Miniaturized cell culture system to mimic perfusion process stage

Abstract

The present disclosure is directed to a method of sub-milliliter perfusion to mimic continuous perfusion in lab-scale bioreactors comprising: establishing a cell culture in a media in at least one well of a multi-well culture plate, wherein the working volume of media is less than 1 mL, and expanding the cell culture in the multi-well culture plate.

Description

Miniaturized Cell Culture System to Mimic Perfusion Process StageFIELD

[0001] The present disclosure is directed to methods for sub-milliliter cell culture that mimic the N-l stage perfusion process or growth phase of an N-stage perfusion process. The methods allow for efficient, high-throughput testing of N-l stage or N-stage parameters.BACKGROUND

[0002] As demand for therapeutic cell products continues to grow, the need for cost- effective production and improved development of the bioproducts remains ever important. N- 1 stage perfusion is one of the key ways to improve product output for a given production process. N-l is a stage of cell growth that takes place prior to the full-scale production bioreactor. The N-l stage is used to increase the inoculation cell concentration of N-stage and thereby increase the product titer.

[0003] Presently, N-l or N-stage perfusion is typically performed with lab-scale bioreactors. Optimization requires multiple bioreactor runs, increasing the time and material costs required to develop an optimized N-l or N-stage process.

[0004] Pseudo-perfusion systems have been used in larger volume experiments to emulate the perfusion culture process performed by the lab-scale bioreactor. Pseudo-perfusion is performed in shake-flasks or microwell plates with discrete media exchange where the cell cultures are centrifuged, aspirated, and re-suspended in fresh media during the discrete media exchange steps.

[0005] As with the standard perfusion process, reductions in culture volumes allow for inherent advantages of reduced material costs and potential for increased throughput. The use of high-throughput processes offers the ability to run experiments in parallel, reducing timescales and cost. Presently, the smallest known volume of pseudo-perfusion has been performed in 24-well microwell plates with a larger working volume of 1 mL (Tregidgo et al., Biochemical Engineering Journal, 2023). -SUMMARY

[0006] In some aspects, the disclosure provides a method of sub-milliliter perfusion to mimic continuous perfusion in lab-scale bioreactors including: establishing a cell culture in a media in at least one well of a multi-well culture plate, wherein the working volume of media is less than 1 mL, and expanding the cell culture in the multi -well culture plate.

[0007] In some aspects, the method further includes maintaining a CO2 concentration of the cell culture at a range of about 1 to about 20%. In some aspects, the method further includes maintaining a CO2 concentration of the cell culture at a range of about 5 to about 10%.

[0008] In some aspects of the method, the media includes an inoculation medium and / or a perfusion medium.

[0009] In some aspects of the method, the media includes at least about 95% inoculation medium at the start of the method.

[0010] In some aspects of the method, the media includes at least about 95% of perfusion medium on a later day of the method.

[0011] In some aspects, the method further includes performing periodic media exchanges, wherein the total percentage of inoculation medium decreases by at least about 10% with every media exchange. In some aspects, the total percentage of inoculation medium decreases by at least about 25% with every media exchange.

[0012] In some aspects of the method, the media exchanges are performed until the total percentage of inoculation medium is less than about 5%.

[0013] In some aspects, the method further includes performing periodic media exchanges, wherein the ratio of inoculation medium to perfusion medium is changed from about 1 :0 to about 0: 1 during a media exchange between the start of the method and a later day of the method.- -

[0014] In some aspects, the method further includes performing periodic media exchanges, wherein the media exchanges include removing a removal volume of medium from the well and adding an addition volume of medium to the well.

[0015] In some aspects of the method, the addition volume is from about 105% to about 125% of the removal volume. In some aspects, the addition volume is from about 106% to about 110% of the removal volume.

[0016] In some aspects of the method, the periodic media exchanges are performed once per day.

[0017] In some aspects of the method, the periodic media exchanges are performed every about 6 to about 12 hours.

[0018] In some aspects of the method, the multi-well culture plate is a 96 deep well culture plate.

