0010] The present invention provides a method for selecting a cell line expressing an asymmetric multispecific antibody. The present invention provides a method for selecting a cell line expressing an asymmetric multispecific antibody comprising a mutein, comprising the steps of: a) establishing at least one clonally derived cell line expressing the antibody; b) establishing at least two cell cultures from one or more clonally derived cell lines, wherein at least one cell culture from each cell line is cultured at a first temperature regime and at least one cell culture from each cell line is cultured at a second temperature regime; c) comparing the expression of at least one product-related impurity comprising an unmatched or mismatched long heavy chain produced by cells cultured at each temperature regime; and (d) selecting a cell line that resulted in a reduced expression of at least one product-related impurity comprising an unmatched or mismatched long heavy chain. In one embodiment, the selected cell line is then cultured at a temperature regime that resulted in a reduced expression of at least one product-related impurity comprising an unmatched or mismatched long heavy chain. In one embodiment, the first temperature regime is selected from about 36°C to about 37°C. In one embodiment, the second temperature regime comprises a temperature shift from a first temperature to a second temperature that is higher or lower than the first temperature. In a related embodiment, the second temperature is selected from about 28°C to about 35°C. In one embodiment, the second temperature is about 1°C to 9°C lower than the first temperature. In one embodiment, the first temperature regime is a single temperature between 36°C to 37°C, and the second temperature regime comprises at least one temperature shift from a first temperature between 36°C to 37°C to a second temperature between 28°C to 35°C. In one embodiment, the product-related impurities comprising unmatched or mismatched long heavy chains result from an imbalance in the ratio of long heavy chains to short heavy chains. In a related embodiment, the product-related impurities comprising unmatched or mismatched long heavy chains result from an increase in the ratio of long heavy chains to short heavy chains. In one embodiment, the product-related impurities comprise unmatched or mismatched long heavy chains and are selected from homodimers, half antibodies, protein aggregates, antibody fragments, combinations of antibody fragments, and unpaired antibody fragments. In one embodiment, the cell culture temperature regime may be further selected for a temperature regime that also regulates expression level, productivity, growth, and / or other desired product quality attributes of the asymmetric multispecific antibody.In one embodiment, the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein linked to one of the two antibody heavy chains of the anti-PD-1 antibody. In one embodiment, the long heavy chain comprises an anti-PD-1 antibody heavy chain to which an IL-21 mutein is linked.

[0011] The present invention provides a method for modulating the production of at least one recombinant asymmetric multispecific antibody product-related impurity comprising an antibody heavy chain. The present invention provides a method for modulating the production of at least one recombinant asymmetric multispecific antibody product-related impurity comprising a long heavy chain. In particular, the present invention provides a method for modulating the production of at least one recombinant asymmetric multispecific antibody product-related impurity comprising an antibody heavy chain conjugated with an IL-21 mutein during cell culture by cell culture temperature, comprising: selecting a temperature regime that reduces the expression of at least one product-related impurity comprising an unpaired or mispaired long heavy chain comprising a long heavy chain; (a) culturing the cell line at the selected temperature regime; and (b) recovering the recombinant asymmetric multispecific antibody. In one embodiment, the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein conjugated to one of the two antibody heavy chains of the anti-PD-1 antibody. In one embodiment, the long heavy chain comprises an anti-PD-1 antibody heavy chain conjugated with an IL-21 mutein.

[0012] The present invention relates to a method for producing isolated and purified recombinant asymmetric multispecific antibodies, comprising the steps of: a) isolating at least one single stably transformed cell expressing the asymmetric multispecific antibody and establishing a clone-derived culture; b) establishing at least two cell cultures from one or more of the clone-derived cultures; c) culturing at least one cell culture from each clone-derived culture at a first temperature regime comprising a single temperature and culturing at least one cell culture from each clone-derived culture at a second temperature regime; and d) detecting at least one product associated with unpaired or mispaired long heavy chains produced by the cells cultured at each temperature regime. e) selecting a cell culture with modulated production of product-related impurities; f) establishing a cell line expressing the asymmetric multispecific antibody from the selected cell culture; g) inoculating a bioreactor with the cell line expressing the asymmetric multispecific antibody; h) culturing the cells to express the asymmetric multispecific antibody at a temperature regime with modulated production of product-related impurities; i) recovering the recombinant asymmetric multispecific antibody from the cell culture; j) processing the recombinant multispecific antibody by one or more chromatographic unit operations; and k) obtaining an isolated and purified recombinant asymmetric multispecific antibody. In one embodiment, an isolated and purified recombinant multispecific antibody by the above method is provided. In one embodiment, a pharmaceutical composition comprising an isolated and purified recombinant multispecific antibody by the above method is provided. In one embodiment, the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein attached to one of the two antibody heavy chains of the anti-PD-1 antibody. In one embodiment, the long heavy chain comprises an anti-PD-1 antibody heavy chain conjugated to an IL-21 mutein.

[0013] The present invention provides a method for controlling the growth of cells in a nanofluidic chamber of a nanofluidic chip, the method comprising: (a) isolating a single cell in a nanofluidic chamber of a nanofluidic chip, the cell comprising an expression construct capable of expressing a recombinant protein; (b) culturing the cell at a first temperature; (c) at a predetermined time point, culturing the cell at a second temperature; and (d) removing the cell from the nanofluidic chamber to a culture container.

[0014] The present invention also provides a method for minimizing cross-contamination during export of cells grown in a nanofluidic chamber of a nanofluidic chip, comprising the steps of: (a) isolating a single cell in a nanofluidic chamber of a nanofluidic chip, the cell comprising an expression construct capable of expressing a recombinant protein; (b) culturing the cell at a first temperature; (c) at a predetermined time point, culturing the cell at a second temperature; and (d) exporting the cells from the nanofluidic chamber to a culture vessel, wherein the number of cells per chamber upon export is lower compared to a single cell cultured under similar conditions at a constant temperature over the culture period.

[0015] The present invention further provides a method for improving clonal selection of cells grown in a nanofluidic chamber of a nanofluidic chip, the method comprising: (a) isolating a single cell in a nanofluidic chamber of a nanofluidic chip, the cell comprising an expression construct capable of expressing a recombinant protein; (b) culturing the cell at a first temperature; (c) reducing the temperature of the culture to a second temperature after the third day of culture; and (d) removing the cells from the nanofluidic chamber at least four days after the temperature shift.

[0016] In one embodiment, the number of cells per chamber at the time of removal is less compared to single cells cultured under similar conditions at a constant temperature during the culture period. In one embodiment, the first temperature is selected from 35°C to 37°C. In one embodiment, the second temperature is selected from 28°C to 34°C. In one embodiment, the second temperature is about 1°C to about 9°C lower than the first temperature. In one embodiment, the first temperature is 36°C and the second temperature is 32°C to 32.5°C. In one embodiment, the predetermined point is the third to fifth day of culture. In one embodiment, the removal is the sixth to eighth day of culture. In one embodiment, the nanofluidic chip comprises 1758 chambers, 3,500 chambers, 11,000 chambers, 14,000 chambers, or 20,000 chambers. In one embodiment, the culture vessel is a multi-well plate. In one embodiment, the protein secretion profiles before and after the temperature shift are compared. [Brief description of the drawings]

[0017] [Figure 1A] (A) shows the increase in % long heavy chain (% LHC) when cells expressing IL-2-anti-PD-1 conjugates are subjected to perfusion culture (P) with temperature shift to 32.5°C compared to fed-batch culture (FB) at constant 36°C (striped bars). (B) and (C) show the results of IL-2-anti-PD-1 conjugate production expressed by cells in perfusion culture (P) with temperature shift from 36°C to 32.5°C subjected to purification by Protein A affinity chromatography (left open bar) and cation exchange chromatography (right open bar) compared to IL-2-anti-PD-1 conjugate production expressed by cells in constant temperature (36°C) fed-batch culture (FB) subjected to Protein A affinity chromatography only (striped bars). (B) % Pre-Peak by nrCE. (C) % Low molecular weight impurities (LMW) by SEC. (D) Characterization of impurities by nrCE-SDS from the CEX pool. Excess long heavy chains could not be separated by CEX. [Figure 1B](A) shows the increase in % long heavy chain (% LHC) when cells expressing IL-2-anti-PD-1 conjugates are subjected to perfusion culture (P) with temperature shift to 32.5°C compared to fed-batch culture (FB) at constant 36°C (striped bars). (B) and (C) show the results of IL-2-anti-PD-1 conjugate production expressed by cells in perfusion culture (P) with temperature shift from 36°C to 32.5°C subjected to purification by Protein A affinity chromatography (left open bar) and cation exchange chromatography (right open bar) compared to IL-2-anti-PD-1 conjugate production expressed by cells in constant temperature (36°C) fed-batch culture (FB) subjected to Protein A affinity chromatography only (striped bars). (B) % Pre-Peak by nrCE. (C) % Low molecular weight impurities (LMW) by SEC. (D) Characterization of impurities by nrCE-SDS from the CEX pool. Excess long heavy chains could not be separated by CEX. [Figure 1C] (A) shows the increase in % long heavy chain (% LHC) when cells expressing IL-2-anti-PD-1 conjugates are subjected to perfusion culture (P) with temperature shift to 32.5°C compared to fed-batch culture (FB) at constant 36°C (striped bars). (B) and (C) show the results of IL-2-anti-PD-1 conjugate production expressed by cells in perfusion culture (P) with temperature shift from 36°C to 32.5°C subjected to purification by Protein A affinity chromatography (left open bar) and cation exchange chromatography (right open bar) compared to IL-2-anti-PD-1 conjugate production expressed by cells in constant temperature (36°C) fed-batch culture (FB) subjected to Protein A affinity chromatography only (striped bars). (B) % Pre-Peak by nrCE. (C) % Low molecular weight impurities (LMW) by SEC. (D) Characterization of impurities by nrCE-SDS from the CEX pool. Excess long heavy chains could not be separated by CEX. [Figure 1D](A) shows the increase in % long heavy chain (% LHC) when cells expressing IL-2-anti-PD-1 conjugates are subjected to perfusion culture (P) with temperature shift to 32.5°C compared to fed-batch culture (FB) at constant 36°C (striped bars). (B) and (C) show the results of IL-2-anti-PD-1 conjugate production expressed by cells in perfusion culture (P) with temperature shift from 36°C to 32.5°C subjected to purification by Protein A affinity chromatography (left open bar) and cation exchange chromatography (right open bar) compared to IL-2-anti-PD-1 conjugate production expressed by cells in constant temperature (36°C) fed-batch culture (FB) subjected to Protein A affinity chromatography only (striped bars). (B) % Pre-Peak by nrCE. (C) % Low molecular weight impurities (LMW) by SEC. (D) Characterization of impurities by nrCE-SDS from the CEX pool. Excess long heavy chains could not be separated by CEX. [Figure 2A] Figure 2 compares the molar ratios of long heavy chain (LHC) to short heavy chain (SHC) identified by rCE and shows that there was a better correlation between fed-batch and perfusion samples when screened at the same temperature. (A) shows fed-batch culture samples grown at 36°C constantly compared to perfusion samples grown with a temperature shift (TS) from 36°C to 32.5°C. Fed-batch (x, constant 36°C) versus perfusion (y, 36°C to 32.5°C). (B) shows fed-batch culture samples compared to perfusion samples, both grown with a temperature shift (TS) from 36°C to 32.5°C. The x-axis is fed-batch culture versus the y-axis is perfusion. There were limited data points for both culture methods grown at 36°C constantly and therefore were not included in the comparison. [Figure 2B]Figure 2 compares the molar ratios of long heavy chain (LHC) to short heavy chain (SHC) identified by rCE and shows that there was a better correlation between fed-batch and perfusion samples when screened at the same temperature. (A) shows fed-batch culture samples grown at 36°C constantly compared to perfusion samples grown with a temperature shift (TS) from 36°C to 32.5°C. Fed-batch (x, constant 36°C) versus perfusion (y, 36°C to 32.5°C). (B) shows fed-batch culture samples compared to perfusion samples, both grown with a temperature shift (TS) from 36°C to 32.5°C. The x-axis is fed-batch culture versus the y-axis is perfusion. There were limited data points for both culture methods grown at 36°C constantly and therefore were not included in the comparison. [Figure 3A] Figure 3 compares the percent low molecular weight (%LMW) measured by SEC and shows that there was a better correlation between fed-batch and perfusion samples when screened at the same temperature. (A) shows fed-batch culture samples grown at 36°C constantly compared to perfusion samples grown with a temperature shift (TS) from 36°C to 32.5°C. x-axis is fed-batch culture vs. y-axis is perfusion. (B) shows fed-batch culture samples compared to perfusion samples, both grown with a temperature shift (TS) from 36°C to 32.5°C. x-axis is fed-batch culture vs. y-axis is perfusion. There were limited data points for both culture methods grown at 36°C constantly and therefore were not included in the comparison. [Figure 3B] Figure 3 compares the percent low molecular weight (%LMW) measured by SEC and shows that there was a better correlation between fed-batch and perfusion samples when screened at the same temperature. (A) shows fed-batch culture samples grown at 36°C constantly compared to perfusion samples grown with a temperature shift (TS) from 36°C to 32.5°C. x-axis is fed-batch culture vs. y-axis is perfusion. (B) shows fed-batch culture samples compared to perfusion samples, both grown with a temperature shift (TS) from 36°C to 32.5°C. x-axis is fed-batch culture vs. y-axis is perfusion. There were limited data points for both culture methods grown at 36°C constantly and therefore were not included in the comparison. [Figure 4A] Figure 4 shows the results of the production of IL-2-anti-PD-1 conjugates expressed by cells in perfusion culture at a constant temperature of 36°C compared to perfusion culture with a temperature shift from 36°C to 32.5°C. (A) shows the molar ratio of long heavy chain (LHC) to short heavy chain (SHC) determined by rCE (constant 36°C, striped bars; temperature shift from 36°C to 32.5°C, white bars). (B) shows the % of Pre-Peak 6 (impurity formed by SHC) purified by Protein A affinity chromatography (striped bars) and cation exchange chromatography (gray bars) and measured by nrCE. (C) shows the % of Pre-Peak 7 (impurity formed upon LHC) purified by Protein A affinity chromatography (striped bars) and cation exchange chromatography (white bars) and measured by nrCE. [Figure 4B] Figure 4 shows the results of the production of IL-2-anti-PD-1 conjugates expressed by cells in perfusion culture at a constant temperature of 36°C compared to perfusion culture with a temperature shift from 36°C to 32.5°C. (A) shows the molar ratio of long heavy chain (LHC) to short heavy chain (SHC) determined by rCE (constant 36°C, striped bars; temperature shift from 36°C to 32.5°C, white bars). (B) shows the % of Pre-Peak 6 (impurity formed by SHC) purified by Protein A affinity chromatography (striped bars) and cation exchange chromatography (gray bars) and measured by nrCE. (C) shows the % of Pre-Peak 7 (impurity formed upon LHC) purified by Protein A affinity chromatography (striped bars) and cation exchange chromatography (white bars) and measured by nrCE. [Figure 4C]Figure 4 shows the results of the production of IL-2-anti-PD-1 conjugates expressed by cells in perfusion culture at a constant temperature of 36°C compared to perfusion culture with a temperature shift from 36°C to 32.5°C. (A) shows the molar ratio of long heavy chain (LHC) to short heavy chain (SHC) determined by rCE (constant 36°C, striped bars; temperature shift from 36°C to 32.5°C, white bars). (B) shows the % of Pre-Peak 6 (impurity formed by SHC) purified by Protein A affinity chromatography (striped bars) and cation exchange chromatography (gray bars) and measured by nrCE. (C) shows the % of Pre-Peak 7 (impurity formed upon LHC) purified by Protein A affinity chromatography (striped bars) and cation exchange chromatography (white bars) and measured by nrCE. [Figure 5A] Figure 5 shows the results of bispecific A clone 1 production in fed-batch cultures with a constant temperature of 36°C (striped bars) and with a temperature shift from 36°C to 32.5°C (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW by SEC. (C) % LMW (impurity formed by LHC) by SEC. [Figure 5B] Figure 5 shows the results of bispecific A clone 1 production in fed-batch cultures with a constant temperature of 36°C (striped bars) and with a temperature shift from 36°C to 32.5°C (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW by SEC. (C) % LMW (impurity formed by LHC) by SEC. [Figure 5C]Figure 5 shows the results of bispecific A clone 1 production in fed-batch cultures with a constant temperature of 36°C (striped bars) and with a temperature shift from 36°C to 32.5°C (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW by SEC. (C) % LMW (impurity formed by LHC) by SEC. [Figure 6A] Figure 6 shows the results of production of bispecific A clones 2 and 3 by simulated perfusion culture with temperature constantly held at 36°C (striped bars) and with temperature shift from 36°C to 32.5°C (white bars). (A) Long heavy chain (LHC) to short heavy chain (SHC) molar ratio determined by rCE-SDS. (B) % of LMW measured by SEC. (C) LHC to SHC transcript ratio measured by ddPCR. [Figure 6B] Figure 6 shows the results of production of bispecific A clones 2 and 3 by simulated perfusion culture with temperature constantly held at 36°C (striped bars) and with temperature shift from 36°C to 32.5°C (white bars). (A) Long heavy chain (LHC) to short heavy chain (SHC) molar ratio determined by rCE-SDS. (B) % of LMW measured by SEC. (C) LHC to SHC transcript ratio measured by ddPCR. [Figure 6C] Figure 6 shows the results of production of bispecific A clones 2 and 3 by simulated perfusion culture with temperature constantly held at 36°C (striped bars) and with temperature shift from 36°C to 32.5°C (white bars). (A) Long heavy chain (LHC) to short heavy chain (SHC) molar ratio determined by rCE-SDS. (B) % of LMW measured by SEC. (C) LHC to SHC transcript ratio measured by ddPCR. [Figure 7A]Figure 7 shows the results of bispecific B clones 1, 2 and 3 production in fed-batch cultures with a constant temperature of 36°C (striped bars) and with a temperature shift from 36°C to 32.5°C (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW2 as determined by SEC. (C) % LMW1 as determined by SEC. (D) Transcript ratio of LHC to SHC as determined by ddPCR. [Figure 7B] Figure 7 shows the results of bispecific B clones 1, 2 and 3 production in fed-batch cultures with a constant temperature of 36°C (striped bars) and with a temperature shift from 36°C to 32.5°C (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW2 as determined by SEC. (C) % LMW1 as determined by SEC. (D) Transcript ratio of LHC to SHC as determined by ddPCR. [Figure 7C] Figure 7 shows the results of bispecific B clones 1, 2 and 3 production in fed-batch cultures with a constant temperature of 36°C (striped bars) and with a temperature shift from 36°C to 32.5°C (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW2 as determined by SEC. (C) % LMW1 as determined by SEC. (D) Transcript ratio of LHC to SHC as determined by ddPCR. [Figure 7D] Figure 7 shows the results of bispecific B clones 1, 2 and 3 production in fed-batch cultures with a constant temperature of 36°C (striped bars) and with a temperature shift from 36°C to 32.5°C (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW2 as determined by SEC. (C) % LMW1 as determined by SEC. (D) Transcript ratio of LHC to SHC as determined by ddPCR. [Figure 8A]FIG. 8 shows the results of bispecific B clones 2 and 3 production by sham perfusion comparing incubation at a constant temperature of 36° C. (striped bars) with incubation at a temperature shift from 36° C. to 32.5° C. (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW2 as determined by SEC. (C) % LMW1 as determined by SEC. [Figure 8B] FIG. 8 shows the results of bispecific B clones 2 and 3 production by sham perfusion comparing incubation at a constant temperature of 36° C. (striped bars) with incubation at a temperature shift from 36° C. to 32.5° C. (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW2 as determined by SEC. (C) % LMW1 as determined by SEC. [Figure 8C] FIG. 8 shows the results of bispecific B clones 2 and 3 production by sham perfusion comparing incubation at a constant temperature of 36° C. (striped bars) with incubation at a temperature shift from 36° C. to 32.5° C. (white bars). (A) Molar ratio of long heavy chain (LHC) to short heavy chain (SHC) as determined by rCE-SDS. (B) % HMW2 as determined by SEC. (C) % LMW1 as determined by SEC. [Figure 9A]FIG. 9 shows that temperature shifts implemented during on-chip culture result in growth inhibition and enable long-term culture. (A) Representative brightfield chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 6 days. Cell counts generated by the instrument software are displayed below each time point. The cell population on day 6 occupied the majority of the chamber contents and grew close to the neck region of the chamber, preventing reliable secretion assessment by the Spotlight™ Human Fc Assay and creating a high risk of cross-contamination in the expected unloading procedure. (B) Representative brightfield chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 3 days and then the temperature was reduced to 32° C. for the remainder of the experimental period. Cell counts generated by the instrument software are displayed below each time point. The temperature shift implemented from day 4 resulted in growth inhibition and prevented cells from growing beyond the maximum recommended height of the chamber. (C) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period. Implementation of a temperature shift from day 4 resulted in growth inhibition as manifested by an increase in mean doubling time and a decrease in mean cell number when compared to control conditions. [Figure 9B]FIG. 9 shows that temperature shifts implemented during on-chip culture result in growth inhibition and enable long-term culture. (A) Representative brightfield chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 6 days. Cell counts generated by the instrument software are displayed below each time point. The cell population on day 6 occupied the majority of the chamber contents and grew close to the neck region of the chamber, preventing reliable secretion assessment by the Spotlight™ Human Fc Assay and creating a high risk of cross-contamination in the expected unloading procedure. (B) Representative brightfield chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 3 days and then the temperature was reduced to 32° C. for the remainder of the experimental period. Cell counts generated by the instrument software are displayed below each time point. The temperature shift implemented from day 4 resulted in growth inhibition and prevented cells from growing beyond the maximum recommended height of the chamber. (C) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period. Implementation of a temperature shift from day 4 resulted in growth inhibition as manifested by an increase in mean doubling time and a decrease in mean cell number when compared to control conditions. [Figure 9C]FIG. 9 shows that temperature shifts implemented during on-chip culture result in growth inhibition and enable long-term culture. (A) Representative brightfield chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 6 days. Cell counts generated by the instrument software are displayed below each time point. The cell population on day 6 occupied the majority of the chamber contents and grew close to the neck region of the chamber, preventing reliable secretion assessment by the Spotlight™ Human Fc Assay and creating a high risk of cross-contamination in the expected unloading procedure. (B) Representative brightfield chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 3 days and then the temperature was reduced to 32° C. for the remainder of the experimental period. Cell counts generated by the instrument software are displayed below each time point. The temperature shift implemented from day 4 resulted in growth inhibition and prevented cells from growing beyond the maximum recommended height of the chamber. (C) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period. Implementation of a temperature shift from day 4 resulted in growth inhibition as manifested by an increase in mean doubling time and a decrease in mean cell number when compared to control conditions. [Figure 10A]Figure 10 shows that performing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform alters monoclonal antibody production profiles. (A) Schematic of the standard cell line development cloning workflow on the Beacon platform (left panel) and the cell line development cloning workflow with temperature shift performed in B and C (right panel). (B) Normalized secretion score data corresponding to Spotlight™ Human Fc Assay performed before temperature shift on day 4 (left panel) and after temperature shift on day 6 (right panel). Cell secretion profiles measured after temperature shift showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. (C) Brightfield and Spotlight™ Human Fc Assay fluorescence images collected throughout the experimental period on the Berkeley Light Bacon platform corresponding to two clonally derived cell lines expressing monoclonal antibodies that underwent a CLD workflow with temperature shift. Cell counts and normalized secretion scores generated by the instrument software are displayed below each time point. Incubation at low temperature conditions resulted in growth arrest of both clones. Both cell lines exhibited comparable secretion profiles on day 4 of the workflow, however, clone 1, but not clone 2, exhibited increased secretion levels in response to the temperature shift. [Figure 10B]Figure 10 shows that performing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform alters monoclonal antibody production profiles. (A) Schematic of the standard cell line development cloning workflow on the Beacon platform (left panel) and the cell line development cloning workflow with temperature shift performed in B and C (right panel). (B) Normalized secretion score data corresponding to Spotlight™ Human Fc Assay performed before temperature shift on day 4 (left panel) and after temperature shift on day 6 (right panel). Cell secretion profiles measured after temperature shift showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. (C) Brightfield and Spotlight™ Human Fc Assay fluorescence images collected throughout the experimental period on the Berkeley Light Bacon platform corresponding to two clonally derived cell lines expressing monoclonal antibodies that underwent a CLD workflow with temperature shift. Cell counts and normalized secretion scores generated by the instrument software are displayed below each time point. Incubation at low temperature conditions resulted in growth arrest of both clones. Both cell lines exhibited comparable secretion profiles on day 4 of the workflow, however, clone 1, but not clone 2, exhibited increased secretion levels in response to the temperature shift. [Figure 10C]Figure 10 shows that performing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform alters monoclonal antibody production profiles. (A) Schematic of the standard cell line development cloning workflow on the Beacon platform (left panel) and the cell line development cloning workflow with temperature shift performed in B and C (right panel). (B) Normalized secretion score data corresponding to Spotlight™ Human Fc Assay performed before temperature shift on day 4 (left panel) and after temperature shift on day 6 (right panel). Cell secretion profiles measured after temperature shift showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. (C) Brightfield and Spotlight™ Human Fc Assay fluorescence images collected throughout the experimental period on the Berkeley Light Bacon platform corresponding to two clonally derived cell lines expressing monoclonal antibodies that underwent a CLD workflow with temperature shift. Cell counts and normalized secretion scores generated by the instrument software are displayed below each time point. Incubation at low temperature conditions resulted in growth arrest of both clones. Both cell lines exhibited comparable secretion profiles on day 4 of the workflow, however, clone 1, but not clone 2, exhibited increased secretion levels in response to the temperature shift. [Figure 11A]Figure 11 shows that implementing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform achieves growth inhibition and alters the recombinant protein production profile of cell lines expressing IgG4 monoclonal and multispecific antibody formats. (A)(D) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period for clonally derived cell lines expressing IgG4 monoclonal and multispecific antibodies, respectively. Temperature settings for each time point of the experiment are outlined at the top of the graph. Implementing a temperature shift from day 4 or day 3 resulted in growth inhibition as measured by increased doubling time and decreased cell number for both IgG4 and multispecific antibody expressing cell lines, respectively. (B)(E) Representative brightfield chamber images collected throughout the experimental period on the Berkeley Light Bacon platform of clonally derived cell lines expressing monoclonal or multispecific antibodies, respectively. Cell counts generated by the instrument software are displayed below each time point. (C)(F) Spotlight™ Human Fc Assay scoring data corresponding to time points of day 4 and day 8 or day 7, respectively. Secretion profiles achieved after extended incubation at reduced temperature showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. [Figure 11B]Figure 11 shows that implementing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform achieves growth inhibition and alters the recombinant protein production profile of cell lines expressing IgG4 monoclonal and multispecific antibody formats. (A)(D) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period for clonally derived cell lines expressing IgG4 monoclonal and multispecific antibodies, respectively. Temperature settings for each time point of the experiment are outlined at the top of the graph. Implementing a temperature shift from day 4 or day 3 resulted in growth inhibition as measured by increased doubling time and decreased cell number for both IgG4 and multispecific antibody expressing cell lines, respectively. (B)(E) Representative brightfield chamber images collected throughout the experimental period on the Berkeley Light Bacon platform of clonally derived cell lines expressing monoclonal or multispecific antibodies, respectively. Cell counts generated by the instrument software are displayed below each time point. (C)(F) Spotlight™ Human Fc Assay scoring data corresponding to time points of day 4 and day 8 or day 7, respectively. Secretion profiles achieved after extended incubation at reduced temperature showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. [Figure 11C]Figure 11 shows that implementing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform achieves growth inhibition and alters the recombinant protein production profile of cell lines expressing IgG4 monoclonal and multispecific antibody formats. (A)(D) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period for clonally derived cell lines expressing IgG4 monoclonal and multispecific antibodies, respectively. Temperature settings for each time point of the experiment are outlined at the top of the graph. Implementing a temperature shift from day 4 or day 3 resulted in growth inhibition as measured by increased doubling time and decreased cell number for both IgG4 and multispecific antibody expressing cell lines, respectively. (B)(E) Representative brightfield chamber images collected throughout the experimental period on the Berkeley Light Bacon platform of clonally derived cell lines expressing monoclonal or multispecific antibodies, respectively. Cell counts generated by the instrument software are displayed below each time point. (C)(F) Spotlight™ Human Fc Assay scoring data corresponding to time points of day 4 and day 8 or day 7, respectively. Secretion profiles achieved after extended incubation at reduced temperature showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. [Figure 11D]Figure 11 shows that implementing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform achieves growth inhibition and alters the recombinant protein production profile of cell lines expressing IgG4 monoclonal and multispecific antibody formats. (A)(D) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period for clonally derived cell lines expressing IgG4 monoclonal and multispecific antibodies, respectively. Temperature settings for each time point of the experiment are outlined at the top of the graph. Implementing a temperature shift from day 4 or day 3 resulted in growth inhibition as measured by increased doubling time and decreased cell number for both IgG4 and multispecific antibody expressing cell lines, respectively. (B)(E) Representative brightfield chamber images collected throughout the experimental period on the Berkeley Light Bacon platform of clonally derived cell lines expressing monoclonal or multispecific antibodies, respectively. Cell counts generated by the instrument software are displayed below each time point. (C)(F) Spotlight™ Human Fc Assay scoring data corresponding to time points of day 4 and day 8 or day 7, respectively. Secretion profiles achieved after extended incubation at reduced temperature showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. [Figure 11E]Figure 11 shows that implementing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform achieves growth inhibition and alters the recombinant protein production profile of cell lines expressing IgG4 monoclonal and multispecific antibody formats. (A)(D) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period for clonally derived cell lines expressing IgG4 monoclonal and multispecific antibodies, respectively. Temperature settings for each time point of the experiment are outlined at the top of the graph. Implementing a temperature shift from day 4 or day 3 resulted in growth inhibition as measured by increased doubling time and decreased cell number for both IgG4 and multispecific antibody expressing cell lines, respectively. (B)(E) Representative brightfield chamber images collected throughout the experimental period on the Berkeley Light Bacon platform of clonally derived cell lines expressing monoclonal or multispecific antibodies, respectively. Cell counts generated by the instrument software are displayed below each time point. (C)(F) Spotlight™ Human Fc Assay scoring data corresponding to time points of day 4 and day 8 or day 7, respectively. Secretion profiles achieved after extended incubation at reduced temperature showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. [Figure 11F]Figure 11 shows that implementing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform achieves growth inhibition and alters the recombinant protein production profile of cell lines expressing IgG4 monoclonal and multispecific antibody formats. (A)(D) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period for clonally derived cell lines expressing IgG4 monoclonal and multispecific antibodies, respectively. Temperature settings for each time point of the experiment are outlined at the top of the graph. Implementing a temperature shift from day 4 or day 3 resulted in growth inhibition as measured by increased doubling time and decreased cell number for both IgG4 and multispecific antibody expressing cell lines, respectively. (B)(E) Representative brightfield chamber images collected throughout the experimental period on the Berkeley Light Bacon platform of clonally derived cell lines expressing monoclonal or multispecific antibodies, respectively. Cell counts generated by the instrument software are displayed below each time point. (C)(F) Spotlight™ Human Fc Assay scoring data corresponding to time points of day 4 and day 8 or day 7, respectively. Secretion profiles achieved after extended incubation at reduced temperature showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. [Figure 12] Figure 12 shows that the export procedure performed at a temperature of 32 °C results in similar clonal recovery when compared to export performed at 36 °C per the standard CLD workflow. (A)(B) Top panels: Schematic of the standard cell line development cloning workflow on the Beacon platform and the cell line development cloning workflow where a temperature shift is performed during the export process, respectively. Bottom panels: Representative images of the 96-well export plate acquired 18 days after the export procedure showing comparable clonal recovery efficiency regardless of the export temperature performed at the single cell cloning step. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Monoclonal antibodies are symmetrical, having two identical polypeptide chains, each pair having one "light" chain (LC) and one "heavy" chain (HC), specific for one target. Multispecific antibodies that bind to, neutralize, and / or interact with more than one target are being engineered in increasingly diverse compositions and configurations and in increasingly diverse formats to meet increasingly challenging therapeutic indications, offering alternatives and improvements over monoclonal antibodies. Many multispecific antibodies share characteristics with monoclonal antibodies, such as having one light chain and one heavy chain, but unlike monoclonal antibodies, the heavy chains of multispecific antibodies can be asymmetric.

