The development of most manufacturing processes for recombinant proteins such as antibodies in mammalian cells usually follows a a sehme of recombinant gene with the necessary transcriptional regulatory elements being transferred to cells, a second gene is transferred that confers to recipient cells a selective advantage so that in the presence of the selection agent only those cells that epxress the selector gene surrive, surivors are then transferred as single cells to a second cultivation vessel and the cultures are expanded to produce clonal populations. Eventually, individual clones are evaluated for recombinant protein epxression, with the highest producers being retained for further cultivation and analysis. (GE Downstream GAB ’04 abstracts, Nice, France Oct 3-5, 2004).

Monoclonal antibody production typically requires up to 9 months to optimize a mamamlian cell line. The steps may differ, but they require the introduction of at least two different gene constructs for expression into a cell, including genes encoiing the two antibdoy chains plus non-antiboic selecable markers. Following selection of expressing lines, the lines are compared for proudction of mAb productivity and often udner further selection for increased mAb production through gene duplicaiton strategies using methotrexate selection that idnetifies cells with the highest number of dihydrofolate reductase or other selectable marker gnees. The emthods require analysis of many spearate rpoduction cell lines and detailed screening for production capability as measured by amount of antibody per cell. Following selection of optimally expressing cell lines, the lines must be optimized for culture conditions, growth characteristics, buffers, nutrients and other variables. Furt, mammalina cell production requires sequential scaling of cultures form a single mater or working cell bank to subseqeuntly seed the next sequential volume production container. Each seeding process puts cultures at risk for contamination . Morton (US 15/110200). 

Isolation of Antibody Genes

A typical outline of isolating and expressing antibody genes is the following: (1) obtaining mRNA from B cells, (2) obtaining cDNA from the mRNA, (3) using a primer exentsion to amplify form this cDNA the gene repertoire corresponding to the heavy chaings and light chains and optionally constant domain of the antibodies, (4) expressing the antibody/fragment in a host cell (see cell expression), (5) identifying the antibody clone desired and 96) isolating the monoclonal antibody/fragment. (Hayday 14/368,749)

Creation of Polyclonal Antibody Cell Mixtures

Haurum (WO2004/061104) discloses a method for manufacturing a recombinant polyclonal protein composition such as a recombinant polyclonal antibody composition by collecting cells transfected with a library of variant nucleic acid sequences wherein each cell in the collection is transfected with and capable of expressing one member of the library which encodes a distinct member of a polyclonal protein that binds a particular antigen (e.g., different antibody clones differing only in the variable region) and which is located at the same single site in the genome of each individual cells in the collection. The nucelic acid sequence is introduced into cells by transfection with a library of fectors for site specific integration (e.g., using a recombinase). See vector design under “biotechnology”). 

Nielsen discloses a method for manufacturing a recombinant polyclonal protein composition such as a polyclonal antibody (composition of different antibody molecules which are capable of binding to several different specific antigenic detemrinants on the same or on different antigens) by collecting cells transfected with a distinct member of the protein which is randomly integrated into the genome of the host cells. The individual cell clones each of which produce an individual member of the polyclonal protein are then mixed in  order to generate a polyclonal manufacturing cell line for the production of a polyclonal protein.  

Creation of Antibody libraries

Bhatt (WO2007/134327) discloses that human influenza antibody libraries can be created from antibodies obtained from convalescent patients of various prior influenza outbreaks. In order to prepare such libraries, blood is collected from individuals suspected to ahve been infected with an influenza virus. Peripheral blood samples, especially grom geographically distant sources, may need to be stabilized prior to transportation and use. Kits for this purpose are well known such as for example BD Vacutainer, CPT cell prepration tube can be used for centrifugal purification of lymphocytes and guanidium Trizol, or RNAlater can be used to stabilize the samples. RT-PCR is performed to rescue heavy and light chain repertoires, using oligo primers known in the art. The PCR repertorie products are combined with linker oligos to generates scFv libraries to clone directly in frame with m13 pIII protein, following procedures known in the art. 

Rasmussen (WO2006/007850) teaches a method for manufacturing an anti-RhD recombinant polyclonal antibody by transfecting a library of cells with anti-RhD anntibody expression vectors wherein each cell line is then capable of expressing from a VH and VL comprising nucleic acid segment, one member of the library. Rasmussen (US2008/0131882) also teaches analytic techniques that can be used to obtain information with respect to the relative proportion of individual members in the samples such as restriction fragment lenght polymorphism (RFLP) analysis, microarray analysis, PCR and nucleic acid sequencing. 

Expression of Multi-subunits

Hsei disclsoes a recombinant expression method for multi-subunit antibody which includes production of subunits in separate host cells (optionaly followed by host cell secretion of subunits into the culture medium), recovery of subunits from the respective host cells and/or culture media, followed by in vitro subunit assembly to form the multi-subunit end product.

Cell Culture Conditions

Initial attempts to maximize culture longevity by feeding cultures with only a few nutrients, such as glucose and glutamine have now evolved into multifeed strategies that result in final antibody titers of 1-2 g/L. Many of these high yielding processes have explited the combined effects of manipulating boht nutrient feed composition and environmental conditions to increase culture longevity and specific secretion rates. (Bibila, Biotechnol. Prog. 1995, 11, 1-13).