[0019] In some aspects, the method includes shaking the multi-well culture plate. In some aspects, the method includes shaking the multi-well culture plate at an RPM in the range of about 350 to about 400 with a “throw,” or orbit diameter or shaking diameter, in the range of about 20 to about 30 mm.

[0020] In some aspects of the method, the working volume of media is less than about 0.7 mL. In some aspects, the working volume of media is less than about 0.5 mL. In some aspects, the working volume of media is less than about 0.3 mL.

[0021] In some aspects of the method, the cell culture is established with an initial concentration in the range of about 1 x 106to about 1 * 108cells / mL.

[0022] In some aspects of the method, the glucose content of from about 2.5 to about 20 g / L during the method.

[0023] In some aspects of the method, the cell culture includes Chinese hamster ovary cells.- i -

[0024] In some aspects of the method, the cell culture is configured to provide or express a therapeutic protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1A is a plot of the viable cell concentration versus time for bisAb clonel (CHOK1SV GS-KO® clone expressing a bispecific antibody) obtained with Protocol VI, with a comparison of cells grown in 96-deep well plates (96-DWPs; squares) and a bioreactor (circles). Fig. IB is a plot of the total cell concentration versus time for bisAb clonel obtained with Protocol VI, with a comparison of cells grown in 96-DWPs (squares) and a bioreactor (circles). Experiments are as described in Example 1A.

[0026] Fig. 2A is a plot of the viable cell concentration versus time for bisAb clonel (CHOK1 SV GS-KO® clone expressing a bispecific antibody) obtained with Protocol V2, with a comparison of cells grown in 96-DWPs (squares) and a bioreactor (circles). Fig. 2B is a plot of the total cell concentration versus time for bisAb clonel obtained with Protocol V2, with a comparison of cells grown in 96-DWPs (squares) and a bioreactor (circles). Experiments are as described in Example 1A.

[0027] Fig. 3A is a bar plot of the viable cell concentration at day 8 for mAb clonel (CHOK1SV GS-KO® clone expressing a monoclonal antibody) obtained with Protocol VI, with a comparison of cells grown in 96-DWPs and a bioreactor as indicated. Fig. 3B is a plot of the total cell concentration versus time for mAb clonel obtained with Protocol VI, with a comparison of cells grown in 96-DWPs and a bioreactor as indicated. Experiments are as described in Example IB.

[0028] Fig. 4A is a bar plot of the viable cell concentration at day 8 for mAb clonel (CHOK1SV GS-KO® clone expressing a monoclonal antibody) obtained with Protocol V2, with a comparison of cells grown in 96-DWPs and a bioreactor as indicated. Fig. 4B is a plot of the total cell concentration versus time for mAb clonel obtained with Protocol V2, with a comparison of cells grown in 96-DWPs and a bioreactor as indicated. Experiments are as described in Example IB.-I-

[0029] Fig. 5A is a plot of the viable cell concentration versus time for mAb clone2 (CHOK1SV GS-KO® clone expressing a monoclonal antibody) obtained with Protocol VI, with a comparison of cells grown in 96-DWPs (squares) and a bioreactor (circles). Fig. 5B is a plot of the total cell concentration versus time for mAb clone2 obtained with Protocol VI, with a comparison of cells grown in 96-DWPs (squares) and a bioreactor (circles). Experiments are as described in Example 1C.

[0030] Fig. 6A is a plot of the viable cell concentration versus time for mAb clone 2 (CHOK1SV GS-KO*' clone expressing a monoclonal antibody) obtained with Protocol V2, with a comparison of cells grown in 96-DWPs (squares) and a bioreactor (circles). Fig. 6B is a plot of the total cell concentration versus time for mAb clone2 obtained with Protocol V2, with a comparison of cells grown in 96-DWPs (squares) and a bioreactor (circles). Experiments are as described in Example 1C.