[0019] An "asymmetric" multispecific antibody comprises heavy chains of different lengths. The heavy chains typically comprise a variable domain (Vh) and a constant domain (C H 1) and two constant domains (C H 2 and / or C H 3), and a hinge region connecting the Fab region and the Fc region. The heavy chain of the asymmetric multispecific antibody may include some or all of these components. In addition, the heavy chain may also include one or more additional components directly and / or indirectly attached to any point of the variable and / or constant domains of the heavy chain. The length of the heavy chain is determined based on the combined size of the amino acid sequences of all components that make up the heavy chain, including any variable domains, constant domains, hinge regions, binding proteins and / or peptides, and any linkers and / or other binding mechanisms. The heavy chain with the longest combined amino acid sequence is called a "long heavy chain". The heavy chain with a shorter combined amino acid sequence is called a "short heavy chain". An asymmetric multispecific antibody has at least one long heavy chain and at least one short heavy chain. An asymmetric multispecific antibody specifically binds to, neutralizes, and / or interacts with at least two different antigens or targets, and / or different epitopes on the same antigen or target.

[0020] In one embodiment, the long heavy chain of the asymmetric antibody comprises a linked mutein. In one embodiment, the asymmetric multispecific antibody is an anti-PD1 antibody comprising a long heavy chain and a short heavy chain. The long heavy chain comprises a single IL-21 mutein linked to one of the two antibody heavy chains of the anti-PD-1 antibody, and the short heavy chain comprises the other anti-PD-1 antibody heavy chain. In one embodiment, the long heavy chain comprises an IL-21 mutein linked to the Fc of the anti-PD-1 antibody. In one embodiment, the long heavy chain comprises an IL-21 mutein linked directly to the Fc of the anti-PD-1 antibody without a linker. In one embodiment, the long heavy chain comprises an IL-21 mutein linked directly to the C-terminus of one of the two antibody heavy chains of the anti-PD-1 antibody with or without a linker.

[0021] The production and purification of asymmetric multispecific antibodies brings its own manufacturing challenges. The engineered origin and asymmetry of these antibodies make them prone to the formation of product-related impurities throughout the manufacturing process, especially during the cell culture step. The type and / or amount of impurities can affect the expression, purification, yield, activity and / or product quality of these asymmetric multispecific antibodies. "Product-related impurities" refer to variants that may share one or more structural components with the desired product, e.g., asymmetric multispecific antibodies. One example of a structural component is the long heavy chain of an asymmetric multispecific antibody. If such impurities are expressed in cell culture, they must be removed in downstream purification steps. The challenge of removing these impurities increases depending on how similar the impurities are to the protein of interest in composition, size, hydrophobicity, isoelectric point (pI), surface charge, etc.

[0022] Balanced expression of the unique components that make up a recombinant asymmetric multispecific antibody is a challenge when expressing these proteins in culture. Imbalance in expression of one or more components can result in the formation of product-related impurities, such as unpaired and / or mispaired protein variants. The degree of success in removing product-related impurities can depend on the properties of the impurity. Furthermore, the removal step results in some product loss. As described herein, it has been found that product-related impurities, including anti-PD-1 long heavy chains, are unexpectedly difficult to remove during purification steps, adversely affecting product quality, yield, and robustness of the overall manufacturing process.

[0023] It was found that the molar ratio of long to short heavy chains of asymmetric multispecific antibodies produced during cell culture is affected by the culture temperature. As described in more detail herein and in the Examples, it was discovered that clonal cells expressing asymmetric multispecific antibodies were subjected to a low set point during the cell culture process as a result of a temperature shift (e.g., from 36° C. to 34° C. or 32.5° C.). The molar ratio of long to short heavy chains expressed by the clonal cells unexpectedly increased at the lower culture temperature. In this case, the clonal cells express asymmetric multispecific antibodies that have been grown and selected using a typical cell line development process that maintains a fixed physiological temperature (e.g., 36° C.) during single cell cloning, scale-up and selection, as is typically done for clonal cells expressing other types of proteins such as monoclonal antibodies and symmetric multispecific antibodies. In one embodiment, the product-related impurities are due to an imbalance in heavy chain expression. In one embodiment, the product-related impurities are due to an imbalance in the ratio of long to short heavy chains. In one embodiment, the product-related impurities are due to an increase in the ratio of long to short heavy chains. No effect of the pH of the cell culture was observed.

[0024] Temperature shifts are commonly used during cell culture processes to affect cell growth and recombinant protein production of symmetric proteins. However, for cells expressing asymmetric multispecific antibodies, such temperature shifts during cell culture can unexpectedly alter the ratio of long and short heavy chains expressed during culture. It has been found that an increase in the ratio of long and short heavy chains affects the formation and type of product-related impurities. This change in the ratio of long and short heavy chains can affect purification, activity, product quality, and the robustness of the entire manufacturing process.

[0025] It has been found that the production quality of asymmetric multispecific antibodies can be regulated by the cell culture temperature. In particular, the present invention provides methods for determining temperature conditions that maintain a more balanced expression of long heavy chains relative to short heavy chains, thereby reducing the formation of product-related impurities having unpaired and / or mispaired long heavy chains. In one embodiment, the cell culture temperature regime can be further selected such that, in addition to regulating heavy chain-related product-related impurities, the temperature also regulates the expression, productivity, growth, and / or other desired product quality attributes of the asymmetric multispecific antibody.

[0026] As described herein, the effect of unexpected temperature-induced imbalance in the expression of long heavy chains of asymmetric multispecific antibodies has been problematic for the manufacture of formulations containing such proteins. For symmetric proteins such as monoclonal antibodies, the balanced expression of heavy chains may not be as important since the chains are identical. However, for asymmetric multispecific antibodies with at least one long heavy chain and at least one short heavy chain, it has been found that the balance of heavy chain expression is a critical component of a robust pharmaceutical manufacturing process and production with desired yield and product quality attributes. The imbalance in the production of long and short heavy chains increased the chance of the formation of product-related impurities resulting from unpaired or mispaired long heavy chains. In one embodiment, the product-related impurities result from an imbalance in heavy chain expression. In one embodiment, the product-related impurities include unpaired or mispaired heavy chains. In one embodiment, the product-related impurities include unpaired or mispaired long heavy chains.

[0027] Product-related impurities include unpaired and / or mispaired heavy chains. Exemplary product-related impurities include unpaired long heavy chains and / or mispaired long heavy chains. Product-related impurities can be in the form of homodimers, half antibodies, aggregates, unpaired components, antibody fragments and / or various combinations of antibody fragments, etc., that contain long heavy chains. "Half antibodies" refers to product-related impurities that may form, for example, due to incomplete association or disruption of interaction between two heavy chain polypeptides. Half antibodies include a single light chain polypeptide and a single heavy chain polypeptide. "Homodimers" refers to product-related impurities that may form, for example, when heavy and light chains with specificity for the same target recombine with each other, instead of pairing with heavy and light chains with specificity for different targets to form the desired asymmetric multispecific heterodimer. This typically occurs during expression in a host cell. Product-related impurities in the form of homodimers, half antibodies, aggregates, antibody fragments and various combinations of antibody fragments, unpaired components, etc., include those that have at least one long heavy chain.

[0028] In one embodiment, the product-related impurity comprises an unpaired long heavy chain. In one embodiment, the product-related impurity comprises a mismatched long heavy chain. In one embodiment, the product-related impurity is selected from homodimers, half antibodies, protein aggregates, antibody fragments, combinations of antibody fragments, and unpaired components with unpaired or mismatched heavy chains. In one embodiment, the product-related impurity is selected from homodimers, half antibodies, protein aggregates, antibody fragments, combinations of antibody fragments, or unpaired antibody fragments with unpaired heavy chains. In one embodiment, the product-related impurity is selected from homodimers, half antibodies, protein aggregates, antibody fragments, or combinations of antibody fragments with mismatched heavy chains. In one embodiment, the product-related impurity is selected from homodimers, half antibodies, protein aggregates, antibody fragments, combinations of antibody fragments, and unpaired components with unpaired or mismatched long heavy chains. In one embodiment, the product-related impurity comprises a homodimer comprising a long heavy chain. In one embodiment, the product-related impurity comprises a half antibody comprising a long heavy chain. In one embodiment, the product related impurities include aggregates comprising long heavy chains. In one embodiment, the product related impurities include antibody fragments comprising long heavy chains. In one embodiment, the product related impurities include unpaired antibody fragments comprising long heavy chains.

[0029] The present invention provides a method for selecting a cell culture temperature regime that modulates the amount of at least one asymmetric multispecific antibody product-related impurity, comprising the steps of: a) establishing at least two cell cultures, each inoculated with the same cell line expressing an asymmetric multispecific antibody; b) culturing at least one cell culture at a first temperature regime consisting of a single temperature and culturing at least one cell culture at a second temperature regime; c) comparing the amount of at least one product-related impurity in the cell cultures at each temperature regime; and d) selecting a temperature regime that reduces the amount of the product-related impurity in the cell culture.

