Expression of Multispecific Antibodies.

Bi- and multispecific antibody formats allow the development of new therapeutic strategies to address previously unmet medical needs. However, due to the increased complexity (e.g., the interface design and the presence of multiple binders), such molecules are generally more challenging to express and purify compared to standard monoclonal antibodies (mAbs). We describe here an optimized methodology to express and purify basic bispecific antibodies using the BEAT® interface. This interface allows to generate antibodies with very high levels of heterodimer product (reported titers exceed 10 g/L) and comes with a built-in purification strategy allowing removal of residual levels of undesired product-related impurities (e.g., homodimers and half molecules).

Benchtop Bioreactors in Mammalian Cell Culture: Overview and Guidelines.

Bioreactors are manufactured apparatuses that allow the generation of a specific environment for the highly controlled cultivation of living cells. Originally used for microbial production systems, they have found widespread applications in fields as diverse as vaccine production, plant cell cultivation, and the growth of human brain organoids and exist in equally diverse designs (Chu and Robinson, Curr Opin Biotechnol 12(2):180-187, 2001; Qian et al., Nat Protoc 13:565-580, 2018). Manufacturing of biologics is currently mostly performed using a stirred tank bioreactor and CHO host cells and represents the most "classical" bioreactor production process. In this chapter, we will therefore use the cultivation of suspension Chinese hamster ovary (CHO) cells for recombinant protein production in a stirred tank bioreactor as an example. However, general guidelines provided in this chapter are transferable to different bioreactor types and host cells (Li et al., MAbs 2(5):466-479, 2010).The preparation and operation of a bioreactor (also referred to as upstream process in a biotechnological/industrial setting) is comprised of three main steps: expansion (generation of biomass), production (batch, fed-batch, or continuous process), and harvest. The expansion of cells can last from few days to weeks depending on the number of cells at the start, the cellular doubling time, and the required biomass to inoculate the production bioreactor. The production phase lasts a few weeks and is a highly sensitive phase as the concentration of different chemicals and physical parameters need to be tightly controlled. Finally, the harvest will allow the separation of the product of interest from large particles and then the desired material (cell culture supernatant or cells) is transferred to the downstream process.The raw materials used during the upstream phase (all three steps) need to be aligned with the final purpose of the manufactured product, as the presence of residual impurities may have an impact on suitability of the final product for a desired purpose.

Mutations in ALDH1A3 represent a frequent cause of microphthalmia/anophthalmia in consanguineous families.

Anophthalmia or microphthalmia (A/M), characterized by absent or small eye, can be unilateral or bilateral and represent developmental anomalies due to the mutations in several genes. Recently, mutations in aldehyde dehydrogenase family 1, member A3 (ALDH1A3) also known as retinaldehyde dehydrogenase 3, have been reported to cause A/M. Here, we screened a cohort of 75 patients with A/M and showed that mutations in ALDH1A3 occurred in six families. Based on this series, we estimate that mutations in ALDH1A3 represent a major cause of A/M in consanguineous families, and may be responsible for approximately 10% of the cases. Screening of this gene should be performed in a first line of investigation, together with SOX2.