[0031] Fig. 7A is a plot of the viable cell concentration versus time for mAb clone3 (CHOK1SV GS-KO® clone expressing a monoclonal antibody) obtained with Protocol VI, with cells grown in 96-DWPs (squares). Fig. 7B is a plot of the total cell concentration versus time for mAb clone3 obtained with Protocol VI, with cells grown in 96-DWPs (squares). Experiments are as described in Example ID.DETAILED DESCRIPTION

[0032] The present disclosure is directed to methods of small volume cell culture that mimic continuous perfusion in laboratory scale bioreactors. In aspects, the methods allow for small volume mimics of the N-l perfusion stage. In embodiments, the methods allow for multi - well small volume models to be used to optimize N-l perfusion stage parameters on a high throughput level with greatly reduced costs. The methods allow for a more efficient and cost- effective way to determine N-l transfer criteria compared to the use of standard N-l perfusion bioreactors and for a similarly beneficial characterization of the initial growth phase of an N stage perfusion process.

[0033] The present disclosure is directed to a method of sub -milliliter cell culture to mimic continuous perfusion in lab-scale bioreactors comprising:- i -establishing a cell culture in a media in at least one well of a multi -well culture plate, wherein the working volume of media is less than 1 mL, and expanding the cell culture in the multi-well culture plate.

[0034] In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at a range of about 1 to about 20%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at a range of about 5 to about 10%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at a range of about 2 to about 18%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at a range of about 5 to about 15%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at a range of about 7 to about 13%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at a range of about 8 to about 12%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at about 5%, 10% or 15%. In aspects, the method further comprises maintaining a CO2 concentration of the cell culture at about 8%, 10% or 12%.

[0035] In aspects of the method the media comprises an inoculation medium and / or a perfusion medium. In aspects, the cell culture is started in an inoculation medium, at day 0. In aspects, the osmolality of the inoculation medium is about 286 to about 307 mOsmol / kg H2O.

[0036] In aspects, subsequent media exchanges are performed following day 0 of the cell culture. In aspects, the media exchanges are performed by adding a mixture of inoculation medium and perfusion medium. In aspects, the media exchanges are performed by adding only perfusion medium. In aspects, the osmolality of the perfusion medium is about 316 to about 474 mOsmol / kg H2O.

[0037] In aspects, the media comprises at least about 95% inoculation medium at the start of the method (day 0). In aspects, the media comprises at least about 90% inoculation medium at the start of the method (day 0). In aspects, the media comprises at least about 93% inoculation medium at the start of the method (day 0). In aspects, the media comprises at least about 97%inoculation medium at the start of the method (day 0). In aspects, the media comprises at least about 99% inoculation medium at the start of the method (day 0).

[0038] In aspects, the media added in a media exchange is a mixture of inoculation medium and perfusion medium. In aspects the media added in a media exchange is 0.75:0.25 inoculation medium to perfusion medium. In aspects the media added in a media exchange is 0.5:0.5 inoculation medium to perfusion medium. In aspects the media added in a media exchange is 0.25:0.75 inoculation medium to perfusion medium. In aspects the media added in a media exchange is 0: 1 inoculation medium to perfusion medium, i.e., only perfusion medium.

[0039] In aspects, the media added in media exchanges has an increased ratio of perfusion medium with subsequent exchanges. In one aspect, the media added in a media exchange is 0.75:0.25 inoculation medium to perfusion medium on day 1; 0.5:0.5 inoculation medium to perfusion medium on day 2; 0.25:0.75 inoculation medium to perfusion medium on day 3; and 0: 1 inoculation medium to perfusion medium on day 4. In aspects of this step-wise method, further media exchanges after day 4 are with 0: 1 inoculation medium to perfusion medium.

[0040] In other aspects, the media added in media exchanges is only perfusion medium. In one aspect, the cell culture is started at day 0 in only inoculation medium and on day 1, only perfusion medium is added (0: 1 inoculation medium to perfusion medium). In aspects of this method, only perfusion medium is added on subsequent days 2, 3, 4, 5 and so on as needed.

[0041] In aspects, the media comprises at least about 95% of perfusion medium on a later day of the method, e g., a day later than day 0. In aspects, the media comprises at least about 90% of perfusion medium on a later day of the method. In aspects, the media comprises at least about 70% of perfusion medium on a later day of the method. In aspects, the media comprises at least about 60% of perfusion medium on a later day of the method. In aspects, the media comprises at least about 50% of perfusion medium on a later day of the method.