[0030] The present invention provides a method for modulating the product quality of a recombinant asymmetric multispecific antibody expressed by cells in cell culture, comprising the steps of: a) selecting a temperature regime that reduces the expression of at least one product-related impurity due to an imbalance in heavy chain expression, the impurity comprising an unpaired or mispaired long heavy chain; culturing the cell line at the selected temperature regime; and recovering the recombinant asymmetric multispecific antibody.

[0031] In one embodiment, the step of selecting a temperature regime comprises: a) establishing at least two cell cultures, each inoculated with the same cell line expressing the asymmetric multispecific antibody; b) culturing at least one cell culture at a first temperature regime consisting of a single temperature and culturing at least one cell culture at a second temperature regime; c) comparing at least one product-related impurity in the cell cultures at each temperature regime; and d) selecting a temperature regime that reduces expression of at least one product-related impurity comprising an unpaired or mispaired long heavy chain.

[0032] The present invention provides a method for modulating the product quality of a recombinant asymmetric multispecific antibody expressed by cells in cell culture, comprising the steps of a) selecting a temperature regime that reduces the expression of at least one product-related impurity comprising an unpaired or mispaired long heavy chain, b) culturing the cell line at the selected temperature regime, and c) recovering the recombinant asymmetric multispecific antibody.

[0033] The present invention provides a method for modulating the production of at least one recombinant asymmetric multispecific antibody product-related impurity comprising a long heavy chain in cell culture via cell culture temperature, the method comprising the steps of: a) selecting a temperature regime that reduces the expression of at least one product-related impurity comprising an unpaired or mispaired long heavy chain; b) culturing the cell line at the selected temperature regime; and c) recovering the recombinant asymmetric multispecific antibody.

[0034] Biotherapeutic development relies on engineering mammalian cells to secrete the desired protein. Successful biotherapeutic development is a lengthy, step-by-step process that includes the development of a clonal cell line expressing the biotherapeutic. The typical mammalian cell line development process is resource intensive and requires a lengthy timeline, due in part to slow cell recovery during selection, single cell cloning, and the need to perform multiple screening assays to identify a suitable production host. Clonal selection is one of the most labor-intensive steps in cell line development due to the need to screen a large number of candidates in various protein production studies. The desired outcome of this process is the identification and isolation of a mammalian clonal cell line expressing a therapeutic protein with the desired product quality attributes while being compatible with the desired manufacturing process and operations with minimal impact.

[0035] The selection of clonal cell lines includes the desired attributes of the expressed recombinant protein, such as titer, product quality, and / or growth characteristics, which may include a more balanced molar ratio of heavy chains. When the clonal cell line candidates selected under a single constant temperature condition were grown in a bioreactor under conditions incorporating at least one temperature change or shift, the titer and viable cell density of the expressed recombinant protein were largely unaffected, but the molar ratio of long to short heavy chains increased, which in turn altered the amount and type of expressed product-associated impurities, which affected the yield, the product quality attributes of the desired protein, downstream purification of the protein, and the robustness of the overall manufacturing process.

[0036] The imbalance in the ratio of asymmetric heavy chains affected the types of product-related impurities formed. It was found that the overproduction of long heavy chains resulted in an increase in protein-related impurities, e.g., homodimers and monomers containing long heavy chains. The imbalanced expression of heavy chains and the need to remove impurities formed due to the imbalanced expression, which was found to be unexpectedly difficult for certain long heavy chain containing impurities, affected the yield and product quality attributes of asymmetric multispecific antibodies. Therefore, in order to maintain the desired and / or required product quality attributes, it is desirable to maintain a more balanced expression of asymmetric heavy chains and reduce unnecessary time, resources, product loss and reduced robustness of the manufacturing process due to excessive efforts to recover the product from undesired long heavy chain impurities.

[0037] Delaying production of the desired drug substance to switch cell lines or rescreen clones to identify new cell lines that can reliably express recombinant asymmetric multispecific antibody drug substance that meets the desired product qualities and attributes and is amenable to established facility practices and / or protocols such as temperature shifting is costly in terms of time, resources, and labor and does not support an efficient and effective manufacturing process. Therefore, the use of temperature as a factor in the selection of clonal cell lines expressing asymmetric multispecific antibodies is beneficial to obtain a production cell line that expresses the desired protein with the desired qualities, thereby reducing and / or eliminating the production of product-related impurities that affect production of the desired protein.

[0038] It has been found that temperature can be used as a tool in cell line development to select desirable cell line candidates expressing recombinant asymmetric multispecific antibodies that meet manufacturing quality as well as manufacturability requirements. Cell culture temperature can affect the product quality of asymmetric proteins, for example, by altering the molar ratio of long and short heavy chains, which affected the management of product-related impurities. It would be beneficial for the cell line development process to incorporate a desired set-point temperature in the screening and selection process that meets not only the attributes of the drug product, but also the requirements of desired facility practices and / or protocols. Clonal screening using temperature as a tool is also beneficial because the response of product quality attributes to temperature appears to be molecule-dependent and / or cell line-dependent. Using temperature during screening and selection of clonal cell lines to modulate product quality reduced the selection of clonal cell lines that were susceptible to the expression of product-related impurities, improving product quality and being more suitable for carrying out the desired process.

[0039] The present invention provides a method for selecting a cell line expressing an asymmetric multispecific antibody, comprising the steps of: establishing at least one clonally derived cell line expressing the asymmetric multispecific antibody; establishing at least two cell cultures from the one or more clonally derived cell lines, where at least one cell culture from each cell line is cultured at a first temperature regime and at least one cell culture from each cell line is cultured at a second temperature regime; comparing the expression of at least one product-related impurity produced by cells cultured at each temperature regime; and selecting the cell line that resulted in a reduced expression of at least one product-related impurity. In one embodiment, the selected cell line is then cultured at a temperature regime that resulted in a reduced expression of at least one product-related impurity.

[0040] The present invention provides a method for establishing a cell line from a clone-derived culture expressing an asymmetric multispecific antibody, comprising the steps of transforming cells with at least one gene encoding the asymmetric multispecific antibody, isolating at least one single stably transformed cell expressing the asymmetric multispecific antibody and establishing a clone-derived culture, establishing at least two cell cultures from the one or more clone-derived cultures, culturing at least one cell culture from each clone-derived culture at a first temperature regime and culturing at least one cell culture from each clone-derived culture at a second temperature regime, comparing the molar ratios of long to short heavy chains produced by the cells at each temperature regime, selecting at least one clone-derived culture that gave rise to a lower molar ratio of long to short heavy chains, and establishing a cell line from the clone-derived culture.

[0041] The present invention relates to a method for producing isolated and purified recombinant asymmetric multispecific antibodies, comprising the steps of: a) isolating at least one single stably transformed cell expressing an asymmetric multispecific antibody and establishing a clone-derived culture; b) establishing at least two cell cultures from one or more clone-derived cultures; c) culturing at least one cell culture from each clone-derived culture at a first temperature regime comprising a single temperature and culturing at least one cell culture from each clone-derived culture at a second temperature regime; and d) isolating at least one single stably transformed cell expressing an asymmetric multispecific antibody and establishing a clone-derived culture from the one or more clone-derived cultures. e) selecting a cell culture that modulates the production of product-related impurities; f) establishing a cell line expressing the asymmetric multispecific antibody from the selected cell culture; g) inoculating a bioreactor with the cell line expressing the asymmetric multispecific antibody; h) culturing the cells to express the asymmetric multispecific antibody at a temperature that modulates the production of product-related impurities; i) recovering the recombinant asymmetric multispecific antibody from the cell culture; j) processing the recombinant asymmetric multispecific antibody by one or more chromatographic unit operations; and k) obtaining an isolated and purified recombinant asymmetric multispecific antibody.

[0042] The present invention provides isolated and purified recombinant asymmetric multispecific antibodies by the methods described herein.

[0043] Cell lines suitable for the production of recombinant asymmetric multispecific antibodies should be stable with respect to product quality and productivity, without the expression or production of, or increased expression or production of, undesirable product-related impurities. These cell lines should be able to produce the desired asymmetric multispecific antibodies over time, with the same quality and attributes every time.

[0044] The generation of a stable cell line begins with the transformation of a host cell that is suitable or desirable for the recombinant production of a protein of interest. Typically, such cells are generated, modified, and / or developed for the production of recombinant proteins.

[0045] The cells may be derived from a multicellular animal. The commonly used animal cell lines for biopharmaceutical manufacturing are mammalian cell lines. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, VA) and distributors. Commonly used cell lines include those derived from Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, mouse myeloma (NS0, Sp2 / 0) cells, baby hamster kidney (BHK) cells, human embryonic kidney (293) cells, fibrosarcoma (HT-1080) cells, human embryonic retina (PER.C6) cells, hybrid kidney and B cells (HKB-11), amniotic cell production of CEVEC (CAP) cells, human liver (HuH-7) cells, and any other cells used or suitable for use in clinical and / or commercial manufacturing.

[0046] The most commonly used cell lines are derived from Chinese Hamster Ovary (CHO) cells. CHO cells are widely used to produce complex recombinant proteins. Dihydrofolate reductase (DHFR) deletion mutant cell lines (Urlaub et al. (1980), Proc Natl Acad Sci USA 77:4216-4220), DXB11 and DG-44 are desirable CHO host cell lines because efficient DHFR-selectable and amplifiable gene expression systems allow high levels of recombinant protein expression in these cells (Kaufman RJ (1990), Meth Enzymol 185:537-566). Also widely used is the glutamine synthetase (GS) knockout CHOK1SV cell line, utilizing GS-based methionine sulfoximine (MSX) selection. CHOK1 cells (ATCC CCL61) are also included. Furthermore, these cells are easy to manipulate as adherent or suspension cultures and exhibit relatively good genetic stability. CHO cells and the proteins recombinantly expressed by them have been extensively characterized and approved for use in clinical and commercial manufacturing by regulatory agencies.

[0047] These cells are transformed with an expression system, such as a vector containing a gene encoding a protein of interest. Also provided herein are expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes containing at least one nucleic acid molecule encoding a protein of interest, as well as host cells containing 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, DNA complex, etc.) suitable for use in transferring and / or transporting protein encoding information to a host cell and / or a specific location and / or compartment within the host cell. Vectors can include viral and non-viral vectors, as well as non-episomal mammalian vectors. Vectors are often referred to as expression vectors, e.g., recombinant expression vectors or cloning vectors. A vector can be introduced into a host cell to allow for replication of itself, thereby amplifying copies of the polynucleotides contained therein. Cloning vectors may contain sequence components, which generally include, but are not limited to, an origin of replication, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selection marker. These elements may be selected as needed by those skilled in the art.

[0048] Transformation refers to a change in the genetic characteristics of a cell. A cell is transformed when it has been modified to contain new DNA or RNA. For example, a cell is transformed when it has been genetically modified from its native state by introducing new genetic material by transfection, transduction, or other techniques. "Transduction" refers to the process of introducing foreign DNA into a cell by a viral vector. Transfection refers to the uptake of foreign or exogenous DNA by a cell. One or more vectors can be inserted into a suitable cell for amplification and / or polypeptide expression. Transformation of an expression vector into a selected cell can be performed by well-known methods including transfection, infection, co-precipitation with calcium phosphate, electroporation, nucleofection, microinjection, DEAE-dextran mediated transfection, cationic lipid-mediated delivery, liposome-mediated transfection, particle bombardment, receptor-mediated gene delivery, polylysine, histone, chitosan, and peptide-mediated delivery. The method selected will be, in part, a function of the type of host cell used. These and other suitable methods are well known to those of skill in the art.

[0049] Following transfection or transduction, the transformed DNA may be recombined into the DNA of the cell by physically integrating into the cell's chromosome, or may be maintained transiently as an episomal element without replication, or may be independently replicated as a plasmid. If the transforming DNA is replicated by cell division, the cell is considered to be "stably transformed." After transfection, cell viability typically declines, and the unamplified pool is usually passaged multiple times to allow recovery. The pool is maintained in constant temperature culture, typically at a physiological temperature such as 35°C to 37°C, typically 36°C.

[0050] The cells can be subjected to selection pressure to select for cells that have internalized the expression system. Other selection genes may be used to amplify the gene to be expressed. Amplification is the process by which genes required for the production of a protein important for growth or cell survival are repeated in tandem in the chromosomes of successive generations of recombinant cells. Examples of selection markers suitable for mammalian cells include the glutamine synthetase (GS) / methionine sulfoximine (MSX) system, dihydrofolate reductase (DHFR) and promoter-less thymidine kinase genes. Mammalian cell transformants are placed under selection pressure in which the transformants are uniquely adapted to survive due to the selection gene present in the vector. Selection pressure is applied by culturing the transformed cells under conditions of successively increasing concentrations of the selection drug in the medium, thereby amplifying both the selection gene and the DNA encoding the protein of interest. As a result, large amounts of the polypeptide of interest are synthesized from the amplified DNA.

[0051] Two common systems used with CHO cells are DHFR-deficient cells (examples include CHO-DXB11 and CHO-DG44) and CHO cells lacking glutamine synthetase (GS), such as CHOK1SV, or other GS knockout cells. For DHFR-deficient cells, selective medium lacking thymidine and hypoxanthine allows cells with sufficient exogenously integrated copies of the DHFR gene to survive. Similarly, for cells lacking GS, selective medium supplemented with L-glutamic acid instead of L-glutamine allows cells with sufficient GS integrated into the cellular genome to survive.

[0052] Cell lines for commercial-scale production of recombinant protein drug substances are derived from single-cell precursors. These clone-derived cultures must be individually grown and assessed for growth, productivity, and quality of the secreted protein product. Stable clonal cell lines are used to establish a master cell bank to stockpile single-cell precursor clones expressing the desired recombinant protein. Cells from the master cell bank are then used to create a working cell bank that supplies the cells used for production. Single-cell cloning is performed by aliquoting amplified or unamplified cells into multi-well vessels, such as 96-well plates, which are maintained at a constant temperature, typically a physiological temperature, e.g., 35°C to 37°C, typically 36°C in culture.

[0053] Techniques are available that aid in the isolation of single cells from a population. These include, but are not limited to, limiting dilution plating, microfluidic encapsulation, FACS-assisted cell sorting, colony sorting in semi-solid media, single cell printers, microfluidic wells or chips, such as optical fluidic technology (Berkley Lights, Emeryville, Calif.). Only colonies arising from single cells are selected for further processing.

[0054] Stable pools generated during the CLD workflow contain a heterogeneous mixture of cells that differ in the number and localization of integrated transgenes, have unique genetic and phenotypic characteristics, and / or differ in cell-specific productivity. These stably transfected pools are used for single cell cloning to generate clonally derived cultures, which are then screened for performance to identify those with the desired qualities and characteristics, e.g., expression, productivity, growth, and / or other product quality attributes, including production of product-related impurities. The transgene copy number can also be amplified using an inhibitor against a selection marker required for the cells to survive. This increases the copy number of the selection marker gene as well as the transgene integrated at the adjacent locus. In one embodiment, the clonally derived cultures can be further selected for the desired expression of the expressed asymmetric multispecific antibody, productivity, growth, and / or other desired product quality characteristics.

[0055] The clones are then scaled up using vessels of increasing size and further maintained in culture at a constant physiological temperature, 35° C. to 37° C., typically 36° C. These vessels can include multi-well plates, e.g., 96-well plates, 24-well plates, 6-well plates.

[0056] Once the cells reach the desired cell number or cell density, the clones are evaluated and ranked under conditions that mimic production conditions and are first evaluated for production and product quality. The clones are transferred to suitable vessels such as deep well plates, spin tubes, and / or agitation shake flasks, grown in suspension in fed-batch culture, and incubated at a constant physiological temperature of 35°C to 37°C, typically 36°C. This is typically a 10-day process, feeding and monitoring culture conditions (e.g., pH, osmolality, lactate / glucose, etc.) every 2-3 days. Typically, at least 100 or more clones are screened. Ranking of pools and clones takes into account factors such as titer, specific productivity (qP), growth, viability, and product quality (PQ) profile. The specific product qualities used for selection usually depend on the modality of the protein expressed, as well as the qualities desired and / or required during manufacturing of the final drug product and / or drug substance. These may include aggregation levels, charge distribution, clips, moieties, and post-translational modifications. The top clones, typically 10 or less, are selected for further development.

[0057] The top clone candidates are typically cultured in a small scale bioreactor (typically 3L or 7L) under some / all of the conditions that will be used during large scale production. For example, if one or more temperature shifts will be implemented during large scale production, these temperature set points are incorporated into the run of the small scale bioreactor. This process is typically a 10-25 day or longer process. In some embodiments, this process is 15-20 days. In some embodiments, this process is at least 15 days. The clones are again evaluated and ranked based on productivity, growth, product quality attributes, etc.

[0058] When this process was used to screen and select clonal cells expressing asymmetric multispecific antibodies, it was observed that the product quality attributes of the proteins expressed by the clonal cells screened and selected at a constant physiological temperature (36° C.) were altered when the cells were subjected to cell culture conditions involving a temperature shift (from 36° C. to 34° C., or from 36° C. to 32.5° C.). Exposure to lower culture temperatures resulted in an increase in the molar ratio of long to short heavy chains. Transcription of both long and short heavy chains continues at the lower temperature, but at different rates, potentially resulting in the biased molar ratios observed. The shift in the ratio of heavy chains affected the types of product-related impurities formed, which in turn affected the yield and activity of the product, as well as the efficiency of downstream purification steps.

[0059] The product quality of these asymmetric multispecific antibodies was affected by temperature during cell culture. To better understand this outcome, clones expressing asymmetric multispecific antibodies were exposed to one or more temperature set points during the initial screening and selection of clones based on product quality attributes. Including temperature as a factor in the screening and selection of clones allowed the identification of clones that were able to maintain a more balanced molar ratio of long to short heavy chains at different temperature set points. Thus, the composition of product-associated impurities was altered, which improved downstream purification and yield of the desired product, as well as contributing to a more robust manufacturing process.

[0060] Temperature regime refers to the temperature or temperature set point used during cell culture. One or more temperature regimes may be used over the course of the cell culture process. Most recombinant therapeutic proteins are expressed by mammalian host cells. To promote optimal growth of the cells, mammalian cell cultures are typically maintained at a temperature of 36°C to 37°C. To increase the efficiency of production of the desired recombinant protein, suboptimal cell growth conditions that promote enhanced production may be desired. Lower culture temperatures can be used to slow or stop cell growth and promote production of the desired recombinant antibody. The use of a temperature shift from an optimal temperature for the growth phase to an optimal temperature for the production phase is often used in cell culture strategies. The growth phase may be carried out at a first temperature of about 28°C to about 37°C, typically 36°C to 37°C, and the production phase may be carried out at a second temperature of about 28°C to about 37°C, typically about 30°C to about 35°C, or about 30°C to about 34°C, preferably 32.5°C.

[0061] This adjustment of temperature can be used throughout the life of the cell culture to achieve the desired production goal. A combination of temperature shifts can also be used to transition from a first growth phase to a first production phase to a second growth phase followed by a second production phase, etc.

[0062] The present invention provides two or more temperature regimes. One embodiment of the present invention includes one or more single temperature regimes. In a single temperature regime, the culture is maintained at a single temperature during the culture period. In one embodiment, the temperature is about 37°C to about 28°C. In one embodiment, the temperature is about 36°C to about 28°C. In one embodiment, the temperature is about 37°C to about 29°C. In one embodiment, the temperature is about 36°C to about 29°C. In one embodiment, the temperature is about 37°C to about 30°C. In one embodiment, the temperature is about 36°C to about 30°C. In one embodiment, the temperature is about 37°C to about 31°C. In one embodiment, the temperature is about 36°C to about 31°C. In one embodiment, the temperature is about 36°C to about 31°C. In one embodiment, the temperature is about 37°C to about 32°C. In one embodiment, the temperature is about 37°C to about 32.5°C. In one embodiment, the temperature is about 36°C to about 32°C. In one embodiment, the temperature is about 36°C to about 32.5°C. In one embodiment, the temperature is about 37°C to about 33°C. In one embodiment, the temperature is about 36°C to about 33°C. In one embodiment, the temperature is about 37°C to about 34°C. In one embodiment, the temperature is about 36°C to about 34°C. In one embodiment, the temperature is about 37°C to about 35°C. In one embodiment, the temperature is about 36°C to about 35°C. In one embodiment, the temperature is about 37°C to about 36°C. In one embodiment, the temperature is about 35°C to about 28°C. In one embodiment, the temperature is about 35°C to about 29°C. In one embodiment, the temperature is about 35°C to about 30°C. In one embodiment, the temperature is about 35°C to about 31°C. In one embodiment, the temperature is about 35°C to about 32°C. In one embodiment, the temperature is about 35°C to about 33°C. In one embodiment, the temperature is about 35°C to about 34°C. In one embodiment, the temperature is about 34°C to about 28°C. In one embodiment, the temperature is about 34°C to about 29°C. In one embodiment, the temperature is about 34°C to about 30°C. In one embodiment, the temperature is about 34°C to about 31°C. In one embodiment, the temperature is about 34°C to about 33°C. In one embodiment, the temperature is about 34°C to about 28°C. In one embodiment, the temperature is about 33°C to about 28°C. In one embodiment, the temperature is about 33°C to about 29°C. In one embodiment, the temperature is about 33°C to about 30°C. In one embodiment, the temperature is about 33°C to about 31°C.In one embodiment, the temperature is about 33°C to about 32°C. In one embodiment, the temperature is about 32°C to about 28°C. In one embodiment, the temperature is about 32°C to about 29°C. In one embodiment, the temperature is about 32°C to about 30°C. In one embodiment, the temperature is about 32°C to about 31°C. In one embodiment, the temperature is about 31°C to about 28°C. In one embodiment, the temperature is about 31°C to about 29°C. In one embodiment, the temperature is about 31°C to about 30°C. In one embodiment, the temperature is about 30°C to about 28°C. In one embodiment, the temperature is about 30°C to about 29°C. In one embodiment, the temperature is 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. In one embodiment, the temperature is 32.5°C.

