A novel measuring chamber and automation platform for mammalian cell culture processes

One of the biggest challenges of biotechnological production is the implementation of Process Analytical Technology (PAT) to obtain a highly automated, monitored and controlled process. Therefore the integration of suitable analytical sensors has to be faced at the earliest opportunity during process development. But sensor implementation for glass or disposable reactors encounters different issues. These systems are not designed to support well-established and standardized analytic ports for more complex probes used for stainless steel reactors. The here presented measuring chamber, combined with an automation platform enable an accelerated integration of PAT during all stages of process development for any reactor type

Hemagglutination inhibition assay.

<p>(A) Schematic representation of hemagglutination using Influenza viral particles. In the absence of agglutinationon inhibitors, the hemagglutinin from viral capsids (1) agglutinates chicken, turkey or human erythrocytes(2). (B) Schematic representation of hemagglutination inhibition. In the presence of neutralizing antibodies (1) that specifically recognize the hemagglutinin from a influenza virus (2), the process of hemagglutination is inhibited proportionally to the concentration and binding affinity of the neutralizing antibodies.</p

Validation of sensitivity against an HI assay.

<p>Normalized absorbance values for fourteen samples with positive anti H1N1/2009 titters based on an HI assay (samples that inhibited hemagglutination of turkey erythrocytes by the Ca/2009/H1N1 influenza virus strain at dilutions equal or higher to 1∶40). Colors indicate HI titter: HI titter = 40 (in blue); HI titter = 80 (in yellow); HI titter = 160 (in orange); HI titter>320 (in red). The proposed positive threshold for the ELISA method is indicated with a solid line (value = 1). One standard deviation is indicated with a dashed line (value = 1.25).</p

Normalized absorbance signals of serum samples from health care and diagnostic personnel in high exposure risk to the Influenza A/H1N1/2009 virus.

<p>Bars 1–22 present signals corresponding to asymptomatic health care workers. Bars 24–29 present signals corresponding to samples from H1N1 molecular diagnostic personnel. Bar 23 (negative reference) illustrates the average and standard deviation of eight samples from non-exposed subjects.</p

Indirect evaluation of proper refolding.

<p>(A) Biorecognition of antibodies from a positive patient observed for different production batches of protein HA_50–274-H1N1. (B) Specific biorecognition ratio (ratio of biorecognition of antibodies from a positive patient serum and a negative subject serum) observed at different refolding batches derived from the same <i>E. coli</i> culture experiment. Variation among batches consisted in minor variations in the dissolution and refolding protocol used.</p

ELISA method designed to evaluate the relative concentration of specific antibodies (Y) anti-influenza A/H1N1/2009 virus in human serum and plasma.

<p>(A) Adsorption of anti-hystidine antibodies to the assay surface on 96-wells micro-assay plates and blockage of the remaining available surface with a commercial blocking solution. (B) Addition of the recombinant protein HA_50–274-H1N1 (semi-circles). (C) Addition of serum samples potentially containing specific antibodies (Y) against the Influenza A H1N1/2009 virus. The left hand panel illustrates a scenario with a higher concentration of specific influenza antibodies. (D) Addition of a peroxidated anti-IgG human antibody (Y) to specifically bind the retained serum antibodies. (E) The addition of peroxidase substrate (S) enables the enzymatic reaction (S→P) with a proportional development of color.</p

Serum from patients infected with Influenza A H1N1/2009 specifically recognize protein HA_50–274-H1N1.

<p>Bars 1–8 (gray) correspond to absorbance signals from non-exposed subjects (samples taken from March to May 2008). Bar 9 (black) shows the average absorbance value of samples 1 to 8. Bars 10 to 14 (blue colors) correspond to absorbance signals from Inf A/H1N1 negative subjects. Bars 15–26 (different colors) correspond to absorbance signals from samples of Inf A H1N1 positive subjects (diagnosed two or three weeks before by RT-PCR). All signals were normalized with respect to the average absorbance signal observed in samples from non-exposed volunteers. Error bars form samples 1–8 and 10–26 represent one standard deviation based on at least three replicates on the assay in the same micro-plate experiment. Error bars form sample 9 represent one standard deviation based on all assays performed to samples from non-exposed volunteers.</p