[0042] In aspects, the media comprises at least about 95% of perfusion medium at the conclusion of the method. In aspects, the media comprises at least about 90% of perfusion medium at the conclusion of the method. In aspects, the media comprises at least about 70% of perfusion medium at the conclusion of the method. In aspects, the media comprises at leastabout 60% of perfusion medium at the conclusion of the method. In aspects, the media comprises at least about 50% of perfusion medium at the conclusion of the method.

[0043] In aspects, where media exchanges are performed, the total percentage of inoculation medium decreases by at least about 10% with every media exchange. In aspects, the total percentage of inoculation medium decreases by at least about 5% with every media exchange. In aspects, the total percentage of inoculation medium decreases by at least about 15% with every media exchange. In aspects, the total percentage of inoculation medium decreases by at least about 20% with every media exchange. In aspects, the total percentage of inoculation medium decreases by at least about 25% with every media exchange. In these aspects, a decrease in the total percentage of inoculation medium in the cell culture media refers to the change in the percentage of inoculation medium in the cell culture media before and after the media exchange. For example - if the total percentage of inoculation medium in the cell culture media is 90% and it decreases 10%, then the total percentage of inoculation medium in the cell culture media becomes 80%.

[0044] In aspects, media exchanges are performed until the total percentage of inoculation medium is less than about 5%. In aspects, media exchanges are performed until the total percentage of inoculation medium is less than about 10%. In aspects, media exchanges are performed until the total percentage of inoculation medium is less than about 15%. In aspects, media exchanges are performed until the total percentage of inoculation medium is less than about 20%. In aspects, media exchanges are performed until the total percentage of inoculation medium is less than about 25%.

[0045] In aspects, media exchanges are performed where the ratio of inoculation medium to perfusion medium is changed from about 1 :0 to about 0: 1 during a media exchange between the start (day 0) and a later day of the method.

[0046] In aspects, media exchanges are performed by removing a removal volume of media from the well of the cell culture plate and adding an addition volume of media to the well. In aspects, the addition volume is greater than the removal volume. In aspect, the addition volume is greater than the removal volume in order to account for evaporation and condensation on the sides of the well and the plate lid. In aspects, the addition volume is from about 105% toabout 125% of the removal volume. Tn aspects, the addition volume is from about 105% to about 150% of the removal volume. In aspects, the addition volume is from about 105% to about 130% of the removal volume. In aspects, the addition volume is from about 106% to about 110% of the removal volume. In aspects, the addition volume is about 101%, 102%,105%, 110%, 112%, 115%, 118%, 120%, 122%, 125%, 127% or 130% of the removal volume.

[0047] In aspects of the method, the periodic media exchanges are performed once per day. In aspects, the periodic media exchanges are performed once every two days. In aspects, the periodic media exchanges are performed once every three days. In aspects, the periodic media exchanges are performed twice a day. In aspects, the periodic media exchanges are performed three times a day. In aspects, the periodic media exchanges are performed four times a day.

[0048] In aspects, the periodic media exchanges are performed every about 6 to about 12 hours. In aspects, the periodic media exchanges are performed every about 2 to about 18 hours. In aspects, the periodic media exchanges are performed every about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours. In aspects, the periodic media exchanges are performed every about 12 hours.

[0049] In aspects, the periodic media exchanges are performed by centrifuging the multi well plate to form a cell pellet and supernatant, removing the supernatant and resuspending the cell pellet in fresh medium. In embodiments, the supernatant is analyzed for metabolites of interest or for proteins of interest, such as a therapeutic protein being produced by the cells in the culture.

[0050] In aspects of the method, the cell culture is inoculated with a seeding cell density of about 5>< 106cells / mL. In aspects, the cell culture is inoculated with a seeding cell density of about I x lO6cells / mL to about l x l08cells / mL. In aspects, the cell culture is inoculated with a seeding cell density of about 1x106cells / mL to about 1x107cells / mL. In aspects, the cell culture is inoculated with a seeding cell density of about I x lO6cells / mL to about 5x l07cells / mL. In aspects, the cell culture is inoculated with a seeding cell density of about 3 * 106cells / mL to about 7x l06cells / mL. In aspects, the cell culture is inoculated with a seeding cell density ofabout 1 * 106cells / mL, 2* 106cells / mL, 3* 106cells / mL, 4* 106cells / mL, 5* 106cells / mL, 6x l06cells / mL, 7x l06cells / mL, 8x l06cells / mL, 9x l06cells / mL or IQx lO6cells / mL.