[0063] An embodiment of the present invention includes one or more temperature regimes including at least one temperature shift. In one embodiment, the temperature shift is from a first temperature to a second temperature that is higher or lower than the first temperature. In one embodiment, the first temperature is about 37°C to about 28°C. In one embodiment, the first temperature is about 36°C to about 28°C. In one embodiment, the first temperature is about 37°C to about 29°C. In one embodiment, the first temperature is about 36°C to about 29°C. In one embodiment, the first temperature is about 37°C to about 30°C. In one embodiment, the first temperature is about 36°C to about 30°C. In one embodiment, the first temperature is about 37°C to about 31°C. In one embodiment, the first temperature is about 36°C to about 31°C. In one embodiment, the first temperature is about 36°C to about 31°C. In one embodiment, the first temperature is about 37°C to about 32°C. In one embodiment, the first temperature is about 37°C to about 32.5°C. In one embodiment, the first temperature is about 36°C to about 32°C. In one embodiment, the first temperature is about 36°C to about 32.5°C. In one embodiment, the first temperature is about 37°C to about 33°C. In one embodiment, the first temperature is about 36°C to about 33°C. In one embodiment, the first temperature is about 37°C to about 34°C. In one embodiment, the first temperature is about 36°C to about 34°C. In one embodiment, the first temperature is about 37°C to about 35°C. In one embodiment, the first temperature is about 36°C to about 35°C. In one embodiment, the first temperature is about 35°C to about 28°C. In one embodiment, the first temperature is about 35°C to about 29°C. In one embodiment, the first temperature is about 35°C to about 30°C. In one embodiment, the first temperature is about 35°C to about 31°C. In one embodiment, the first temperature is about 35°C to about 32°C. In one embodiment, the first temperature is about 35°C to about 32.5°C. In one embodiment, the first temperature is about 35°C to about 33°C. In one embodiment, the first temperature is about 35°C to about 34°C. In one embodiment, the first temperature is about 34°C to about 28°C. In one embodiment, the first temperature is about 34°C to about 29°C. In one embodiment, the first temperature is about 34°C to about 30°C. In one embodiment, the first temperature is about 34°C to about 31°C. In one embodiment, the first temperature is about 34°C to about 32°C. In one embodiment, the first temperature is about 34°C to about 32.5°C.In one embodiment, the first temperature is about 34°C to about 28°C. In one embodiment, the first temperature is about 33°C to about 28°C. In one embodiment, the first temperature is about 33°C to about 29°C. In one embodiment, the first temperature is about 33°C to about 30°C. In one embodiment, the first temperature is about 33°C to about 31°C. In one embodiment, the first temperature is about 33°C to about 32°C. In one embodiment, the first temperature is about 33°C to about 32.5°C. In one embodiment, the first temperature is about 32°C to about 28°C. In one embodiment, the first temperature is about 32°C to about 29°C. In one embodiment, the first temperature is about 32°C to about 30°C. In one embodiment, the first temperature is about 32.5°C to about 28°C. In one embodiment, the first temperature is about 32.5°C to about 29°C. In one embodiment, the first temperature is about 32.5°C to about 30°C. In one embodiment, the first temperature is about 32.5°C to about 31°C. In one embodiment, the first temperature is about 32.5°C to about 32°C. In one embodiment, the first temperature is about 32.5°C to about 33°C. In one embodiment, the first temperature is about 32.5°C to about 34°C. In one embodiment, the first temperature is about 32.5°C to about 35°C. In one embodiment, the first temperature is about 31°C to about 28°C. In one embodiment, the first temperature is about 31°C to about 29°C. In one embodiment, the first temperature is about 31°C to about 30°C. In one embodiment, the first temperature is about 30°C to about 28°C. In one embodiment, the first temperature is about 30°C to about 29°C. In one embodiment, the first temperature is 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. In one embodiment, the first temperature is 32.5°C.

[0064] In one embodiment, the second temperature is about 1°C to 9°C lower than the first temperature. In one embodiment, the second temperature is about 2°C to 9°C lower than the first temperature. In one embodiment, the second temperature is about 3°C ​​to 9°C lower than the first temperature. In one embodiment, the second temperature is about 4°C to 9°C lower than the first temperature. In one embodiment, the second temperature is about 5°C to 9°C lower than the first temperature. In one embodiment, the second temperature is about 6°C to 9°C lower than the first temperature. In one embodiment, the second temperature is about 7°C to 9°C lower than the first temperature. In one embodiment, the second temperature is about 8°C to 9°C lower than the first temperature. In one embodiment, the second temperature is about 1°C to 8°C lower than the first temperature. In one embodiment, the second temperature is about 2°C to 8°C lower than the first temperature. In one embodiment, the second temperature is about 3°C ​​to 8°C lower than the first temperature. In one embodiment, the second temperature is about 4°C to 8°C lower than the first temperature. In one embodiment, the second temperature is about 5°C to 8°C lower than the first temperature. In one embodiment, the second temperature is about 6°C to 8°C lower than the first temperature. In one embodiment, the second temperature is about 7°C to 8°C lower than the first temperature. In one embodiment, the second temperature is about 1°C to 7°C lower than the first temperature. In one embodiment, the second temperature is about 2°C to 7°C lower than the first temperature. In one embodiment, the second temperature is about 3°C ​​to 7°C lower than the first temperature. In one embodiment, the second temperature is about 4°C to 7°C lower than the first temperature. In one embodiment, the second temperature is about 5°C to 7°C lower than the first temperature. In one embodiment, the second temperature is about 6°C to 7°C lower than the first temperature. In one embodiment, the second temperature is about 1°C to 6°C lower than the first temperature. In one embodiment, the second temperature is about 2°C to 6°C lower than the first temperature. In one embodiment, the second temperature is about 3°C ​​to 6°C lower than the first temperature. In one embodiment, the second temperature is about 4°C to 6°C lower than the first temperature. In one embodiment, the second temperature is about 5°C to 6°C lower than the first temperature. In one embodiment, the second temperature is about 1°C to 5°C lower than the first temperature. In one embodiment, the second temperature is about 2°C to 5°C lower than the first temperature. In one embodiment, the second temperature is about 3°C ​​to 5°C lower than the first temperature. In one embodiment, the second temperature is about 4°C to 5°C lower than the first temperature. In one embodiment, the second temperature is about 1°C to 4°C lower than the first temperature.In one embodiment, the second temperature is about 2°C to 4°C lower than the first temperature. In one embodiment, the second temperature is about 3°C ​​to 4°C lower than the first temperature. In one embodiment, the second temperature is about 1°C to 3°C lower than the first temperature. In one embodiment, the second temperature is about 2°C to 3°C lower than the first temperature. In one embodiment, the second temperature is about 2.5°C to 3.5°C lower than the first temperature. In one embodiment, the second temperature is about 1°C to 2°C lower than the first temperature.

[0065] In one embodiment, the second temperature is about 37°C to about 28°C. In one embodiment, the second temperature is about 36°C to about 28°C. In one embodiment, the second temperature is about 37°C to about 29°C. In one embodiment, the second temperature is about 36°C to about 29°C. In one embodiment, the second temperature is about 37°C to about 30°C. In one embodiment, the second temperature is about 36°C to about 30°C. In one embodiment, the second temperature is about 37°C to about 31°C. In one embodiment, the second temperature is about 36°C to about 31°C. In one embodiment, the second temperature is about 36°C to about 31°C. In one embodiment, the second temperature is about 36°C to about 31°C. In one embodiment, the second temperature is about 37°C to about 32°C. In one embodiment, the second temperature is about 37°C to about 32.5°C. In one embodiment, the second temperature is about 36°C to about 32 ...7°C to about 33°C. In one embodiment, the second temperature is about 36°C to about 33°C. In one embodiment, the second temperature is about 37°C to about 34°C. In one embodiment, the second temperature is about 36°C to about 34°C. In one embodiment, the second temperature is about 37°C to about 35°C. In one embodiment, the second temperature is about 36°C to about 35°C. In one embodiment, the second temperature is about 35°C to about 28°C. In one embodiment, the second temperature is about 35°C to about 29°C. In one embodiment, the second temperature is about 35°C to about 30°C. In one embodiment, the second temperature is about 35°C to about 31°C. In one embodiment, the second temperature is about 35°C to about 32°C. In one embodiment, the second temperature is about 35°C to about 32.5°C. In one embodiment, the second temperature is about 35°C to about 33°C. In one embodiment, the second temperature is about 35°C to about 34°C. In one embodiment, the second temperature is about 34°C to about 28°C. In one embodiment, the second temperature is about 34°C to about 29°C. In one embodiment, the second temperature is about 34°C to about 30°C. In one embodiment, the second temperature is about 34°C to about 31°C. In one embodiment, the second temperature is about 34°C to about 32°C. In one embodiment, the second temperature is about 34°C to about 32.5°C. In one embodiment, the second temperature is about 34°C to about 28°C. In one embodiment, the second temperature is about 33°C to about 28°C. In one embodiment, the second temperature is about 33°C to about 29°C.In one embodiment, the second temperature is about 33°C to about 30°C. In one embodiment, the second temperature is about 33°C to about 31°C. In one embodiment, the second temperature is about 33°C to about 32°C. In one embodiment, the second temperature is about 33°C to about 32.5°C. In one embodiment, the second temperature is about 32°C to about 28°C. In one embodiment, the second temperature is about 32°C to about 29°C. In one embodiment, the second temperature is about 32°C to about 30°C. In one embodiment, the second temperature is about 32.5°C to about 28°C. In one embodiment, the second temperature is about 32.5°C to about 29°C. In one embodiment, the second temperature is about 32.5°C to about 30°C. In one embodiment, the second temperature is about 32.5°C to about 31°C. In one embodiment, the second temperature is about 31°C to about 28°C. In one embodiment, the second temperature is about 31°C to about 29°C. In one embodiment, the second temperature is about 31°C to about 30°C. In one embodiment, the second temperature is about 30°C to about 28°C. In one embodiment, the second temperature is about 30°C to about 29°C. In one embodiment, the second temperature is 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. In one embodiment, the second temperature is 32.5°C.

[0066] In one embodiment, the first temperature regime is a single temperature between 36°C and 37°C, and the second temperature regime includes at least one temperature shift from a first temperature between 36°C and 37°C to a second temperature between 28°C and 35°C.

[0067] "Asymmetric multispecific antibodies" include asymmetric proteins engineered to bind, neutralize, and / or specifically interact with at least two different antigens or targets, or at least two different epitopes on the same antigen or target. In one embodiment, asymmetric multispecific antibodies can be engineered to target immune effectors, stimulate or induce immune responses, and deliver cytotoxic agents to tumors or infectious agents. These asymmetric multispecific antibodies have been found to be useful in a variety of applications, for example, in cancer immunotherapy by redirecting immune effector cells to tumor cells, blocking signaling pathways to alter cell signaling, targeting tumor angiogenesis, blocking cytokines, and as vehicles for pretargeted delivery of drugs, for example, chemotherapeutic agents, radiolabels (to improve detection sensitivity), and nanoparticles (directed to specific cells / tissues, such as cancer cells).

[0068] Asymmetric multispecific antibodies may be of scientific and / or commercial interest. Asymmetric multispecific antibodies can be produced in a variety of ways, most commonly by recombinant animal cell lines using cell culture techniques. Asymmetric multispecific antibodies may be produced intracellularly or secreted into the culture medium from which they can be recovered and / or harvested, and may be referred to as "recombinant asymmetric multispecific antibodies". The term "isolated recombinant asymmetric multispecific antibodies" refers to asymmetric multispecific antibodies that have been purified to remove proteins, polypeptides, DNA, and / or other contaminants or impurities that would interfere with their therapeutic, diagnostic, prophylactic, research, or other uses. Asymmetric multispecific antibodies of interest include, among others, those that exert a therapeutic effect by binding to two or more targets, particularly targets derived from, related to, and modifications thereof, among those listed herein. Asymmetric multispecific antibodies may have two or more heavy chains that differ in length from one another, for example, a long heavy chain and a short heavy chain. In a related embodiment, the asymmetric multispecific antibody is an asymmetric bispecific antibody.Various bispecific antibody formats, including asymmetric bispecific antibodies, are known in the art (Spiess et al., Mol Immunol 67, 95-106, 2015; Sedykh et al., Drug Design, Development and Therapy 18(12), 195-208, 2018; Fan et al., J Hematol&Oncology 8:130-143, 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; 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.

[0069] Asymmetric multispecific antibodies also include those with an odd number of active components with unique peptide and / or protein sequences that collectively result in an asymmetric multispecific antibody with specificity for more than one target. Antibodies include human, humanized, chimeric, multispecific, monoclonal, polyclonal, bispecific, and oligomers or antigen-binding fragments thereof. Antibodies also include both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, such as IgG1, IgG2, IgG3, or IgG4. Antibodies with antigen-binding fragments or antigen-binding regions are also included, particularly antibody antigen-binding fragments that compete with intact antibodies for specific binding. Also included are peptide bodies, antibody derivatives, antibody analogs, fusion proteins (and proteins made using Xmab® technology, bispecific T cell engagers with extended half-life, e.g., BiTE® molecules).

[0070] Modified asymmetric multispecific antibodies are also included, e.g., antibodies that are chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds. Also included are antibodies that further comprise one or more post-translational modifications that may be made by cellular modification systems, or modifications introduced in vitro by enzymatic and / or chemical methods, or that are otherwise introduced. In particular, antibodies are modified using such methods to attach one or more proteins of interest to the antibody.

[0071] In some embodiments the asymmetric multispecific antibodies specifically bind, neutralize and / or interact with 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, relaxin, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins and immunoadhesins.

[0072] In some embodiments, the asymmetric multispecific antibody is directed to, 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, CD174, e.g., HER receptor family proteins, including HER2, HER3, HER4, EGF receptor EGFRvIII, cell adhesion molecules, e.g., LFA-1, Mol, p150,95, VLA- 4. ICAM-1, VCAM and αv / β3 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-α), erythropoietin (EPO), nerve growth factors such as NGF-β, platelet derived growth factor (PDGF), fibroblast growth factors including, for example, aFGF and bFGF, epidermal growth factor (EGF), cryopreservatives, and the like. transforming growth factors (TGFs), including TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5, among others; 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 and insulin-related proteins, including, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding proteins; factor VIII, tissue factor, von Willebrand factor, among others; coagulation and coagulation-related proteins such as Willebrand factor, plasminogen activators such as protein C, alpha-1-antitrypsin, urokinase and tissue plasminogen activator ("t-PA"), bombadin, thrombin, thrombopoietin, thrombopoietin receptor, colony stimulating factors (CSFs) including M-CSF, GM-CSF, and G-CSF, among others, albumin, other blood and serum proteins including, but not limited to, IgE and blood group antigens, e.g., flk2 / flt3 receptor, obesity (OB) receptor, growth hormone receptor,and T cell receptors, 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); relaxin A chain, relaxin B chain, and prorelaxin, interferons including, for example, interferon-α, -β, and -γ, interleukins (IL), for example, IL-1 through IL-10, IL-12, IL-15, IL-17, IL-23, IL-12 / 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, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-71, IL-72, IL-73, IL-74, IL-75, IL-76, IL-77, IL-78, IL-79, IL-80, IL-81, IL-82, IL-83, IL -23, IL-2Ra, IL1-R1, IL-6 receptor, IL-4 receptor and / or IL-13RA2, which is a receptor for IL-13, or IL-1RAP, which is a receptor for IL-17, viral antigens including, but not limited to, AIDS envelope virus antigen, lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-α and -β, enkephalinase, BCMA, IgKappa, ROR-1, ERBB2, mesothelin, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptide, Dnase, FR-α, 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, 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 (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 β (TFGβ), STEAP 1, zona pellucida sperm binding protein 3 (ZP-3), TWEAK, platelet derived growth factor receptor alpha (PDGFRα), sclerostin, and one or more of the bioactive fragments or variants of any of the foregoing. In some embodiments, the asymmetric multispecific antibody comprises an antibody or immunoglobulin, e.g., a mutein bound to an IL-2 variant immunocytokine. An example is CEA-IL-2v (sergituzumab amnaleukin), in which the IL-2v moiety is fused to one heavy chain of the Fc portion of the antibody, Schneider Electric et al.,2019,Biotechnology Bioengineering 116:2503-2513;and Anti-FAP-IL-2(fibroblast activation protein(FAP)targeted interleukin-2 variant(IL-2v)、Soerensen MM,et al.Safety,PK / PD,and anti-tumor activity of RO6874281,an engineered variant of interleukin-2(IL-2v)targeted to tumor-associated fibroblasts via binding to fibroblast activation protein(FAP),Nicolini V, et al.Combining CEA-IL2v and FAP-IL2v immunocytokines with PD-L1 checkpoint blockade.Annual Meeting of the American Association for Cancer Research;New Orleans:2016. ,

[0073] In some embodiments, the asymmetric multispecific antibody is a bifunctional fusion protein. In some embodiments, the bifunctional fusion protein comprises an IL-21 mutein. In some embodiments, the bifunctional fusion protein comprises a conjugate of an IL-21 mutein linked to the C-terminus of one of the two antibody heavy chains of an anti-PD-1 antibody. The IL-21-anti-PD-1 conjugate comprises a "short heavy chain" (anti-PD1 heavy chain) and a "long heavy chain" (anti-PD1 heavy chain linked to an IL-21 mutein). Such antibodies are described in WO 2019 / 028316, which is incorporated by reference in its entirety.

[0074] Interleukin-21 (IL-21) is a cytokine expressed by T cells, B cells, NK cells, and myeloid cells that regulates the activity of both innate and adaptive immune cells and improves T cell survival and effector function. IL-21 has a four-helix bundle structure and exists as a monomer. In some embodiments, the asymmetric protein comprises an IL-21 mutein that comprises at least one amino acid substitution compared to the wild-type IL-21 amino acid sequence shown herein as SEQ ID NO:1. In an exemplary aspect, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO:2, where X is any amino acid, and the amino acid sequence of the IL-21 mutein differs from the amino acid sequence of human IL-21 (SEQ ID NO:1) by at least one amino acid. In various aspects, the IL-21 mutein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO:1) by 3, 4, 5, 6, or 7 amino acids. In various aspects, an IL-21 mutein comprises an amino acid sequence that differs by one or two amino acids from the amino acid sequence of human IL-21 (SEQ ID NO: 1). In an exemplary aspect, an IL-21 mutein comprises an amino acid substitution at a position according to Table 1 with an amino acid.

