Peptonics: A new family of cell-protecting surfactants for the recombinant expression of therapeutic proteins in mammalian cell cultures.

Polymer surfactants are key components of cell culture media as they prevent mechanical damage during fermentation in stirred bioreactors. Among cell-protecting surfactants, Pluronics are widely utilized in biomanufacturing to ensure high cell viability and productivity. Monodispersity of monomer sequence and length is critical for the effectiveness of Pluronics-since minor deviations can damage the cells-but is challenging to achieve due to the stochastic nature of polymerization. Responding to this challenge, this study introduces Peptonics, a novel family of peptide and peptoid surfactants whose monomer composition and sequence are designed to achieve high cell viability and productivity at a fraction of chain length and cost of Pluronics. A designed ensemble of Peptonics was initially characterized via light scattering and tensiometry to select sequences whose phase behavior and tensioactivity align with those of Pluronics. Selected sequences were evaluated as cell-protecting surfactants using Chinese hamster ovary (CHO) cells expressing therapeutic monoclonal antibodies (mAb). Peptonics IH-T1010, ih-T1010, and ih-T1020 afforded high cell density (up to 3 × 107 cells mL-1 ) and viability (up to 95% within 10 days of culture), while reducing the accumulation of ammonia (a toxic metabolite) by ≈10% compared to Pluronic F-68. Improved cell viability afforded high mAb titer (up to 5.5 mg mL-1 ) and extended the production window beyond 14 days; notably, Peptonic IH-T1020 decreased mAb fragmentation and aggregation ≈5%, and lowered the titer of host cell proteins by 16% compared to Pluronic F-68. These features can improve significantly the purification of mAbs, thus increasing their availability at a lower cost to patients.

Application of platform process development approaches to the manufacturing of Mabcalin™ bispecifics.

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.

Development of a platform-based approach for the clinical production of HIV gp120 envelope glycoprotein vaccine candidates.

Preclinical development of vaccine candidates is an important link between the discovery and manufacture of vaccines for use in human clinical trials. Here, an exploratory clinical study utilizing multiple gp120 envelope proteins as vaccine antigens was pursued, which required a harmonized platform development approach for timely and efficient manufacture of the combined HIV vaccine product. Development of cell lines, processes, and analytical methods was initiated with a transmitted founder envelope protein (CH505TF), then applied to produce three subsequent gp120 Env (envelope) variants. Cell lines were developed using the commercially available Freedom CHO DG44 kit (Life Technologies). The fed-batch cell culture production process was based on a commercially-available medium with harmonized process parameters across the variants. A platform purification process was developed utilizing a mixed mode chromatography capture step, with ceramic hydroxyapatite and ion exchange polishing steps. A suite of analytical methods was developed to establish and monitor the Quality Target Profile (QTP), release and long-term stability testing of the vaccine products. The platform development strategy was successfully implemented to produce four gp120 envelope protein variants. In some cases, minor changes to the platform were required to optimize for a particular variant; however, baseline conditions for the processes (cell line type, media & feed system, chromatography resins, and analytical approaches) remained constant, leading to successful transfer and manufacture of all four proteins in a cGMP facility. This body of work demonstrates successful pursuit of a platform development approach to manufacture important vaccine candidates and can be used as a model for other vaccine glycoproteins, such as HIV gp140 trimers or other viral glycoproteins with global health implications. Clinical trial identifier. NCT03220724, NCT03856996.

Modulation of high mannose levels in N-linked glycosylation through cell culture process conditions to increase antibody-dependent cell-mediated cytotoxicity activity for an antibody biosimilar.

The regulatory approval of a biosimilar product is contingent on the favorable comparability of its safety and efficacy to that of the innovator product. As such, it is important to match the critical quality attributes of the biosimilar product to that of the innovator product. The N-glycosylation profile of a monoclonal antibody (mAb) can influence effector function activities such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity. In this study, we describe efforts to modulate the high-mannose (HM) levels of a biosimilar mAb produced in a Chinese hamster ovary cell fed-batch process. Because the HM level of the mAb was observed to impact ADCC activity, it was desirable to match it to the innovator mAb's levels. Several cell culture process related factors known to modulate the HM content of N-glycosylation were investigated, including osmolality, ammonium chloride (NH4 Cl) addition, glutamine concentration, monensin addition, and the addition of alternate sugars and amino sugars to the feed medium. The process conditions evaluated varied in impact on HM levels, process performance and product quality. One condition, the addition of alternate sugars and amino sugars to feed medium, was identified as the preferred method for increasing HM levels with minimal disruptions to process performance or other product quality attributes. Interestingly, a secondary interaction between sugar and amino sugar supplemented feeds and osmolality was observed during process scale-up. These studies demonstrate sugar and amino sugar concentrations and osmolality are critical variables to evaluate to match HM content in biosimilar and their innovator mAbs.

Evolving trends in mAb production processes.

Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. The establishment of robust manufacturing platforms are key for antibody drug discovery efforts to seamlessly translate into clinical and commercial successes. Several drivers are influencing the design of mAb manufacturing processes. The advent of biosimilars is driving a desire to achieve lower cost of goods and globalize biologics manufacturing. High titers are now routinely achieved for mAbs in mammalian cell culture. These drivers have resulted in significant evolution in process platform approaches. Additionally, several new trends in bioprocessing have arisen in keeping with these needs. These include the consideration of alternative expression systems, continuous biomanufacturing and non-chromatographic separation formats. This paper discusses these drivers in the context of the kinds of changes they are driving in mAb production processes.

A comparison of protein A chromatographic stationary phases: performance characteristics for monoclonal antibody purification.

Protein A chromatography remains the dominant capture step used during the downstream purification of monoclonal antibodies (mAbs). With the recent expiry of the Repligen patent on recombinant Protein A, a variety of new Protein A resins have been introduced in the market. Given productivity limitations during downstream processing that have come into sharper focus with the recent increase in cell culture titers for mAbs, the selection of an appropriate Protein A resin has direct implications on the overall process economics of mAb production. The performance of seven different Protein A chromatographic resins was compared with respect to static binding capacity and dynamic binding capacity as a function of flow rate. This data was translated into a comparison of productivity (g mAb purified per unit resin volume per unit time) for the seven stationary phases. In addition, elution pH and host cell protein impurity levels after product capture on each of these resins were determined. The current article provides an effective methodology and dataset for the selection of the optimal Protein A chromatographic resin.

Multimodal chromatography: Characterization of protein binding and selectivity enhancement through mobile phase modulators.

The unique selectivity of mixed mode chromatography resins is driving increasing utilization of these novel selectivities into bioprocess applications. There is a need for improved fundamental understanding of protein binding to these stationary phases to enable the development of efficient and robust purification processes. A panel of four monoclonal antibodies and two model proteins were employed to characterize protein interaction with a mixed-mode chromatographic resin comprising a hydrophobic ligand with cation-exchange functionality. Binding of these proteins was studied as a function of salt concentration and pH in the presence of various mobile phase modulators. This knowledge was applied towards screening mobile phase modulators that could selectively decrease host cell protein levels during monoclonal antibody purification.

High-throughput miniaturized bioreactors for cell culture process development: reproducibility, scalability, and control.

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr™) is an automated micro-bioreactor system with miniature single-use bioreactors with a 10-15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in-line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr™ was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr™ system as a high throughput system for cell culture process development.

Strategies for improved dCO2 removal in large-scale fed-batch cultures.

Carbon dioxide buildup in large-scale reactors can be detrimental to cell growth and productivity. In case of protein X, a therapeutic glycoprotein, when cultures were scaled up from bench scale to the pilot plant, there was a 40% loss of specific productivity. The dissolved CO(2) (dCO(2)) level was 179 +/- 9 mmHg at the pilot plant scale and 68 +/- 13 mmHg at bench scale. The authors proposed a comprehensive approach to maintain dCO(2) levels between 40 and 120 mmHg throughout the 14-day fed-batch process. A cell-free experiment was used to investigate the impact of the following parameters on dCO(2) removal: (1) sparge rate, (2) agitator speed, (3) bubble size, (4) bicarbonate concentration, (5) impeller position, and (6) aeration rate at the headspace of bioreactor. dCO(2) was measured using a fiber optic based probe. dCO(2) removal rate was a strong function of sparge rate and a weak function of agitator speed. Bubble size was modulated by the presence or absence of a sparge stone (10 microm pore size, 1 cm pipe i.d.). Open pipe provided 3- to 4-fold better dCO(2) removal for the same mass transfer coefficient (k(L)a) value. A mathematical model and a bench-scale experiment indicated that the benefit of a lower level of sodium bicarbonate in the culture medium was transient for batch and fed-batch cultures. Thus, this strategy was not used at pilot scale. Decreasing top impeller position improved k(L)a of dCO(2) by 2-fold. Changing headspace aeration rate from 0.02 to 0.04 vvm had no impact on dCO(2) removal. Two pilot runs were conducted using (A) open pipe and (B) antifoam in the presence of sparge stone, both in conjunction with lower impeller position. The presence of antifoam may interfere in product purification; however, demonstration of antifoam removal can be difficult. Open pipe allowed an alternative to using antifoam, as foam level with open pipe was significantly less. Both strategies successfully reduced dCO(2) level by 2.5-fold (179 +/- 9 vs 72 +/- 9 mmHg). Titer at day 10 of culture improved by 1.5-fold. Specific productivity improved by 41%. Historically, cultures were harvested around day 9-11 because of the high amount of foam; both strategies allowed the cultures to be extended up to day 14, resulting in 2-fold higher titer compared to that of the historical control without compromising protein quality.