[0051] In aspects of the method, the multi-well culture plate is a 96 deep well culture plate. In aspects, the multi-well culture plate is a 96 well culture plate. In aspects, the multiwell culture plate is a 24 well culture plate. In aspects, the multi-well culture plate is a 12 well culture plate. In aspects, the multi-well culture plate is a 6 well culture plate.

[0052] In aspects, the method comprises shaking the multi-well culture plate. In aspects, the shaking is performed using an orbital shaker. In aspects, the shaking is performed using a microplate orbital shaker.

[0053] In aspects, the method comprises shaking the multi-well culture plate at an RPM in the range of about 350 to about 400. In aspects, the method comprises shaking the multi -well culture plate at an RPM in the range of about 300 to about 400. In aspects, the method comprises shaking the multi-well culture plate at an RPM in the range of about 300 to about 500. In aspects, the method comprises shaking the multi-well culture plate at an RPM in the range of about 350 to about 450. In aspects, the method comprises shaking the multi -well culture plate at an RPM in the range of about 350 to about 500. In aspects, the method comprises shaking the multi-well culture plate at an RPM of about 350, 375, 400, 425 or 450.

[0054] In aspects, the shaking of the multi well plate is maintained throughout the entire method. In aspects, the shaking of the multi well plate is periodic during the entire method. In aspects, the RPM of the shaking of the multi well plate is the same for the entire method. In aspects, the RPM of the shaking of the multi well plate is varied throughout the method.

[0055] In aspects, the method comprises shaking the multi-well culture plate with a throw in the range of about 20 to about 30 mm. In aspects, the method comprises shaking the multiwell culture plate with a throw in the range of about 10 to about 50 mm. In aspects, the method comprises shaking the multi-well culture plate with a throw in the range of about 20 to about 40 mm. In aspects, the method comprises shaking the multi-well culture plate with a throw of about 10, 20, 30, 40 or 50 mm.

[0056] In aspects, the method comprises shaking the multi-well culture plate at an RPM in the range of about 350 to about 400 with a throw in the range of about 20 to about 30 mm.

[0057] In aspects, the temperature of the cell culture is an optimal temperature for the purpose of the cells being cultured. In aspects, the temperature of the cell culture is about 37 °C. In aspects, the temperature of the cell culture is about 30 °C. In aspects, the temperature of the cell culture is about 35 °C. In aspects, the temperature of the cell culture is about 36 °C. In aspects, the temperature of the cell culture is about 38 °C.

[0058] In aspects, the working volume of media of the cell culture is less than about 0.7 mL. In aspects, the working volume of media is less than about 0.9 mL. In aspects, the working volume of media is less than about 0.8 mL. In aspects, the working volume of media is less than about 0.6 mL. In aspects, the working volume of media is less than about 0.5 mL. In aspects, the working volume of media is less than about 0.4 mL. In aspects, the working volume of media is less than about 0.3 mL. In aspects, the working volume of media is less than about 0.2 mL.

[0059] In aspects, the cell culture media comprises a glucose content of from about 2.5 to about 20 g / L during the method. In aspects, the cell culture media comprises a glucose content of from about 5 to about 15 g / L during the method. In aspects, the cell culture media comprises a glucose content of from about 8 to about 12 g / L during the method. In aspects, the cell culture media comprises a glucose content of about 10 g / L during the method. In aspects, the cell culture media comprises a glucose content of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 g / L during the method.

[0060] In aspects, the cell culture is supplemented with 2.5 g / L of glucose during the method. In aspects, the cell culture is supplemented with 5.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 2.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 3.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 4.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 6.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 7.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 8.0 g / L of glucose during the method. In aspects, the cell culture issupplemented with 9.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 10.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 11.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 12.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 13.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 14.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 15.0 g / L of glucose during the method. In aspects, the cell culture is supplemented with 2.5 g / L of glucose on day 3 and 5.0 g / L glucose on day 5 of the method.