[0075] [Table 1]

[0076] In various aspects, the asymmetric multispecific antibody comprises an IL-21 mutein comprising one amino acid substitution compared to SEQ ID NO:1, optionally comprising one of SEQ ID NOs:3-198, 249-254 and 283. In some embodiments, the asymmetric multispecific antibody comprises an IL-21 mutein comprising two amino acid substitutions compared to SEQ ID NO:1, optionally comprising one of SEQ ID NOs:199-248 and 255. In some embodiments, the asymmetric multispecific antibody comprises an IL-21 mutein comprising amino acid substitutions at any two of positions 5, 9, 73 and 76 of SEQ ID NO:1. In one embodiment, the amino acid substitutions are selected from A, E or Q at position 5, E or A at position 9, A or Q at position 73 and A, D or E at position 76. In one embodiment, the amino acid substitutions are at positions 5 and 73 of SEQ ID NO:1. In one embodiment, the amino acid substitutions are at positions 5 and 76 of SEQ ID NO:1. In one embodiment, the amino acid substitutions are at positions 9 and 73 of SEQ ID NO:1. In one embodiment, the amino acid substitutions are at positions 9 and 76 of SEQ ID NO: 1. In one embodiment, the IL-21 mutein comprises the amino acid sequence of any of SEQ ID NOs: 233-245. In one embodiment, a single IL-21 mutein of SEQ ID NO: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, or 245 is linked to the Fc of an anti-PD-1 antibody. In one embodiment, a single IL-21 mutein of SEQ ID NO: 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, or 245 is linked to the Fc of an anti-PD-1 antibody. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 233. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 234. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 235. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 236. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 237. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 238. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 239.In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 240. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 241. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 242. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 243. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 244. In one embodiment, the IL-21 mutein comprises the amino acid sequence of SEQ ID NO: 245.

[0077] In some embodiments the asymmetric multispecific antibody comprises a conjugate of a mutein and an antibody or immunoglobulin. In some embodiments the conjugate comprises an IL-21 mutein. In some embodiments the conjugate comprises an IL-21 mutein bound to an antibody or immunoglobulin.

[0078] In some embodiments, the asymmetric multispecific antibody comprises an IL-21 mutein bound to an antibody or immunoglobulin via a non-covalent or covalent bond, such as a peptide bond, a disulfide bond, or via physical forces such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions. A variety of non-covalent binding systems can be used, such as biotin-avidin, ligand / receptor, enzyme / substrate, nucleic acid / nucleic acid binding protein, lipid / lipid binding protein, cell adhesion molecule partner, or any binding partner or fragment thereof that has affinity for each other. In some embodiments, the binding can be via a direct covalent bond by reacting the target amino acid residues of the IL-21 mutein with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues of these target amino acids.

[0079] In some embodiments, the link between the IL-21 mutein and the antibody or immunoglobulin may be via a linker. In some embodiments, the linker comprises a chain of atoms of 1 to about 60, or 1 to 30 or more, 2 to 5, 2 to 10, 5 to 10, or 10 to 20 atoms in length. In some embodiments, the chain of atoms is all carbon atoms. In some embodiments, the chain of atoms in the backbone of the linker is selected from the group consisting of carbon (C), oxygen (O), nitrogen (N) and sulfur (S). The chain of atoms and the linker may be selected according to their expected solubility (hydrophilicity) to result in a more soluble conjugate. In some embodiments, the linker provides a functional group that undergoes cleavage by an enzyme or other catalytic or hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the length of the linker is sufficient to reduce the possibility of steric hindrance. When the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide, the entire conjugate may be a fusion protein. Such peptidyl linkers may be of any length. Exemplary peptidyl linkers are about 1 to 50 amino acids, 5 to 50 amino acids, 3 to 5 amino acids, 5 to 10 amino acids, 5 to 15 amino acids, or 10 to 30 amino acids in length and are flexible or rigid. In some embodiments, the linker is a peptide comprising about 2 to about 20 amino acids. In some embodiments, the linker is a peptide comprising about 2 to about 15 amino acids, about 2 to about 10 amino acids, or about 2 to about 5 amino acids. Suitable peptide linkers are known in the art. See, for example, Chen et al., Adv Drug Delivery Reviews 65(10):1357-1369(2013); Arai et al., Protein Eng Des Sel 14(8):529-532(2001); and Wriggers et al., Curr Trends in Peptide Science 80(6):736-746(2005). In one embodiment, the linker is a peptide comprising the amino acid sequence GGGGS (SEQ ID NO:262).

[0080] The asymmetric multispecific antibody comprises an IL-21 mutein directly linked to the Fc of the antibody. Alternatively, the IL-21 mutein is linked to the Fc of the antibody via a linker, such as a peptide comprising the amino acid sequence of SEQ ID NO: 262. In various examples, the asymmetric multispecific antibody comprises a single IL-21 mutein, which is linked to the C-terminus of one of the two antibody heavy chains.

[0081] In various aspects, the antibody heavy chain comprises one or more amino acid modifications compared to the naturally occurring counterpart to improve half-life / stability or to make the antibody more amenable to expression / manufacturability (e.g., as an asymmetric multispecific antibody comprising an IL-21 mutein).

[0082] In a typical example, the antibody heavy chain is designed to prevent or reduce the interaction between the antibody and the Fc receptor. In a typical example, the antibody is a Stable Effector Functionless (SEFL) antibody that comprises a constant region that lacks the ability to interact with Fcγ receptors. SEFL antibodies are known in the art. See, for example, Liu et al., J Biol Chem 292:1876-1883 (2016); and Jacobsen et al., J.Biol.Chem.292:1865-1875 (2017). In an exemplary embodiment, the SEFL antibody comprises one or more of the following mutations, numbered according to the EU system: L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. In an exemplary embodiment, the SEFL antibody comprises N297G. In an exemplary embodiment, the SEFL antibody comprises A287C, N297G, and L306C. In another exemplary embodiment, the SEFL antibody comprises R292C, N297G, and V302C (i.e., SEFL2-2).

[0083] The antibody may include other half-life extension (HLE) modifications. In a typical example, the HLE modifications occur in the heavy chain constant region and include one or more of the following mutations numbered according to the EU system: M252Y, S254T, and T256E. In a typical example, the antibody includes one or two of M252Y, S254T, and T256E. In a typical example, the antibody includes all three of M252Y, S254T, and T256E. In an exemplary embodiment, the heavy chain constant region includes the amino acid sequence of SEQ ID NO: 545 or SEQ ID NO: 547 or SEQ ID NO: 549. In a typical example, the HLE modifications occur in the heavy chain constant region and include one or more of the following mutations numbered according to the EU system: L309D, Q311H, and N434S. In a typical example, the antibody includes one, two, or all three of L309D, Q311H, and N434S. In a typical example, the antibody comprises all three of L309D, Q311H and N434S. In an exemplary embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:544 or SEQ ID NO:546 or SEQ ID NO:548.

[0084] In exemplary embodiments, the antibody comprises a SEFL2-2 modification and an HLE modification. In some examples, the HLE modification comprises one or two or all three of M252Y, S254T, and T256E. In exemplary embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:551 or SEQ ID NO:553 or SEQ ID NO:555. In some examples, the HLE modification comprises one or two or all three of L309D, Q311H, and N434S. In exemplary embodiments, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:550 or SEQ ID NO:552 or SEQ ID NO:554. In exemplary embodiments, the heavy chain further comprises a charge pair mutation as described below.

[0085] Generally, the C-terminal lysine of an antibody is subject to cleavage by carboxypeptidase during expression. A heavy chain constant region lacking a C-terminal Lys advantageously prevents carboxypeptidase from acting on the heavy chain of the antibody. In an exemplary embodiment, the antibody comprises a heavy chain constant region lacking a C-terminal Lys and further comprises a linker, such as a peptide comprising the amino acid sequence of SEQ ID NO:262.

[0086] In an exemplary embodiment, when the asymmetric multispecific antibody contains only one IL-21 mutein, the Fc of the antibody contains modifications designed to drive heterodimerization of the two heavy chains (one heavy chain fused to the IL-21 mutein and one heavy chain lacking the IL-21 mutein). Such modifications include Fc mutations such as knobs-into-holes, DuoBody, Azymetric, charge pair, HA-TF, SEEDbody, and modifications with different Protein A affinities. See, for example, Spiess et al., Molecular Immunology, 67(2, Part A), 2015, pp. 95-106. Knobs-into-holes mutations include T366W in the first heavy chain and T366S, L368A, and / or Y407V in the second heavy chain. See, e.g., Ridgway et al., Protein Eng., 9 (1996), pp. 617-621; and Atwell et al., J. Mol. Biol., 270 (1997), pp. 26-35. DuoBody mutations include F405L in the first heavy chain and K409R in the second heavy chain. See, e.g., Labrijn et al., Proc. Natl. Acad. Sci. USA, 110 (2013), pp. 5145-5150. Azymetric mutations include T350V, L351Y, F405A, and / or Y407V in the first heavy chain and T350V, T366L, K392L, and / or T394W in the second heavy chain. See, e.g., Von Kreudenstein et al., mAbs, 5 (2013), pp. 646-654. HA-TF mutations include S364H and / or F405A in the first heavy chain and Y349T and / or T394F in the second heavy chain. See, e.g., Moore et al., mAbs, 3 (2011), pp. 546-557. SEEDbody mutations include IgG / A chimeric mutations in the first heavy chain and IgG / A chimeric mutations in the second heavy chain. See, e.g., Davis et al., Protein Eng. Des. Sel., 23 (2010), pp. 195-202.Mutations with different Protein A affinities include H435R in one heavy chain and no mutation in the other heavy chain, see, e.g., U.S. Patent No. 8,586,713.

[0087] In a particular example, the mutation is a charge pair mutation. The following are examples of such charge pair mutations numbered according to the EU system. The charge pair mutations include K409D in the first heavy chain and D399K in the second heavy chain, K392D in the first heavy chain and E356K in the second heavy chain, or both K409D and K392D in the first heavy chain and both D399K and E356K in the second heavy chain (the latter is designated herein as "V1"). See, for example, Gunasekaran et al., J Biol Chem 285:19637-19646 (2010). In another particular example, the charge pair mutations include K439D, K392D, and K409D in the first heavy chain and E356K and D399K in the second heavy chain (designated herein as "V103"). In yet another particular example, the charge pair mutations include K360E, K370E, K392E, and K409D in the first heavy chain and E357K and D399K in the second heavy chain (referred to herein as "V131"). The charge pair mutations can also include K370D in the first heavy chain and E357K in the second heavy chain, or all three of K409D, K392D, and K370D in the first heavy chain and all three of D399K, E357K, and E356K in the second heavy chain (the latter of which is denoted herein as "V4"). Further charge pair mutations include D221E, P228E, and / or L368E in the first heavy chain and D221R, P228R, and / or K409R in the second heavy chain. See, e.g., Strop et al., J. Mol. Biol., 420 (2012), pp. 204-219.

[0088] In embodiments where the asymmetric multispecific antibody comprises only one IL-21 mutein (i.e. the asymmetric multispecific antibody comprises an IL-21 mutein monomer) and the heavy chain comprises a V1 charge pair mutation, the IL-21 mutein may bind to a heavy chain that comprises K409D and K392D mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 294, 296 or 298) or may bind to a heavy chain that comprises D399K and E356K mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 295, 297 or 299). In a specific embodiment, the IL-21 mutein is bound to a heavy chain that contains D399K and E356K mutations.

[0089] In embodiments where the asymmetric multispecific antibody comprises only one IL-21 mutein (i.e. the asymmetric multispecific antibody comprises an IL-21 mutein monomer) and the heavy chain comprises a V4 charge pair mutation, the IL-21 mutein may bind to a heavy chain that comprises K409D, K392D and K370D mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 288, 290 or 292) or may bind to a heavy chain that comprises D399K, E357K and E356K mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 289, 291 or 293). In a particular embodiment, the IL-21 mutein is bound to a heavy chain that contains D399K, E357K and E356K mutations.

[0090] In embodiments where the asymmetric multispecific antibody comprises only one IL-21 mutein (i.e. the asymmetric multispecific antibody comprises an IL-21 mutein monomer) and the heavy chain comprises the V103 charge pair mutation, the IL-21 mutein may bind to a heavy chain that comprises K439D, K392D and K409D mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 472, 474 or 476) or may bind to a heavy chain that comprises E356K and D399K mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 473, 475 or 477). In a particular embodiment, the IL-21 mutein is bound to a heavy chain that contains E356K and D399K mutations.

[0091] In embodiments where the asymmetric multispecific antibody comprises only one IL-21 mutein (i.e. the asymmetric multispecific antibody comprises an IL-21 mutein monomer) and the heavy chain comprises the V131 charge pair mutation, the IL-21 mutein may bind to a heavy chain that comprises K360E, K370E, K392E and K409D mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 478, 480 or 482) or may bind to a heavy chain that comprises E357K and D399K mutations (e.g. the IL-21 mutein binds to a heavy chain that comprises SEQ ID NO: 479, 481 or 483). In a particular embodiment, the IL-21 mutein is bound to a heavy chain that contains E357K and D399K mutations.

[0092] In an exemplary embodiment, one or both heavy chains of the asymmetric multispecific antibody comprise a constant region comprising a peptide linker (e.g., SEQ ID NO: 262), a SEFL or SEFL2-2 mutation, an HLE modification, a clipped C-terminal Lys, a charge pair mutation, or any combination thereof. In a typical example, one or both heavy chains of the asymmetric multispecific antibody comprise a constant region comprising the amino acid sequence of any one of SEQ ID NOs: 265, 266, 267, 282, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, and 472-495.

[0093] In some embodiments the asymmetric multispecific antibody comprises a single IL-21 mutein linked to an antibody that binds to a protein of an immune checkpoint pathway selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TEV13, BTLA, or the costimulatory receptors: ICOS, OX40, 41BB, CD27, GITR.

[0094] In a related embodiment, the antibody is an anti-PD-1 antibody. Suitable PD-1 antibodies are known in the art and include, but are not limited to, nivolumab (BMS-936558), pembrolizumab (MK-3475), BMS936558, BMS-936559, TSR-042 (Tesaro), ePDR001 (Novartis), and pidilizumab (CT-011), as well as any of the anti-PD-1 antibodies disclosed in WO 2019 / 140196, which are incorporated herein by reference. In various examples, the anti-PD-1 antibody comprises: (a) a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence selected from the group consisting of SEQ ID NOs: 312, 322, 332, 342, 352, 362, 372, and 382; (b) a HC CDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 313, 323, 333, 343, 353, 363, 373, and 383; (c) a HC CDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 314, 324, 334, 344, 354, 364, 374, and 384; (d) a light chain (LC) CDR1 amino acid sequence selected from the group consisting of SEQ ID NOs: 315, 325, 335, 345, 355, 365, 375, and 385, (e) a LC CDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 316, 326, 336, 346, 356, 366, 376, and 386, (f) a LC CDR3 amino acid sequence selected from the group consisting of SEQ ID NOs: 317, 327, 337, 347, 357, 367, 377, and 387, or (g) a combination of any two, three, four, five, or six of (a)-(f). In some embodiments, the anti-PD-1 antibody protein product comprises a HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3 according to Table 2.

[0095] [Table 2]

[0096] In another embodiment, the anti-PD-1 antibody comprises two light chains comprising LC CDR1, LC CDR2, and LC CDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 385, 386, and 387, respectively, and two heavy chains comprising HC CDR1, HC CDR2, and HC CDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 382, ​​383, and 384, respectively. In a related embodiment, each light chain variable region comprises the amino acid sequence of SEQ ID NO: 389 and each heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 388. In a related embodiment, each light chain comprises the amino acid sequence of SEQ ID NO: 391 and each heavy chain comprises the amino acid sequence of SEQ ID NO: 390.

[0097] In some embodiments, the asymmetric multispecific antibody is an anti-PD-1 antibody comprising an IL-21 mutein attached to the C-terminus of one of the two antibody heavy chains of the anti-PD-1 antibody. In some embodiments, the single IL-21 mutein is attached to the Fc of one of the two antibody heavy chains of the anti-PD-1 antibody. In some embodiments, the single IL-21 mutein is directly attached to the Fc of the anti-PD-1 antibody, e.g., the single IL-21 mutein is attached to the Fc of the anti-PD-1 antibody without a linker. In some embodiments, the single IL-21 mutein is attached to the Fc of the anti-PD-1 antibody by a linker (e.g., a peptide linker comprising SEQ ID NO: 262), e.g., the peptide linker connects the single IL-21 mutein to the Fc of the anti-PD-1 antibody.

[0098] In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 391, a single heavy chain bound to an IL-21 mutein comprising the amino acid sequence of any one of SEQ ID NOs: 501-506, and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 556-558. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 391, one heavy chain comprising the amino acid sequence of SEQ ID NO: 556, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 501. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 391, one heavy chain comprising the amino acid sequence of SEQ ID NO: 557, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 502. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 391, one heavy chain comprising the amino acid sequence of SEQ ID NO: 558, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 503. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 391, one heavy chain comprising the amino acid sequence of SEQ ID NO: 556, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 504. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 391, one heavy chain comprising the amino acid sequence of SEQ ID NO: 557, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 505. In some embodiments, the asymmetric multispecific antibody comprises two light chains, each comprising the amino acid sequence of SEQ ID NO: 391, one heavy chain comprising the amino acid sequence of SEQ ID NO: 558, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 506.

[0099] In another embodiment, the anti-PD-1 antibody comprises two light chains comprising LC CDR1, LC CDR2, and LC CDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 365, 366, and 367, respectively, and two heavy chains comprising HC CDR1, HC CDR2, and HC CDR3, respectively, comprising the amino acid sequences of SEQ ID NOs: 362, 363, and 364. In a related embodiment, each light chain variable region comprises the amino acid sequence of SEQ ID NO: 369, and each heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 368. In a related embodiment, each light chain comprises the amino acid sequence of SEQ ID NO: 371, and each heavy chain comprises the amino acid sequence of SEQ ID NO: 370.

[0100] In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, a single heavy chain bound to an IL-21 mutein comprising the amino acid sequence of any one of SEQ ID NOs: 513-518, and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 559-561. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, one heavy chain comprising the amino acid sequence of SEQ ID NO: 559, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 513. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, one heavy chain comprising the amino acid sequence of SEQ ID NO: 560, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 514. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, one heavy chain comprising the amino acid sequence of SEQ ID NO: 561, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 515. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, one heavy chain comprising the amino acid sequence of SEQ ID NO: 559, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 516. In some embodiments, the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, one heavy chain comprising the amino acid sequence of SEQ ID NO: 560, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 517. In some embodiments the asymmetric multispecific antibody comprises two light chains each comprising the amino acid sequence of SEQ ID NO: 371, one heavy chain comprising the amino acid sequence of SEQ ID NO: 561, and one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO: 518.

[0101] The invention provides a method for modulating the product quality of a recombinant asymmetric multispecific antibody comprising a mutein, comprising the steps of: a) establishing at least two cell cultures, each inoculated with the same cell line expressing the asymmetric multispecific antibody; b) culturing at least one cell culture at a first temperature regime consisting of a single temperature and at least one cell culture at a second temperature regime; c) comparing at least one product-related impurity in the cell cultures at each temperature regime; and d) selecting a temperature regime that reduces the expression of at least one product-related impurity comprising unpaired or mispaired long heavy chains, wherein the amount of the at least one product-related impurity resulting from an imbalance in the ratio of long to short heavy chains in the harvested cell culture is reduced compared to the amount of the same product-related impurity in a harvest from a cell culture exposed to a temperature of 32°C-34°C at some point during the culture.

[0102] The present invention provides a method for modulating the product quality of an asymmetric multispecific anti-PD-1 antibody comprising an IL-21 mutein attached to the C-terminus of one of two antibody heavy chains of the anti-PD-1 antibody expressed by cells in cell culture, the method comprising the steps of: a) establishing a cell culture inoculated with a cell line expressing the antibody, culturing the cells at 36±1°C for a culture period, and recovering the antibody, wherein the amount of at least one product-related impurity due to an imbalance in the ratio of long to short heavy chains in the harvested cell culture is reduced compared to the amount of the same product-related impurity in the harvest from a cell culture exposed to a temperature of 32°C-34°C at some point during culture.

[0103] The present invention relates to a method for establishing cell lines from clone-derived cultures expressing asymmetric multispecific antibodies, comprising the steps of: a) transforming cells with genes encoding an asymmetric multispecific antibody; b) isolating at least one single stably transformed cell expressing the asymmetric multispecific antibody and establishing a clone-derived culture; c) establishing at least two cell cultures from one or more of the clone-derived cultures; and d) culturing at least one cell culture from each clone-derived culture at a first temperature regime and isolating at least one single stably transformed cell expressing the asymmetric multispecific antibody and establishing a clone-derived culture. at a second temperature regime; e) culturing at least one cell culture at a first temperature regime consisting of a single temperature and culturing at least one cell culture at a second temperature regime; f) comparing the molar ratios of long heavy chains to short heavy chains produced by the cells at each temperature regime; g) selecting at least one clonally derived culture that produced a balanced or lower molar ratio of long heavy chains to short heavy chains; and h) establishing a cell line from the clonally derived culture.

[0104] The present invention relates to a method for producing isolated and purified recombinant asymmetric multispecific antibodies, comprising the steps of: a) isolating at least one single stably transfected cell expressing the asymmetric multispecific antibody and establishing a clonally derived culture; b) establishing at least two cell cultures from one or more of the clonally derived cultures; c) culturing at least one cell culture from each clonally derived culture at a first temperature regime comprising a single temperature and culturing at least one cell culture from each clonally derived culture at a second temperature regime; and d) detecting at least one product-associated antibody comprising unpaired or mispaired long heavy chains produced by the cells cultured at each temperature regime. e) selecting a cell culture with modulated production of product-related impurities, f) establishing a cell line expressing the asymmetric multispecific antibody from the selected clone-derived culture, g) inoculating a bioreactor with the cell line expressing the asymmetric multispecific antibody, h) culturing host cells to express the asymmetric multispecific antibody at a temperature regime that modulates production of product-related impurities, i) recovering the recombinant asymmetric multispecific antibody from the cell culture, j) processing the recombinant multispecific antibody by one or more chromatographic unit operations, and k) obtaining an isolated and purified recombinant asymmetric multispecific antibody. In one embodiment, an isolated and purified recombinant asymmetric multispecific antibody produced according to the above method is provided. In one embodiment, a pharmaceutical composition is provided comprising an isolated and purified recombinant asymmetric multispecific antibody produced according to the above method.

[0105] Also provided herein is a method for controlling cell growth in a nanofluidic chamber of a nanofluidic chip via one or more temperature shifts to enable extending the cell culture period, collecting additional measurements of protein secretion levels, and minimizing the risk of cell cross-contamination during the unloading procedure performed under low temperature conditions. Single cells expressing recombinant proteins such as monoclonal antibodies, multispecific antibodies, asymmetric proteins, etc. are placed on the nanofluidic chip and subjected to either constant temperature culture, maintaining a constant temperature for the duration of the culture, or biphasic culture, applying a temperature shift to one or more lower temperatures after an initial incubation at a first temperature condition. The temperature reduction resulted in growth inhibition, preventing cell overgrowth, reducing cell cross-contamination risk, and enabling long-term culture, while altering the recombinant protein production profile.

[0106] The method utilizes nanofluidic cell culture to screen thousands of clonally derived cell lines that are grown and evaluated for secretion of the desired therapeutic biologic. In one embodiment, the nanofluidic cell culture system is the Beacon® Optofludic system (Berkeley Lights, Inc., Emeryville, Calif.), a fully integrated nanofluidic cell culture system that utilizes a nanofluidic chip to enable the isolation of thousands of clonal cell lines. The Beacon® Optofludic platform is suitable for cell line development workflows, allowing for growth and evaluation of desired secretion profiles at the single cell or small number of cells level. Cells can be loaded into the nanofluidic chamber of the nanofluidic chip and simultaneously cultured and assayed for recombinant protein secretion. Candidate clones are selected based on the desired growth and secretion profiles, exported from the chip, and subjected to a typical scale-up workflow. Additional single cell sorting and analysis platforms are also contemplated for use with the disclosed methods. In one embodiment, a lithography-based microarray or nanowell-assisted cell patterning platform (Love et al., Nat. Biotechnol., 24(6) 703 (2006), and Ozkumur et al., Materials Views, 11(36), 4643-4650 (2015)) is contemplated.

[0107] As used herein, a "nanofluidic chamber of a nanofluidic chip" refers to a portion or section of a nanofluidic device capable of isolating a single cell. By way of example, the nanofluidic chamber may be a NanoPen™ chamber associated with Berkeley Light's Beacon® technology platform described herein. In some embodiments, the nanofluidic chip or device includes hundreds or thousands of individual chambers, each capable of isolating a single cell. In some embodiments, the nanofluidic device or chip includes 1758 chambers, 3,500 chambers, 11,000 chambers, 14,000 chambers, or 20,000 chambers. Such nanofluidic chips are known in the art and are commercially available, including the BLI OptoSelect™ chip, BLI OptoSelect™ chip 1750b, BLI OptoSelect™ chip 3500, BLI OptoSelect™ chip 11k, BLI OptoSelect™ chip 14K, and BLI OptoSelect™ chip 20k.

[0108] Methods for producing selected biomolecules using the method are also contemplated by the present disclosure. For example, in one embodiment, biomolecules grown under the conditions of the method are exported to multi-well plates, such as 96-well, 12-well or 24-well plates. The effects of stationary and shifting temperatures on recombinant proteins are analyzed and used to aid early clonal selection. Cell lines secreting recombinant proteins with desired properties and product quality characteristics are isolated and transferred to a vessel for culturing under conditions that allow recombinant protein production in the vessel and as described herein.

[0109] The present invention provides a method for controlling the growth of cells in a nanofluidic chamber of a nanofluidic chip, the method comprising: (a) isolating a single cell in a nanofluidic chamber of a nanofluidic chip, the cell comprising an expression construct capable of expressing a recombinant protein; (b) culturing the cell at a first temperature; (c) culturing the cell at a second temperature at a predetermined time point; and (d) exporting the cell from the nanofluidic chamber to a culture vessel. In one embodiment, the number of cells per chamber upon export is lower compared to a cell line derived from a single cell cultured under similar conditions at a constant temperature for the duration of the culture.

[0110] The present invention also provides a method for minimizing cell cross-contamination during export of cells grown in a nanofluidic chamber of a nanofluidic chip, comprising the steps of: (a) isolating a single cell in a nanofluidic chamber of a nanofluidic chip, said cell comprising an expression construct capable of expressing a recombinant protein; (b) culturing the cell at a first temperature; (c) culturing the cell at a second temperature at a predefined time point; and (d) exporting the cells from the nanofluidic chamber into a culture vessel at a lower temperature setting applied during the export procedure, wherein the number of cells per chamber upon export is lower compared to single cells cultured under similar conditions at a constant temperature throughout the culture period.

[0111] The present invention further provides a method for improving clonal selection of cells grown in a nanofluidic chamber of a nanofluidic chip, the method comprising: (a) isolating a single cell in a nanofluidic chamber of a nanofluidic chip, the cell comprising an expression construct capable of expressing a recombinant protein; (b) culturing the cell at a first temperature; (c) reducing the temperature of the culture to a second temperature after the third day of culture; and (d) removing the cells from the nanofluidic chamber at least 1 to 4 days after the temperature shift.

[0112] In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 2-4 days after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 3-4 days after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 2-3 days after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 1-2 days after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 1, 2, 3, or 4 days after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 1 day after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 2 days after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 3 days after the temperature shift. In one embodiment, the step of removing the cells from the nanofluidic chamber is at least 4 days after the temperature shift.

[0113] In one embodiment, the first temperature is selected from 35° C. to 37° C. In one embodiment, the first temperature is 35° C., 36° C., or 37° C. In one embodiment, the first temperature is 35° C. In one embodiment, the first temperature is 36° C. In one embodiment, the first temperature is 37° C.

[0114] In one embodiment, the second temperature is selected from 28°C to 34°C. In one embodiment, the second temperature is selected from 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, or 34°C±0.5°C. In one embodiment, the second temperature is selected from 28°C. In one embodiment, the second temperature is selected from 29°C. In one embodiment, the second temperature is selected from 30°C. In one embodiment, the second temperature is selected from 31°C. In one embodiment, the second temperature is selected from 32°C. In one embodiment, the second temperature is selected from 33°C. In one embodiment, the second temperature is selected from 34°C.

[0115] In one embodiment, the second temperature is lower than the first temperature by about 1° C. to about 9° C. In one embodiment, the first temperature is 36° C. and the second temperature is 32° C. to 32.5° C.

[0116] In one embodiment, the predetermined time point is between day 3 and day 5 of the culture. In one embodiment, the predetermined time point is between day 3 and day 4 of the culture. In one embodiment, the predetermined time point is between day 4 and day 5 of the culture. In one embodiment, the predetermined time point is the third, fourth or fifth day of the culture. In one embodiment, the predetermined time point is the third day of the culture. In one embodiment, the predetermined time point is the fourth day of the culture. In one embodiment, the predetermined time point is the fifth day of the culture.

[0117] In one embodiment, the export occurs between day 6 and day 8 of the culture. In one embodiment, the export occurs between day 6 and day 7 of the culture. In one embodiment, the export occurs between day 7 and day 8 of the culture. In one embodiment, the export occurs on day 6, day 7 or day 8 of the culture. In one embodiment, the export occurs on day 6 of the culture. In one embodiment, the export occurs on day 7 of the culture. In one embodiment, the export occurs on day 8 of the culture.

[0118] In one embodiment, the nanofluidic chip includes 1758 chambers, 3,500 chambers, 11,000 chambers, 14,000 chambers, or 20,000 chambers.

[0119] In one embodiment, the culture vessel is a multi-well plate. In one embodiment, protein secretion profiles before and after a temperature shift are compared. In one embodiment, the nanofluidic chip contains 1758 chambers.

[0120] "Culturing" or "culture" 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 and refer to media suitable for the growth of host cells in in vivo cell culture. Typically, cell culture media contain buffers, salts, an energy source, amino acids, vitamins and essential trace 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, STEMCELL Technologies Inc., 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 concentrations higher than their usual recommended concentrations.

[0121] Various media formulations can be used at all stages of cell culture from clonal selection and cell line generation to large-scale production cell culture. Different media formulations can be used during selection to facilitate the transition from one stage (e.g., growth stage or growth phase) to another stage (e.g., production stage or production phase) and / or to optimize conditions during cell culture (e.g., enriched media can be provided during perfusion culture and fed media can be provided during fed-batch culture). Selection media can be formulated to apply selection pressure to newly transformed cells. Growth media formulations can be used to promote cell growth and minimize protein expression. Production media formulations can be used to promote production of the protein of interest and maintenance of the cells while minimizing new growth of the cells. Feed media, which is consumed during the production phase of the cell culture, usually contains more concentrated components such as nutrients and amino acids, can be used to supplement and maintain active cultures, especially cultures operated in fed-batch, semi-perfusion, or perfusion modes. Such concentrated feed media may contain most of the components of the 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 even about 1000x their normal amounts.

[0122] The cell cultures can be operated in batch, fed-batch, continuous, semi-continuous, perfusion mode or any combination therein.

[0123] Temperature shifts are commonly used in Chinese Hamster Ovary (CHO) cell culture to affect culture performance and recombinant expression of proteins. CHO cells are typically grown at physiological temperatures of about 36° C. to 37° C. Temperature shifts to hypothermic conditions of about 30° C. to 34° C. are used to control aspects of the culture such as cell growth, improve recombinant protein production, improve yields, and / or maintain cell viability.

[0124] Additionally, chemical inducers of protein production, such as caffeine, butyrate, and hexamethylene bisacetamide (HMBA), can be added to the cell culture. If there is a temperature shift, the inducers can be added simultaneously with the temperature shift, before the temperature shift, and / or after the temperature shift. If the inducers are added after the temperature shift, they can be added 1 hour to 5 days after the temperature shift, optionally 1 to 2 days after the temperature shift. The pH can also be shifted during the culture, either independently or in combination with other methods.

[0125] Host cells can be cultured in suspension or in an attached form attached to a solid substrate. Cells can be cultured in multi-well plates, such as 96, 24, or 6-well and deep-well plates. Cell cultures can be established in fluidized bed bioreactors, gas-permeable culture bags, gas-permeable bioreactors, hollow fiber bioreactors, roller bottles, spin tubes, shake flasks, or stirred tank bioreactors, with or without microcarriers. Mammalian cells, such as CHO cells, can be cultured in bioreactors on a small scale, less than 100 ml to less than 1000 ml. Alternatively, larger scale bioreactors containing 1000 ml to more than 20,000 liters of medium can be used. Large scale cell cultures, such as clinical and / or commercial scale biomanufacturing of protein therapeutics, can be maintained for weeks or even months while the cells produce the desired protein.

[0126] The term "unit operation" refers to a functional step that is performed as part of a process to purify a recombinant protein of interest. For example, a unit operation may include steps in an operation such as, but not limited to, recovery, capture, purification, polishing, viral inactivation, viral filtration, concentration, and / or formulation of a recombinant protein of interest. A unit operation can be designed to accomplish a single objective or multiple objectives, such as a combination of capture and viral inactivation steps. A unit operation may also include a holding or storage step between processing steps.

[0127] Recombinant protein is recovered from cell culture medium.Methods for recovering protein from suspended cells are known in the art, and include, but are not limited to, acid precipitation, accelerated sedimentation such as flocculation, separation using gravity, centrifugation, sonication, filtration, including membrane filtration using ultrafiltration membrane, microfiltration membrane, tangential flow filtration membrane, alternative tangential flow filtration membrane, depth filtration membrane, and sand filtration membrane (alluvial filter).Recombinant protein expressed by prokaryotic organisms is recovered from inclusion bodies in the cytoplasm by a process incorporating redox folding process known in the art.

[0128] The recovered proteins can then be purified or partially purified using one or more unit operations to remove any impurities, such as residual cell culture medium, cell extracts, undesired components, host cell proteins, improperly expressed proteins, etc. By "purifying" it is meant increasing the purity of the asymmetric multispecific antibodies in a composition by removing (partially or completely) at least one impurity or contaminant from the composition. Recovery and purification of the multispecific antibodies is accomplished by downstream unit operations, particularly those involving chromatography, resulting in a more "homogeneous" asymmetric multispecific antibody composition that meets yield and product quality goals.

[0129] The capture unit operation may include capture chromatography, which utilizes resins and / or membranes containing agents that bind and / or interact with at least one desired protein, impurity, or contaminant. Examples of capture chromatography include affinity chromatography, size-exclusion chromatography, ion-exchange chromatography, hydrophobic interaction chromatography (HIC), immobilized metal affinity chromatography (IMAC), and the like. Such materials are known in the art and commercially available. Affinity chromatography is commonly used in biomanufacturing processes as an initial capture step to isolate and concentrate recombinant proteins of interest having Fc components. Examples of such affinity chromatography materials include those utilizing staphylococcal proteins such as Protein A, Protein G, Protein A / G, Protein L, substrate-bound capture mechanisms, antibody or antibody fragment-bound capture mechanisms, aptamer-bound capture mechanisms, and cofactor-bound capture mechanisms. Immobilized metal affinity chromatography can be used to capture proteins that have, or have been engineered to have, affinity for metal ions. Affinity chromatography materials are commercially available from a number of sources. For example, MABSELECT™ SURE Protein A, Protein A Sepharose FAST FLOW™ (Cytiva, Marlborough, Massachusetts), PROSEP-A™ (Merck Millipore, UK), TOYOPEARL™ 650M Protein A (TosoHass Co., Philadelphia, Pennsylvania).

[0130] Intermediate and / or polishing unit operations utilize various chromatographic techniques to continuously purify the protein of interest and remove contaminants and impurities such as DNA, host cell proteins; product-specific impurities, variant products and aggregates, viral adsorption, etc. These chromatographic unit operations utilize resins and / or membranes containing agents that can be used either in a flow-through mode where the protein of interest is included in the eluate and the contaminants and impurities bind to the chromatographic medium; in a frontal or overload chromatography mode where the solution containing the protein of interest is loaded onto the column until the adsorption sites are occupied and the species with the least affinity for the stationary phase (protein of interest) begins to elute; or in a bind and elute mode where the protein of interest is bound to the chromatographic medium and eluted after the contaminants and impurities have passed through or been washed off from the chromatographic medium; or in other manners. Examples of such chromatographic methods include, inter alia, 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.

[0131] Multiple chromatography unit operations (usually one, two, or three, each performing a different function) are combined depending on the requirements of the manufacturing process. Ion exchange chromatography separates the protein of interest from impurities based on electrostatic interactions between charged surfaces, and on differential adsorption and desorption. "Cation exchange chromatography" refers to chromatography performed on a solid phase medium that is negatively charged and has free cations for exchange with cations in aqueous solution passed over or through the solid phase. The charge may be provided by attaching one or more charged ligands to the solid phase, e.g., by covalent bonds. Alternatively, or in addition, the charge may be an inherent property of the solid phase (e.g., as in the case of silica, which has an overall negative charge). Cation exchange chromatography is usually performed in bind-elute mode, where the high pI of many proteins of interest allows binding to the chromatographic material. Cation exchange chromatography can also be performed in flow-through mode. CEX chromatography is typically used for the removal of high molecular weight (HMW) contaminants, removal of process-related impurities, and / or virus clearance.Commercially available cation exchange media are available, examples of which include sulfopropyl (SP) immobilized on agarose (e.g., SP-SEPHAROSE FAST FLOW™, SP-SEPHAROSE FAST FLOW XL™, or SP-SEPHAROSE HIGH PERFORMANCE™, CAPTO S™, CAPTO SP ImpRes™, CAPTO S ImpAct™ (Cytiva), FRACTOGEL-SO3™, FRACTOGEL-SE HICAP™, and FRACTOPREP™ (EMD Merck, Darmstadt, Germany), TOYOPEARL™ XS, TOYOPEARL™ HS (Tosh Bioscience, King of Prussia, PA), UNOsphere™ (BioRad, Hercules, CA), S Ceramic Hyper DF (Pall, Port Washington, NY), POROS™ (ThermoFisher, Waltham, MA).

[0132] Anion exchange chromatography refers to chromatography performed on a solid phase medium that is positively charged and has free cations for exchange with anions in an aqueous solution passed over or through the solid phase. Anion exchange chromatography is typically performed in flow-through mode. Many proteins have high pIs and therefore do not bind to AEX chromatography materials. AEX chromatography is used, for example, in viral clearance and impurity removal. Commercially available anion exchange media are available, including, but not limited to, sulfopropyl (SP) immobilized on agarose (e.g., Source 15 Q, Capto™ Q, Q-SEPHAROSE FAST FLOW™ (Cytiva), FRACTOGEL EDM TMAE™, FRACTOGEL EDM DEAE™ (EMD Merck), TOYOPEARL Super Q® (Tosh Bioscience), POROS HQ™, POROS XQ™, (ThermoFisher).

[0133] Mixed-mode or multimodal chromatography (MMC) refers to chromatography performed on solid-phase media that utilizes a combination of interaction mechanisms such as ion exchange (CEX or AEX) and hydrophobic interactions. Commercially available multimodal chromatography media are available, including Capto™ Adhere (Cytiva).

[0134] Hydrophobic interaction chromatography refers to chromatography on a solid phase medium that exploits the interactions between hydrophobic ligands and hydrophobic residues on the surface of the protein of interest. Commercially available hydrophobic interaction chromatography media include, but are not limited to, Phenyl Sepharose™ (Cytiva), Tosoh hexyl (Tosoh Bioscience), and Capto™ phenyl (Cytiva).

[0135] Hydroxyapatite chromatography refers to chromatography performed on a solid phase medium that utilizes positively charged calcium and negatively charged phosphate, which act as cations or anions depending on the pI of the protein and the pH of the buffer.

[0136] Unit operations involving inactivation, reduction, and / or elimination of viral contaminants may include processes that manipulate the environment and / or filtration. A variety of methods can be employed for viral inactivation, including heat inactivation / pasteurization, UV and gamma irradiation, high intensity broad spectrum ionization, and the like. white These include the use of light, chemical inactivation agents, addition of surfactants, solvent / surfactant treatments, etc. Surfactants, such as detergents, can be very effective in specifically inactivating enveloped viruses because they solubilize the membrane. One method to achieve viral inactivation is incubation at low pH (e.g., pH<4). Low pH viral inactivation can be followed by a neutralization unit operation that readjusts the virus-inactivated solution to a pH that is 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. Virus filtration can be performed using microfiltration or nanofiltration membranes, such as those available from Asahi Kasei (Plavona®) and EDM Millipore (VPro®).

[0137] Unit operations may also include concentration of the product, and buffer exchange of the protein of interest into the desired formulation buffer for bulk storage of the drug substance can be accomplished using known methods for ultrafiltration and diafiltration (UF / DF). Unit operations related to fill / finish of the drug product may follow.

[0138] Key attributes and performance parameters of purified asymmetric multispecific antibodies 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 medium 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 during cell culture. Key attributes such as specific productivity, viable cell density, pH, osmolality, appearance, color, aggregation, yield and titer can be monitored during appropriate stages in the manufacturing process. Monitoring and measurements can be done using known techniques and commercially available equipment.

[0139] Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: buffers such as neutral buffered saline, phosphate buffered saline, carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), and preservatives, sterile diluents such as water for injection, saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that can serve as a solvent or suspending medium, polyethylene glycol, glycerin, propylene glycol, or other solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium sulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and agents for adjusting isotonicity such as sodium chloride or dextrose. Parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple dose vials.

[0140] The terminology used in this application is standard in the art, but definitions of certain terms are provided herein to ensure clarity and clarity to the meaning of the claims. Units, prefixes, and symbols may be indicated in their SI accepted form. Numerical ranges described herein are inclusive of the numbers defining the range, and include and support each integer within the defined range. Unless otherwise indicated, the methods and techniques described herein are generally performed according to conventional methods well known in the art and are described in various general and more specific references cited and discussed throughout this specification. All documents or portions of documents cited in this application, including but not limited to patents, patent applications, papers, books, and academic papers, are expressly incorporated herein by reference.