[0061] In aspects, the cell culture comprises Chinese hamster ovary cells. In aspects, the cell culture comprises NS0 cells. In aspects, the cell culture comprises human embryonic kidney cells (such as HEK-293), human embryonic retinal cells (such as Per.C6 cells) or human amniocyte cells. In aspects the cell culture comprises Hela, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC 12, PER.C6, BHK (baby hamster kidney cell), VERO, SP2 / 0, NS0, YB2 / 0, Y0, EB66, C127, L cell, COS, COS1, COS7, QC1-3, CHOK1, CHOK1SV, CHO GS knockout, CHOK1SV GS-KO, CHOS, CHO DG44, CHO DXB11, or CHOZN cell.

[0062] In aspect, the cells in the cell culture are engineered to express a therapeutic protein. In aspects, the therapeutic protein is a therapeutic antibody or a binding molecule having antibody binding domains or a bispecific antibody. In aspects, the therapeutic protein is insulin. In aspects, the therapeutic protein is a hormone.EXAMPLES

[0063] Having now generally described the above aspects, the same will be more readily understood through reference to the following examples. The following examples illustrate various methods for compositions in the diagnostic or treatment methods of the disclosure. The examples are intended to illustrate, but in no way limit, the scope of the appended claims.Example 1- Performance comparison between 96-Deep Well Plate and Bioreactor cultures

[0064] Tests were performed to determine the performance of cells grown in a 96-Deep Well Plate (96-DWP) at reduced volumes compared with the same cells grown in a lab-scale4 -perfusion bioreactor. Suspension Chinese Hamster Ovary (CHO) cells expressing recombinant or bispecific antibodies were revived and sub-cultured in CM76 medium (proprietary medium formulation, Lonza AG, CH). Inoculation medium was CM157 and perfusion medium was either CM158 or a combination of CM157 and CM158. Both CM157 and CM158 are proprietary media (Lonza AG, CH). Pre-sterilized polypropylene 96-DWP (Thermo Fisher Scientific Inc, USA) were used with maximum total volume of 2000 pL and with a Deutz lid (Kuhner Shaker AG, CH). The bioreactor was a 3 L stirred tank perfusion bioreactor.

[0065] The cells were grown in a bioreactor over an 8 day period. Lab scale stirred tank bioreactors of working volume of either 2 L or 3 L were used. They were seeded at a cell concentration of 0.5 x 106cells / mL in inoculation medium (CM157). After 72 hours of batch operation the cultures were shifted to perfusion mode wherein the perfusion medium (CM158) was continuously added and spent medium was removed via a hollow fiber filter of 0.2 pm pore size (XCell ATF2, Repligen Inc, USA). The temperature was maintained at 36.5 °C. The pH was maintained at 6.9 using CO2-bicarbonate buffer system. Glucose was monitored and was supplemented as continuous feed throughout culture to maintain at level of > 3 g / L. At Day 3, the bioreactors exhibited total and viable cell concentrations around 5* 106cells / mL. Thus, cells were seeded at 5x l06cells / mL in 96-DWP, and grown over a 5 day period according to protocols VI and V2. The 96-DWP cultures were maintained with a working volume of 300 pL with an 8% CO2, 80% humidity, and 37 °C. The 96-DWPs were agitated at 375 RPM and a throw of 25 mm.

[0066] A discrete perfusion process was used to culture the cells in the 96-DWP as the plates are not suitable for standard perfusion methods. Media exchanges were performed periodically by centrifuging the 96-DWP. Supernatant was removed, and cell pellets were resuspended in fresh media. Two media exchange protocols, VI and V2, were studied. Protocol VI involved exclusively using inoculation medium to begin the method, maintaining use of exclusively inoculation medium, switching to exclusively using perfusion medium at a later day during the method, and then maintaining use of exclusively perfusion medium for the rest of the method. In other words, the ratio of inoculation medium to perfusion medium was changed from about 1 :0 to about 0: 1 during a media exchange after the start of the method and- -maintained thereafter. Specifically, for protocol VI, the ratio was changed upon the first media exchange following the initial seeding. Protocol V2 involved exclusively using inoculation medium to begin the method, and replacing a portion of the inoculation medium with perfusion medium in stepwise fashion. Specifically, the total percentage of inoculation medium was decreased by about 25% with every media exchange, and the total percentage of perfusion medium was increased by about 25% with every media exchange. Both Protocol VI and V2 involved media exchanges performed once per day.