[0141] The present invention is not limited in scope by the specific embodiments described herein, which are intended as illustrations of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Any embodiment of the invention described may be combined with other embodiments 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 description and accompanying drawings. Such modifications are intended to be included within the scope of the appended claims.

[0142] The following examples, including the experiments conducted and results achieved, are provided for illustrative purposes only and should not be construed as limiting the scope of the appended claims. EXAMPLES

[0143] Example 1 CHO cells were transfected with plasmid DNA encoding a bifunctional fusion protein containing a conjugate of an IL-21 mutein linked to the C-terminus of one of the two antibody heavy chains of an anti-PD-1 antibody. The IL-21-anti-PD-1 conjugate contained a "short heavy chain" (anti-PD1 heavy chain) and a "long heavy chain" (anti-PD1 heavy chain linked to an IL-21 mutein). The weight, length and amino acid composition differed between the two heavy chains. After transfection, a pooled population stably expressing the conjugate was generated through repeated passaging in selective growth medium. When the pool reached >90% viability as measured by Trypan Blue Exclusion and maintained a consistent doubling time, single cell clones were isolated from the pool.

[0144] Single cell clones were expanded and cultured in 96-well plates (Corning, Corning, NY) maintained at 36°C, 5% CO2, and 85% relative humidity. Cells were maintained by passaging until the desired cell viability density was reached.

[0145] The clones were then plated at 8 × 10 in 24-well plates (Axygen, Union City, CA) containing a working volume of 3.5 ml of chemically defined serum-free culture medium. 5 Cells / ml were inoculated. Plates were maintained at 36°C, 5% CO2, 85% relative humidity, shaken at 225 rpm for 10 days and fed on days 3, 6, and 8 with 7% of the post-inoculation volume. Cultures were harvested on day 10. In-process samples were taken to monitor culture conditions. Viable cell density (VCD) and viability (%) were determined using a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Indianapolis, IN). Glucose, lactate, and NH4+ concentrations were determined using a BioProfile Flex Analyzer (Nova Biomedical, Waltham, MA).

[0146] Samples from each culture were taken for titer determination and product quality analysis. Titers were measured by high performance liquid chromatography (HPLC) with affinity chromatography (Protein A, Waters, Milford, MA). Integrated viable cell density (IVCD) was calculated by the trapezoidal rule of VCD versus culture time. Cell-specific productivity (q p ) was calculated by dividing the titer by the IVCD. The harvested material was used for product quality evaluation. The top four clones were selected for bioreactor cultivation.

[0147] The top four clones (clones 1-4) were inoculated at 10E5 cells / ml in 2 x 7 L bioreactors in 4.4 L working volume of chemically defined serum-free culture medium. The bioreactors were maintained at 36 °C, 5% CO2, 6.90 pH, and 315 rpm. Perfusion was started on day 3 and an alternating tangential flow filter system (Repligen, Waltham, MA) with 30,000 NMWC filters (GE Healthcare, Westboro, MA) was used at a rate of 0.5 reactor volumes / day on days 3-6, 0.75 reactor volumes / day on days 7-8, and 1.0 reactor volumes / day on days 9-15, except for clone 2, which was stopped on day 14. Daily samples were taken to monitor culture conditions (e.g., pH, osmolality, lactate / glucose, etc.) as previously described for 24-well plates.

[0148] When the viable cell density (VCD) exceeded 300E5 cells / ml, the temperature was shifted to 32.5°C and the cultures were maintained at that temperature until harvest. Clones were harvested from days 12 to 15 using 750,000 NMWC filters, GE Healthcare, except for clone 2, which was harvested from days 12 to 14. Culture conditions and harvested culture supernatants were monitored using the same methods as previously described for 24-well plates.

[0149] Unexpectedly, samples exposed to lower incubation temperatures (32.5° C.) showed an increase in the molar ratio of long heavy chain (LHC) to short heavy chain (SHC) for all clones tested.

[0150] A second experiment was performed to investigate the cause of the increased molar ratio of long to short heavy chains. A 10-day fed-batch culture was performed to examine the response of clone 4 grown at 36°C constantly and under conditions incorporating a temperature shift (from 36°C to 32.5°C) during culture. Cells from clone 4 were plated at 8 × 10 in two 24-deep-well plates (Axygen, Union City, CA) containing a working volume of 3.5 ml of chemically defined serum-free culture medium. 5 Cultures were seeded at 1000 cells / ml. Plates were maintained at 36°C, 5% CO2, 85% relative humidity and shaken at 225 rpm. One plate was temperature shifted to 32.5°C, 5% CO2, 225 rpm. Both cultures were fed at 7% of the post-inoculation volume on days 3, 6, and 8. Cells from both cultures were harvested on day 10. Analytical measurements were performed throughout the 10-day culture as previously described. The harvested material was then purified using Protein A affinity chromatography.

[0151] Cells from clones 1-3 were also tested in perfusion culture for 15 days in a 7 L bioreactor to examine the protein response of the clones under perfusion conditions with a constant temperature of 36°C maintained throughout. Clone 4 was tested in perfusion culture for 15 days in a 7 L bioreactor to examine the protein response of the clones under perfusion conditions with a temperature shift (36°C to 32.5°C).

[0152] Test clones and controls were inoculated at 10E5 cells / ml in 2 x 7 L bioreactors with a working volume of 4400 ml of chemically defined serum-free culture medium. Bioreactors were maintained at 36°C, 5% CO2, 6.90 pH, and 315 rpm. Perfusion was initiated on day 3 and used an alternating tangential flow filter system (Repligen, Waltham, MA) with 30,000 NMWC filters (GE Healthcare, Westboro, MA) at a rate of 0.5 reactor volumes / day on days 3-6, 0.75 reactor volumes / day on days 7-8, and 1.0 reactor volumes / day on days 9-15, except for clone 2, which was stopped on day 14. Daily samples were taken to monitor culture conditions as previously described. Clones 1 and 3 were harvested from days 12 to 15 using 750,000 NMWC filters, GE Healthcare, except for clone 2, which was harvested from days 12 to 14. Culture conditions were monitored throughout. Analytical measurements were performed throughout the cultures as previously described. Harvested material was purified using Protein A affinity chromatography followed by cation exchange chromatography in bind and elute mode.

[0153] The molar ratio of long to short heavy chains for both fed-batch and perfusion samples was determined by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS). The average Pre-Peak % was determined by non-reduced capillary electrophoresis-sodium dodecyl sulfate (nrCE-SDS). The average LMW % was determined using size-exclusion ultra-high performance liquid chromatography (SE-UHPLC). The molar ratio of long to short heavy chains for both fed-batch and perfusion samples was determined by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS).

[0154] The increase in the percentage of long heavy chains (% of LHC) when cells expressing IL-2-anti-PD-1 conjugates were subjected to perfusion culture (P) with a temperature shift to 32.5°C was compared with fed-batch culture (FB) at a constant temperature of 36°C ( StripesThe ratios of the temperature-shifted perfusion cultures (P) and the protein A affinity chromatography (P) fractions subjected to purification by protein A affinity chromatography (left open bar) and cation exchange chromatography (right open bar) were compared (see Fig. 1A). This data confirms that the change in ratio is due to the temperature shift and not due to the switching of the cultivation method. It was found that the partially reduced species produced during the perfusion cultivation with the temperature shift from 36°C to 32.5°C could not be removed in the downstream purification. Stripes A comparison of Pre-Peak from a constant temperature fed-batch culture (FB) subjected to a temperature shift (bars 0.01 and 0.01) is shown in Figure 1B. The perfusion culture sample subjected to a temperature shift had a higher percentage of LMW than the fed-batch culture sample subjected to a constant temperature (see Figure 1C). Notably, the excess long heavy chains could not be separated by CEX purification. A chromatogram of the Pre-Peak impurity characterized by nrCEX-SDS from the CEX pool is shown in Figure 1D. The excess long heavy chain species from the perfusion sample could not be separated by CEX chromatography.

[0155] Comparison of the molar ratios of long and short heavy chains identified by rCE showed a better correlation between fed-batch and perfusion samples when both were subjected to the same temperature regime. Figure 2A shows a fed-batch culture sample grown at 36°C all the time compared to a perfusion sample grown with a temperature shift from 36°C to 32.5°C. Figure 2B shows a fed-batch culture sample compared to a perfusion sample, both grown with a temperature shift from 36°C to 32.5°C. Data points for both culture methods grown at 36°C all the time were limited and therefore not included in the comparison.

[0156] Comparing the % of LMW measured by SEC showed a better correlation between fed-batch and perfusion samples when subjected to the same temperature regime. Figure 3A shows a fed-batch culture sample grown at 36°C all the time compared to a perfusion sample grown with a temperature shift from 36°C to 32.5°C. Figure 3B shows a fed-batch culture sample compared to a perfusion sample, both grown with a temperature shift from 36°C to 32.5°C. Data points for both culture methods grown at 36°C all the time were limited and therefore not included in the comparison.

[0157] When cultures were subjected to a temperature shift from 36°C to 32.5°C, an unexpected shift in the molar ratio of long heavy chain (LHC) to short heavy chain (SHC) was observed compared to cultures grown at a constant temperature of 36°C (Figure 4A, constant temperature of 36°C, Stripes bar, temperature shift from 36°C to 32.5°C, white Bars at 0°C). This imbalance affected downstream purification (Protein A affinity chromatography and cation exchange chromatography) and product quality. Cultivation at lower temperature (32.5°C) resulted in a decrease in impurities formed by short heavy chains (Pre-Peak 6) and an increase in impurities formed by long heavy chains (Pre-Peak 7) as measured by nrCE. The short heavy chain impurities could be removed by Protein A and CEX chromatography purification (Figure 4B), whereas the long heavy chain impurities were difficult to remove by the same purification system (Figure 4C). Protein A affinity chromatography ( Stripes bar) and cation exchange chromatography ( white bar).

[0158] This result showed that cell culture temperature could affect product quality by affecting the ratio of the two heavy chains, which affected the impurities removed by the purification process. Depending on the characteristics of the impurities, different levels of purity could be achieved. In this example, higher purity was achieved at higher cell culture temperatures.

[0159] These results indicated that there was a better correlation between the product quality of IL-2-anti-PD-1 conjugates when clones were screened and selected at the same temperature regime that the resulting cell lines were expected to encounter. The molar ratio of long to short heavy chains was found to be affected by the temperature of the culture, with lower temperatures resulting in increased expression of long heavy chains. The increase in the ratio of long to short heavy chains affected the formation and type of product-related impurities. Impurities containing long heavy chains were found to be surprisingly difficult to remove in downstream purification steps compared to impurities containing short heavy chains. This change in the ratio of long to short heavy chains affected purification, activity, product quality, as well as the robustness of the overall manufacturing process.

[0160] Example 2: In this experiment, bispecific A, an asymmetric bispecific antibody containing two heavy chains, a "long heavy chain" with a longer pair of amino acid sequences and a "short heavy chain" with a shorter amino acid sequence, was investigated to determine the effect of temperature during clone selection and on product quality at the transcriptional level.

[0161] Fed-batch culture A clone expressing a bispecific antibody (Bispecific A) was selected from the group of clones from the initial clonal cell screen (Clone 1). A 10-day fed-batch clone screen was performed in shake flasks to examine the transcript and protein response of the clones during cell culture, with the screening protocol comparing the performance of the clones when cultured at a constant temperature (36° C.) versus culture with a temperature shift (36° C. to 32.5° C.).

[0162] Started a 1 L shake flask containing 250 ml of working volume of chemically defined serum-free culture medium. Add 8 x 10 5 The shake flasks were incubated at 36°C and 5% CO 2、 They were maintained at 160 rpm for 7 days and fed on days 3 and 6 with 7% of the post-inoculation volume.

[0163] On day 7, the culture was split into two 250 ml shake flasks containing 60 ml working volume of chemically defined serum-free culture medium. One flask was maintained at 36°C, while the culture temperature of the other flask was reduced to 32.5°C. Both flasks were cultured until day 10 with 5% CO2 and agitation at 160 rpm. Cultures were fed on day 8 with 7% of the post-inoculation volume. Analytical measurements were performed throughout the 10-day culture as described in the previous examples. Integrated viable cell density (IVCD) was measured at harvest using Nova CDV (Nova Biomedical). Titers were measured at harvest using HPLC analysis. Transcript ratios were determined using ddPCR according to the manufacturer's recommendations (DROPLET DIGITAL™ PCR (ddPCR™) system (Bio Rad, Hercules, CA)). Molar ratios of long and short heavy chains were determined by rCE-SDS.

[0164] An increase in the molar ratio of long heavy chains (LHC) to short heavy chains (SHC) was observed in samples temperature-shifted to 32.5 °C, indicating preferential expression of long heavy chains compared to short heavy chains at lower temperatures (Figure 5A, 36 °C ( Stripes bar), 32.5℃( white (bars). This correlated with a decrease in % high molecular weight (HMW) measured by SEC in samples cultured at 36°C (Figure 5B) and an increase in % LMW measured by SEC in temperature-shifted samples (see Figure 5C). HMW impurities were formed by short heavy chains and LMW impurities by long heavy chains. The integrated viable cell density was relatively unchanged and titers were comparable between the two culture conditions. The transcript ratio of long to short heavy chains was also increased at the lower temperature.

[0165] Simulated perfusion culture Two clones expressing bispecific A (clone 2 and clone 3) were selected from the group of clones from the initial clonal cell screening and tested in small-scale simulated perfusion cultures to investigate the transcript and protein response times to temperature changes in a culture process that mimics high cell density perfusion culture in a bioreactor. Clones were cultured at constant temperature (36°C) and also with temperature shifts (from 36°C to 32.5°C).

[0166] For each clone, 200 x 10 cells were cultured in two 24-deep well plates. 5 Cells / ml / well were seeded. On day 0, plates were incubated at 36°C, 5% CO2. On day 1, the temperature was reduced to 32.5°C for one plate from each clone while the other plate from each clone was maintained at 36°C. Plates were harvested on day 3. Daily medium changes were performed using chemically defined serum-free medium, removing spent medium and adding fresh medium to mimic perfusion culture. Analytical measurements were performed daily.

[0167] After a 24-h temperature shift from 36°C to 32.5°C, the molar ratio of long heavy chain (LHC) to short heavy chain (SHC) at lower temperatures increased by 9% (clone 2) and 13% (clone 3) compared with cultures held at a constant temperature of 36°C (Figure 6A; 36°C was Stripes bar, 32.5℃ white Bars). This indicated that at lower temperatures there was preferential expression of long heavy chains compared to short heavy chains. The increase in molar ratio coincided with an increased % of LMW containing long heavy chain homodimer and monomeric impurities (Figure 6B). Transcription ratios are shown in Figure 11C. Both cell growth and titer were lower for clones subjected to temperature shift.

[0168] As can be seen from experiment 1, the molar ratio of long to short heavy chains in bispecific 1 was also found to be affected by incubation temperature. The increase in the molar ratio of long to short heavy chains was consistent with the formation of increased impurities containing long heavy chains at lower temperatures.

[0169] Example 3 In this experiment, we investigated the effect of temperature during clonal selection at the transcriptional level and on product quality for an asymmetric bispecific antibody, Bispecific C. Bispecific C has two heavy chains, a "long heavy chain" with a longer amino acid sequence and a "short heavy chain" with a shorter amino acid sequence.

[0170] Fed-batch shake flask culture Three clones (Clone 1, Clone 2, Clone 3) were selected from the initial clonal screen of bispecific B. A 10-day fed-batch temperature study was performed in shake flasks as described above in Example 2 to examine the transcriptional and protein responses of the clonal cells in a process at a constant temperature of 36° C. and in a process involving a temperature shift from 36° C. to 32.5° C.

[0171] One 1 L shake flask (Corning, Corning, NY) containing 250 ml working volume of chemically defined serum-free culture medium was initiated for each clone. On day 0, each flask was cultured with 8 × 10 5 Shake flasks were maintained at 36° C., 5% CO2, 160 rpm for 7 days and fed on days 3 and 6 with 7% of the post-inoculation volume.

[0172] On day 7, each flask was split into two flasks containing a working volume of 60 ml of chemically defined serum-free culture medium. One flask was maintained at 36°C and the other flask at 32.5°C. Both were cultured at 5% CO2 with agitation at 160 rpm until day 10. Cultures were fed on day 8 with 7% of the post-inoculation volume. Analytical measurements were performed throughout the 10 days of culture.

[0173] The molar ratio of long to short heavy chains increased at lower temperatures, suggesting preferential expression of long heavy chains over short heavy chains (see FIG. 7A, 36° C. Stripes bar, 32.5℃ whiteBars). This was consistent with an increase in the % of HMW2 containing long heavy chain homodimer impurities (Figure 7B) and a decrease in the % of LMW1 containing short heavy chain homodimer impurities (Figure 7C). Transcription ratios are shown in Figure 7C. Similar titers were observed between the two temperature regimes. There was a small difference (10%) in the integrated viable cell density (IVCD) between the two temperature regimes.

[0174] Simulated perfusion culture As mentioned above, bispecific B clones 2 and 3 were also tested in small scale simulated perfusion cultures to examine transcript and protein response times to temperature changes in a culture process that mimics high cell density perfusion culture in a bioreactor.

[0175] For each of clones 2 and 3, place 200 x 10 cells in two 24-well deep well plates. 5 Cells / ml / well were seeded and incubated at 36°C, 5% CO2 on day 0. On day 1, the temperature of one plate of each clone was lowered to 32.5°C, while the other plate was maintained at 36°C. On day 3, the plates were harvested. Chemically defined serum-free medium was used for culture. Daily medium changes were performed to remove spent medium and add fresh medium. Analytical measurements were performed daily as described in the previous examples.

[0176] The molar ratio of long to short heavy chains increased by 17% for clone 2 and 25% for clone 3 24 hours after a temperature shift from 36°C to 32.5°C compared to a constant temperature of 36°C (see Figure 8A, 36°C is Stripes bar, 32.5℃ white Bars at 32.5°C). This indicated that at lower temperatures, the long heavy chain was preferentially expressed compared to the short heavy chain. The increase in molar ratio was consistent with the increased % of HMW2 measured by SEC and the decreased % of LMW1 measured by SEC (see Figures 8B and 8C). HMW2 impurities contained long heavy chain homodimers and LMW1 impurities contained short heavy chain homodimers. Slightly lower titers were seen at 32.5°C for both clones on day 2.

[0177] As seen in experiments 1 and 2, the molar ratio of long to short heavy chains in bispecific 2 was also found to be affected by incubation temperature. The increase in the molar ratio of long to short heavy chains was consistent with the formation of increased impurities containing long heavy chains at lower temperatures.

[0178] As demonstrated in the experiments, the expression of long heavy chains of asymmetric multispecific proteins was affected by temperature, as indicated by the increased molar ratio of long to short heavy chain expression at lower temperatures. This change in ratio affected the formation of product-related impurities and the difficulty of separating them from the desired product. Impurities containing long heavy chains proved surprisingly difficult to remove in downstream purification steps compared to impurities containing short heavy chains. Using temperature as a means to balance the expression of long and short heavy chains positively impacts the purification, activity, and product quality of asymmetric proteins.

[0179] Example 4 These experiments provide a method to control cell growth on nanofluidic chips by temperature shift to extend the culture period and the measurement of protein secretion levels, and to minimize the risk of cell cross-contamination during the unloading procedure performed under low temperature settings. Single cells expressing either monoclonal or multispecific antibodies were placed on the nanofluidic chips and subjected to a constant temperature culture mode, maintaining the same temperature for the duration of the culture, or a biphasic culture mode, applying a temperature shift (reducing the temperature) after an initial incubation at the first temperature condition. The temperature reduction resulted in growth inhibition, preventing cell overgrowth, and thus altering the recombinant protein production profile, while reducing the cell cross-contamination risk and allowing long-term culture. The effects of constant and shift temperatures on recombinant protein productivity were analyzed and used to aid early clone selection.

[0180] CHO cell lines were generated by transfecting CHO hosts with plasmid DNA encoding monoclonal or multispecific antibodies. After transfection, stable expressing pooled populations were generated through repeated passaging in selective medium that also applied MSX selection stringency until the cells reached >90% viability and maintained consistent doubling times.

[0181] Throughout this process, cells were cultured in either T-175 flasks (Corning, Corning, NY) or 50 mL spin tubes (TPP, Trazerdingen, Switzerland) in selective growth medium with MSX selection stringency at 36° C., 5% CO2 and 85% humidity. Cells were maintained by passaging multiple times per week at the target seeding density.