[0067] Cell culture that was not removed from the bioreactor for seeding the 96-DWPs was cultured in the bioreactor under the initial conditions to the end of the 8 day period.

[0068] The viable cell concentration and total cell concentration were tracked with cell counts carried out in an automated cell counter Vi-Cell XR (Beckman Coulter, Inc, USA). On day 8, both the bioreactor and 96-DWP reached total and viable cell concentrations suitable for transfer as shown below.Example 1A: bisAb clonel

[0069] The aforementioned experiment was performed with bisAb clonel clones using both Protocol VI and Protocol V2. For Protocol VI : A plot of the viable cell concentration versus time is shown in Fig. 1A, and a plot of the total cell concentration versus time is shown in Fig. IB. For Protocol V2: A plot of the viable cell concentration versus time is shown in Fig. 2A, and a plot of the total cell concentration versus time is shown in Fig. 2B. The data for the 96-Deep Well Plates (squares in all figures) are shifted along the X-axis by 3 days to align with the bioreactor data (circles in all figures) as cells were seeded in the 96-DWP at 5* 106cells / mL which equates to the concentration of day 3 of the cells in the bioreactor.

[0070] As can be seen in Figs. 1 and 2, the cells grown in the 96-DWP according to the above method approximately matched the transfer criteria of bioreactor grown cells by reaching similar total and viable cell concentrations. Furthermore, both protocols VI and V2 in the 96- DWP achieved similar performance to the bioreactor.Example IB: mAb clonel

[0071] The aforementioned experiment was performed with mAh clone 1 clones using both Protocol VI and Protocol V2. For Protocol VI : A bar plot of the viable cell concentration at day 8 is shown in Fig. 3A, and a bar plot of the total cell concentration at day 8 is shown in Fig. 3B. The measured cell viability was 98.9% for the bioreactor and 93.3% for the 96-DWP. Average live cell diameter was 18.3 um for the bioreactor and 17.8 um for the 96-DWP. For Protocol V2: A bar plot of the viable cell concentration at day 8 is shown in Fig. 4A, and a bar plot of the total cell concentration at day 8 is shown in Fig. 4B. The measured cell viability was 98.9% for the bioreactor and 93.2% for the 96-DWP. Average live cell diameter was 18.3 um for the bioreactor and 17.8 um for the 96-DWP.

[0072] For this experiment, the pre-sterilized polypropylene 96-DWP (Thermo Fisher Scientific Inc, USA) were used with a gas permeable rayon membrane in place of the Deutz lid.

[0073] As can be seen in Figs. 3 and 4, the cells grown in the 96-DWP according to the above method and protocol V2 approximately matched the transfer criteria of bioreactor grown cells by reaching similar total and viable cell concentrations. Cells grown under these conditions according to protocol VI had reduced performance compared to the bioreactor. This suggests that the clone is sensitive to drastic changes in osmolality in VI and hence gradual increase in osmolality seems to support normal growth.Example 1C: mAb clone2

[0074] The aforementioned experiment was performed with mAb clone2 clones using both Protocol VI and Protocol V2. For Protocol VI : A plot of the viable cell concentration versus time is shown in Fig. 5A, and a plot of the total cell concentration versus time is shown in Fig. 5B. For Protocol V2: A plot of the viable cell concentration versus time is shown in Fig. 6A, and a plot of the total cell concentration versus time is shown in Fig. 6B. As in Example 1 A, the data for the 96-Deep Well Plates (squares in all figures) are shifted along the X-axis by 3 days to align with the bioreactor data (circles in all figures).

[0075] As can be seen in Figs. 5 and 6, the cells grown in the 96-DWP according to the above method approximately matched the transfer criteria of bioreactor grown cells by reaching- ii -similar total and viable cell concentrations. Furthermore, both protocols VI and V2 in the 96- DWP achieved similar performance to the bioreactor.Example ID: mAb clone3

[0076] The aforementioned experiment was performed with mAb clone3 clones. This experiment was conducted only with Protocol VI. This experiment was conducted to show if the DWP model would predict data for clones whose behavior was not known in the bioreactor. A plot of the viable cell concentration versus time is shown in Fig. 7A, and a plot of the total cell concentration versus time is shown in Fig. 7B. As in Examples 1 A and 1C, the data for the 96-Deep Well Plates (squares) are shifted along the X-axis by 3 days. The data may be plotted against mAb clone2 performance in the bioreactor.

[0077] As can be seen in Figs. 7A and 7B, the cells grown in the 96-DWP according to the above method approximately matched the transfer criteria of bioreactor grown mAb clone2 cells by reaching similar total and viable cell concentrations.

[0078] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While specific aspects are described herein, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the aspects.

Claims

WHAT IS CLAIMED IS:

1. A method of sub-milliliter perfusion to mimic continuous perfusion in lab-scale bioreactors comprising: establishing a cell culture in a media in at least one well of a multi-well culture plate, wherein the working volume of media is less than 1 mL, and expanding the cell culture in the multi-well culture plate.

2. The method of claim 1, further comprising maintaining a CO2 concentration of the cell culture at a range of about 1 to about 20%.

3. The method of claim 1, further comprising maintaining a CO2 concentration of the cell culture at a range of about 5 to about 10%.

4. The method of any of claims 1 to 3, wherein the media comprises an inoculation medium and / or a perfusion medium.

5. The method of claim 4, wherein the media comprises at least about 95% inoculation medium at the start of the method.

6. The method of claim 4, wherein the media comprises at least about 95% of perfusion medium on a later day of the method.

7. The method of claim 4, further comprising performing periodic media exchanges, wherein the total percentage of inoculation medium decreases by at least about 10% with every media exchange.

8. The method of claim 4, further comprising performing periodic media exchanges, wherein the total percentage of inoculation medium decreases by at least about 25% with every media exchange.

9. The method of any of claims 1-8, wherein the media exchanges are performed until the total percentage of inoculation medium is less than about 5%.- -10. The method of any of claims 1-3, further comprising performing periodic media exchanges, wherein the ratio of inoculation medium to perfusion medium is changed from about 1 :0 to about 0: 1 during a media exchange between the start of the method and the later day of the method.

11. The method of any of claims 1-3, further comprising performing periodic media exchanges, wherein the media exchanges comprise removing a removal volume of medium from the well and adding an addition volume of medium to the well.

12. The method of claim 11, wherein the addition volume is from about 105% to about 125% of the removal volume.

13. The method of claim 11, wherein the addition volume is from about 106% to about 110% of the removal volume.

14. The method of any of claims 7 to 13, wherein the periodic media exchanges are performed once per day.

15. The method of any of claims 7 to 13, wherein the periodic media exchanges are performed every about 6 to about 12 hours.

16. The method of any preceding claim, wherein the multi -well culture plate is a 96 deep well culture plate.

17. The method of any preceding claim, wherein the method comprises shaking the multiwell culture plate.

18. The method of claim 17, wherein the method comprises shaking the multi-well culture plate at an RPM in the range of about 350 to about 400 with a throw in the range of about 20 to about 30mm.

19. The method of any preceding claim, wherein the working volume of media is less than about 0.7 mL.

20. The method of any preceding claim, wherein the working volume of media is less than about 0.5 mL.

21. The method of any preceding claim, wherein the working volume of media is less than about 0.3 mL.

22. The method of any preceding claim, wherein the cell culture is established with an initial concentration in the range of about 1 x 106to about 1 x 108cells / mL.

23. The method of any preceding claim, wherein the glucose content of from about 2.5 to about 20 g / L during the method.

24. The method of any preceding claim, wherein the cell culture comprises Chinese hamster ovary cells.

25. The method of any preceding claim, wherein the cell culture is configured to provide / express a therapeutic protein.

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