[0182] Steady-state single temperature culture (control) CHO cell lines were single cell loaded onto OptoSelect™ chips (Design1750, Berkeley Light, Emeryville, CA) using a Beacon Instrument (Berkeley Light, Emeryville, CA). Scripts for OptoElectroPositioning (OEP™), local electric field gradient settings, and cell loading and unloading were provided by the manufacturer. Cells were cultured on the OptoSelect™ chips for up to 6 days at 36°C using proprietary growth media and the manufacturer's recommended settings. Repetitive imaging and cell counting were performed using the Beacon Instrument's integrated 4X microscope and camera. Evidence of clonal origin was confirmed using a NanoPen™ chamber (0.0481 mm 2Images of single cells within a 100-μm (30-μm) area, 1.70 nL volume were obtained. Secretion assays were performed using the Spotlight™ Human Fc Assay (Berkeley Light, Emeryville, CA) using the supplied method script and assay analyzer software (Cell Analysis Suite, build 30552). Selected pens were used to eject cells from the chamber using the OEP™ setting, followed by flushing the tip and harvesting into 96-well microtiter plates pre-filled with proprietary single-cell cloning medium. Clonal derived cell lines were harvested after ejection via culture in 96-well plates (Corning, Corning, NY) maintained at 36° C., 5% CO2, and 85% relative humidity. Cells were monitored for proliferation by repeated imaging using a CellMetric imaging system.

[0183] Stationary single-temperature culture with temperature shift during unloading procedure CHO cell lines were single cell loaded onto OptoSelect™ chips (Design1750, Berkeley Light, Emeryville, CA) using a Beacon Instrument (Berkeley Light, Emeryville, CA). OptoElectroPositioning settings and scripts for loading and unloading cells were provided by the manufacturer. Cells were cultured on the OptoSelect™ chips at 36°C using proprietary growth medium and the manufacturer's recommended settings for up to 4 days. On day 4 during the unloading procedure, the temperature was shifted to 32°C and maintained until the end of the unloading procedure. Stationary culture at 36°C was maintained throughout the 4-day culture period. The script for unloading was provided by the manufacturer and modified such that the unloading procedure was performed with a temperature shift setting of 32°C. Repetitive imaging and cell counting were performed using the Beacon Instrument's integrated 4X microscope and camera. Evidence of clonal origin was obtained using a NanoPen™ chamber (0.0481 mm 2Images of single cells within a 1.70 nL volume (area, 1.70 nL volume) were obtained. Secretion assays were performed using the Spotlight™ Human Fc Assay (Berkeley Light, Emeryville, CA) using the supplied method script and assay analyzer software (Cell Analysis Suite, build 30552). Selected chambers were pen-ejected with cells using the OEP™ setting, followed by flushing the tip and collecting cells into 96-well microtiter plates pre-filled with proprietary single cell cloning medium.

[0184] Temperature shift incubation CHO cell lines were single cell loaded onto OptoSelect™ chips (Design1750, Berkeley Light, Emeryville, CA) using a Beacon Instrument (Berkeley Light, Emeryville, CA). OptoElectroPositioning settings and scripts for loading and unloading cells were provided by the manufacturer. Cells were cultured on OptoSelect™ chips at 36°C for up to 3, 4 or 5 days (as indicated) using proprietary growth medium and manufacturer's recommended settings. A temperature shift to 32°C or 32.5°C was applied on days 3, 4 or 5, and cultures were maintained at the lower temperature until days 7 or 8. Scripts for unloading were provided by the manufacturer, and unloading procedures were performed with either the manufacturer's recommended settings or the 32°C temperature shift settings. Repetitive imaging and cell counting were performed using the Beacon Instrument's integrated 4X microscope and camera. Evidence of clonal origin was obtained using a NanoPen™ chamber (0.0481 mm 2Images of single cells within a 100-well plate (1.70 nL volume, 1.70 nL area) were obtained. Secretion assays were performed using the Spotlight™ Human Fc Assay (Berkeley Light, Emeryville, CA) using the supplied method script and assay analyzer software (Cell Analysis Suite, build 30552). Selected chambers were then stained with OEP™ to remove cells from the chamber, followed by flushing the chip and collecting cells into 96-well microtiter plates pre-filled with proprietary single cell cloning medium.

[0185] result FIG. 9 shows that temperature shifts implemented during on-chip culture result in growth inhibition and enable long-term culture. (A) Representative brightfield NanoPen™ chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 6 days. Cell counts generated by the instrument software are displayed below each time point. The cell population on day 6 occupied the majority of the chamber contents and grew close to the neck region of the chamber, preventing reliable secretion assessment by the Spotlight™ Human Fc Assay and creating a high risk of cross-contamination in the expected unloading procedure. (B) Representative brightfield chamber images collected throughout the experimental period on a Berkeley Light Bacon platform where single cells expressing monoclonal antibodies were loaded into individual chambers on the chip and the chip was cultured at 36° C. for 3 days and then the temperature was reduced to 32° C. for the remainder of the experimental period. Cell counts generated by the instrument software are displayed below each time point. The temperature shift implemented from day 4 resulted in growth inhibition and prevented cells from growing beyond the maximum recommended height of the chamber. (C) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period. The implementation of the temperature shift from day 4 resulted in growth inhibition as manifested by an increase in mean doubling time and a decrease in mean cell number when compared to the control condition.

[0186] Figure 10 shows that performing a temperature shift during a standard cell line development (CLD) workflow on the Berkeley Light Beacon platform alters monoclonal antibody production profiles. (A) Schematic of the standard cell line development cloning workflow on the Beacon platform (left panel) and the cell line development cloning workflow with temperature shift performed in B and C (right panel). (B) Normalized secretion score data corresponding to Spotlight™ Human Fc Assay performed before temperature shift on day 4 (left panel) and after temperature shift on day 6 (right panel). Cell secretion profiles measured after temperature shift showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines. (C) Brightfield and Spotlight™ Human Fc Assay fluorescence images collected throughout the experimental period on the Berkeley Light Bacon platform corresponding to two clonally derived cell lines expressing monoclonal antibodies that underwent a CLD workflow with temperature shift. Cell counts and normalized secretion scores generated by the instrument software are displayed below each time point. Incubation at low temperature conditions resulted in growth arrest of both clones. Both cell lines showed comparable secretion profiles on day 4 of the workflow, however clone 1, but not clone 2, showed increased secretion levels in response to the temperature shift.

[0187] Figure 11 shows that implementing a temperature shift during the CLD workflow on the Berkeley Light Beacon platform achieves growth inhibition and alters the recombinant protein production profile of cell lines expressing IgG4 monoclonal and multispecific antibody formats. (A)(D) Mean doubling time (upper panel) and mean number of cells per chamber (lower panel) measured daily throughout the experimental period for clonally derived cell lines expressing IgG4 monoclonal and multispecific antibodies, respectively. Temperature settings for each time point of the experiment are outlined at the top of the graph. Implementing a temperature shift from day 4 or day 3 resulted in growth inhibition as measured by increased doubling time and decreased cell number for both IgG4 and multispecific antibody expressing cell lines, respectively. (B)(E) Representative brightfield chamber images collected throughout the experimental period on the Berkeley Light Bacon platform of clonally derived cell lines expressing monoclonal or multispecific antibodies, respectively. Cell counts generated by the instrument software are displayed below each time point. (C)(F) Spotlight™ Human Fc Assay scoring data corresponding to time points of day 4 and day 8 or day 7, respectively. Secretion profiles achieved after extended incubation at reduced temperature showed a more diverse distribution, allowing better discrimination between highly productive clones and poorly secreting cell lines.

[0188] Figure 12 shows that the export procedure performed at a temperature of 32 °C results in similar clonal recovery when compared to export performed at 36 °C per the standard CLD workflow. (A)(B) Top panels: Schematic of the standard cell line development cloning workflow on the Beacon platform and the cell line development cloning workflow where a temperature shift is performed during the export process, respectively. Bottom panels: Representative images of the 96-well export plate acquired 18 days after the export procedure showing comparable clonal recovery efficiency regardless of the export temperature performed at the single cell cloning step.

[0189] conclusion We investigated alternative cell line development workflow conditions on the Berkeley Light technology platform to better control cell growth, minimize the risks associated with cell cross-contamination during the export procedure, achieve higher secretion levels, and improve early clone selection. We demonstrated that a temperature shift introduced after day 3 during the CLD workflow results in lower proliferation and extended cell doubling times allowing for longer culture periods. Growth inhibition at lower temperatures can be exploited during the export procedure, as dividing cells that may be present near the chamber inlet / outlet can leak into the channel area and cross-contaminate the fluidic pathway, thus compromising export purity. We provide evidence that implementing low temperatures during the export process does not affect clone recovery during clonal expansion at the 96-well plate stage. Furthermore, implementing a temperature shift allows for secretion assays to be performed at later time points to better discriminate between highly expressing clones and eliminate poorly performing cell lines. Finally, our data show that cultivation at low temperature can alter recombinant protein secretion profiles that could potentially be exploited to identify clones with unique phenotypes.

Claims

1. 1. A method for modulating the product quality of a recombinant asymmetric multispecific antibody expressed by cells in cell culture, comprising: a) establishing at least two cell cultures each inoculated with the same cell line expressing said asymmetric multispecific antibody; b) culturing at least one cell culture at a first temperature regime consisting of a single temperature and culturing at least one cell culture at a second temperature regime; c) comparing at least one product-related impurity in said cell cultures at each temperature regime; d) selecting said temperature regime that reduces the expression of at least one product-related impurity comprising an unmatched or mismatched long heavy strand; e) culturing said cell line at said selected temperature regime; f) recovering the recombinant asymmetric multispecific antibody; A method comprising:

2. The method of claim 1, wherein the first temperature regime is selected from 36°C to 37°C.

3. The method of claim 1 , wherein the second temperature regime comprises a temperature shift from a first temperature to a second temperature that is higher or lower than the first temperature.

4. The method of claim 3, wherein the second temperature is selected from 28°C to 35°C.

5. The method of claim 3, wherein the second temperature is about 1° C. to about 9° C. lower than the first temperature.

6. 2. The method of claim 1, wherein the first temperature regime is a single temperature between 36°C and 37°C, and the second temperature regime includes at least one temperature shift from a first temperature between 36°C and 37°C to a second temperature between 28°C and 35°C.

7. 2. The method of claim 1, wherein the product-related impurities result from an imbalance in the ratio of long to short heavy chains.

8. 2. The method of claim 1, wherein the product-related impurities comprise unmatched or mismatched long heavy strands.

9. 9. The method of claim 8, wherein the product-related impurities comprise unpaired or mispaired long heavy chains and are selected from homodimers, half antibodies, protein aggregates, antibody fragments, combinations of antibody fragments, and unpaired antibody fragments.

10. 2. The method of claim 1 , wherein the cell culture temperature regime can be further selected to modulate expression level, productivity, growth, yield, and / or other desired product quality attributes of the asymmetric multispecific antibody.

11. The method of claim 1 , wherein the asymmetric multispecific antibody comprises a mutein.

12. 2. The method of claim 1, wherein the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein attached to one of the two antibody heavy chains of the anti-PD-1 antibody.

13. The method of claim 7, wherein the long heavy chain comprises an anti-PD-1 antibody heavy chain having an IL-21 mutein attached thereto.

14. 1. A method for modulating the product quality of a recombinant asymmetric multispecific antibody comprising a mutein expressed by a cell in cell culture, comprising: a) establishing at least two cell cultures each inoculated with the same cell line expressing said asymmetric multispecific antibody; b) culturing at least one cell culture at a first temperature regime consisting of a single temperature and culturing at least one cell culture at a second temperature regime; c) comparing at least one product-related impurity, including unmatched or mismatched long heavy strands, produced by the cell cultures at each temperature regime; d) selecting a temperature regime that reduces the expression of at least one product-related impurity comprising an unmatched or mismatched long heavy strand; A method comprising:

15. The method of claim 14, wherein the mutein is an IL-21 mutein.

16. 16. The method of claim 15, wherein the IL-21 mutein comprises amino acid substitutions at any two of positions 5, 9, 73 and 76 of SEQ ID NO:1, wherein the amino acid substitutions are selected from A, E or Q at position 5, E or A at position 9, A or Q at position 73, and A, D or E at position 76.

17. The method of claim 15, wherein the IL-21 mutein comprises the amino acid sequence of any of SEQ ID NOs: 233-245.

18. The method of claim 14, wherein the asymmetric multispecific antibody comprises a mutein attached to the C-terminus of one of the two antibody heavy chains of an anti-PD-1 antibody.

19. The anti-PD-1 antibody, two light chains each comprising an LC CDR1, an LC CDR2 and an LC CDR3 each comprising the amino acid sequences of SEQ ID NOs: 385, 386 and 387, respectively; two heavy chains each comprising an HC CDR1, an HC CDR2 and an HC CDR3 each comprising the amino acid sequences of SEQ ID NOs: 382, ​​383 and 384, respectively; 20. The method of claim 18, comprising:

20. 19. The method of claim 18, wherein the anti-PD-1 antibody comprises two light chains comprising the amino acid sequence of SEQ ID NO:389 and two heavy chains comprising the amino acid sequence of SEQ ID NO:

388.

21. The anti-PD-1 antibody, two light chains each comprising an LC CDR1, an LC CDR2 and an LC CDR3 each comprising the amino acid sequences of SEQ ID NOs: 365, 366 and 367, respectively; two heavy chains each comprising an HC CDR1, an HC CDR2 and an HC CDR3 each comprising the amino acid sequences of SEQ ID NOs: 362, 363 and 364, respectively; 20. The method of claim 18, comprising:

22. 20. The method of claim 18, wherein the anti-PD-1 antibody comprises two light chains comprising the amino acid sequence of SEQ ID NO:369 and two heavy chains comprising the amino acid sequence of SEQ ID NO:

368.

23. The anti-PD-1 antibody, (i) two light chains, each of which comprises the amino acid sequence of SEQ ID NO: 391, a heavy chain bound to an IL-21 mutein comprising the amino acid sequence of any one of SEQ ID NOs: 501-506, and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 556-558; or (ii) two light chains, each comprising the amino acid sequence of SEQ ID NO: 371, a heavy chain bound to a single IL-21 mutein comprising the amino acid sequence of any one of SEQ ID NOs: 513-518, and a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 559-561.

20. The method of claim 18, comprising:

24. The anti-PD-1 antibody, two light chains each comprising the amino acid sequence of SEQ ID NO: 391; one heavy chain comprising the amino acid sequence of SEQ ID NO:556; one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO:501; 20. The method of claim 18, comprising:

25. The anti-PD-1 antibody, two light chains each comprising the amino acid sequence of SEQ ID NO: 371; one heavy chain comprising the amino acid sequence of SEQ ID NO:559; one heavy chain bound to an IL-21 mutein comprising the amino acid sequence of SEQ ID NO:513; 20. The method of claim 18, comprising:

26. The method of claim 14, wherein the asymmetric multispecific antibody comprises an IL-21 mutein linked to the C-terminus of one of the two antibody heavy chains of an anti-PD-1 antibody.

27. The method of claim 14, wherein the long heavy chain comprises an anti-PD-1 antibody heavy chain having an IL-21 mutein attached thereto.

28. 1. A method for regulating the product quality of a conjugate of an IL-21 mutein linked to the C-terminus of one of two antibody heavy chains of an anti-PD-1 antibody expressed by cells in cell culture, comprising: a) establishing a cell culture inoculated with an antibody-expressing cell line; b) culturing the cells at 36±1° C. for a culture period; c) recovering the antibody; Including, wherein the amount of at least one product-related impurity resulting from an imbalance in the ratio of long heavy chains to short heavy chains in the harvested cell culture is reduced compared to the amount of the same product-related impurity in a harvest from a cell culture that was exposed to a temperature of 32°C to 34°C at some point during said culturing.

29. A method for selecting a cell line expressing an asymmetric multispecific antibody comprising a mutein, comprising: a) establishing at least one clonally derived cell line expressing said antibody; b) establishing at least two cell cultures from one or more of said clonally derived cell lines, wherein at least one cell culture from each cell line is cultured at a first temperature regime and at least one cell culture from each cell line is cultured at a second temperature regime; c) comparing the expression of at least one product-related impurity, including unpaired or mismatched long heavy strands, produced by cells cultured at each temperature regime; d) selecting cell lines that result in reduced expression of at least one product-related impurity comprising an unmatched or mismatched long heavy chain; A method comprising:

30. 30. The method of claim 29, wherein the selected cell line is then cultured in a temperature regime that resulted in reduced expression of at least one product-related impurity comprising an unmatched or mismatched long heavy strand.

31. 30. The method of claim 29, wherein the first temperature regime is selected from about 36°C to about 37°C.

32. 30. The method of claim 29, wherein the second temperature regime comprises a temperature shift from a first temperature to a second temperature that is higher or lower than the first temperature.

33. 33. The method of claim 32, wherein the second temperature is selected from about 28°C to about 35°C.

34. 30. The method of claim 29, wherein the second temperature is about 1° C. to 9° C. lower than the first temperature.

35. 30. The method of claim 29, wherein the first temperature regime is a single temperature between 36°C and 37°C, and the second temperature regime includes at least one temperature shift from a first temperature between 36°C and 37°C to a second temperature between 28°C and 35°C.

36. 30. The method of claim 29, wherein the product-related impurities comprising unpaired or mismatched long heavy strands result from an imbalance in the ratio of long to short heavy strands.

37. 36. The method of claim 35, wherein the product-related impurities comprising unpaired or mismatched long heavy strands result from an increased ratio of long heavy strands to short heavy strands.

38. 30. The method of claim 29, wherein the product-related impurities comprising unpaired or mispaired long heavy chains are selected from homodimers, half antibodies, protein aggregates, antibody fragments, combinations of antibody fragments, and unpaired antibody fragments.

39. 30. The method of claim 29, wherein the cell culture temperature regime may further be selected for a temperature regime that also regulates expression level, productivity, growth and / or other desired product quality attributes of the asymmetric multispecific antibody.

40. 30. The method of claim 29, wherein the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein attached to one of the two antibody heavy chains of the anti-PD-1 antibody.

41. 30. The method of claim 29, wherein the long heavy chain comprises an anti-PD-1 antibody heavy chain having an IL-21 mutein attached thereto.

42. 1. A method for modulating the production of at least one recombinant asymmetric multispecific antibody product-related impurity comprising an antibody heavy chain having an IL-21 mutein attached thereto during cell culture by cell culture temperature, comprising: a) selecting a temperature regime that reduces the expression of at least one product-related impurity comprising a long heavy strand, an unmatched or mismatched long heavy strand; b) culturing said cell line at said selected temperature regime; c) recovering the recombinant asymmetric multispecific antibody; A method comprising:

43. 43. The method of claim 42, wherein the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein attached to one of the two antibody heavy chains of the anti-PD-1 antibody.

44. 43. The method of claim 42, wherein the long heavy chain comprises an anti-PD-1 antibody heavy chain having an IL-21 mutein attached thereto.

45. 1. A method for producing an isolated and purified recombinant asymmetric multispecific antibody, comprising: a) isolating at least one single stably transformed cell expressing an asymmetric multispecific antibody and establishing a clonally derived culture; b) establishing at least two cell cultures from one or more of said clonally derived cultures; c) culturing at least one cell culture from each clone-derived culture in a first temperature regime comprising a single temperature and culturing at least one cell culture from each clone-derived culture in a second temperature regime; d) comparing the amount of at least one product-related impurity, including unpaired or mismatched long heavy strands, produced by the cells cultured at each temperature regime; e) selecting said cell cultures that have modulated production of said product-related impurities; f) establishing cell lines expressing said asymmetric multispecific antibodies from said selected cell cultures; g) inoculating a bioreactor with said cell lines expressing said asymmetric multispecific antibodies; h) culturing said cells to express said asymmetric multispecific antibodies in said temperature regime that modulates the production of said product-related impurities; i) recovering said recombinant asymmetric multispecific antibody from said cell culture; j) processing said recombinant multispecific antibody through one or more chromatographic unit operations; k) obtaining an isolated and purified recombinant asymmetric multispecific antibody; A method comprising:

46. 46. ​​The isolated and purified recombinant multispecific antibody of claim 45.

47. 46. ​​A pharmaceutical composition comprising the isolated and purified recombinant multispecific antibody of claim 45.

48. 46. ​​The method of claim 45, wherein the asymmetric multispecific antibody is an asymmetric multispecific anti-PD-1 antibody comprising a single IL-21 mutein attached to one of the two antibody heavy chains of the anti-PD-1 antibody.

49. 49. The method of claim 48, wherein the long heavy chain comprises an anti-PD-1 antibody heavy chain having an IL-21 mutein attached thereto.

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