DESC:BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 illustrates a process or/schematic to determine porcine trypsin, according to an embodiment herein;
FIG. 2 and 3 illustrate ELISA assay protocols of applicant’s kit and comparator’s kit respectively, according to an embodiment herein;

DETAILED DESCRIPTION
The embodiment herein discloses a method and system for effective determination of the residual porcine trypsin from a variety of intermediary entities present in the cell culture immunoassays and various biological and biopharmaceutical samples.
The method and system herein specifically refer to an Enzyme-Linked Immunosorbent Assay (ELISA) to determine absence or presence, and accordingly quantify, porcine trypsin.
In an embodiment, the ELISA of the present embodiment is based on the principle where the captured antibody binds with the target antigen that is porcine trypsin. In an embodiment, the captured antibodies are specifically developed as antibodies in a number of New Zealand rabbits when injected with the porcine trypsin.
Further, a primary detection antibody is introduced to this complex of porcine trypsin anti-trypsin antibody. The primary detection antibody is labelled with Biotin that has affinity for binding with a secondary detection agent. Biotin is the most preferred labelling agent as it binds to an antibody without disrupting the activity. Also, it is known for its strong non-covalent interaction with the streptavidin or avidin, both known for enzymatic interaction with biotin in ELISA assays. The Biotin-streptavidin conjugation has been known to also increase the Limit of detection (LOD). The biotin labelled antibodies lead to strong interaction and enhanced detection of the conjugate.
The secondary detection agents are the enzyme labelled antibodies. These are labelled with the streptavidin alkaline phosphatase or any other enzymes which will produce color when the enzyme specific substrate is being catalysed by the enzyme. The chromogenic substrates include HRP(horseradish peroxidase) specific substrate, TMB (tetramethylbenzidine) substrate and others based on the enzyme. The ELISA technique is specifically known for its high throughput and specificity. But the presence or the non-specific binding by intermediary biomolecules is crucial in the ELISA being performed. To eliminate the chances of such contamination, blocking agents are added. BSA (Bovine serum albumin) and Tween 20 are some of the blocking agents being which are added during the stages of ELISA protocol to prevent undesired binding by non-specific entities. The blocking agent or solutions are prepared with the aim to reduce background interference and noise and increase the assay efficiency.
The invention aims to provide the method to detect the porcine trypsin in any of the biological samples, cultures which are obtained in the manufacturing process such as cell assays, passages, insulin downstream processing or during any therapeutic production stages which involves trypsinization. The end product must be free of the residual trypsin or within acceptable percentage as mentioned under suitable regulatory guidelines.
One of the embodiments of the invention mentions the capture antibody which aims to bind to the residual porcine trypsin with high affinity. The figure 1 exhibits the schematic for the production of the antibody in the rabbits against the antigen introduced. The antigen being the porcine trypsin. As the antibodies developed are protein specific, they conjugate at epitope sites in a biomolecular lock mechanism.
As indicated, the embodiments of the invention are directed towards the highly specific and sensitive detection of the porcine trypsin. The following examples are illustrative of the invention:-
EXAMPLE 1:
Preparation of Reagents
Applicant’s Assay (Assay 1)Method Reagents:
The assay reagents are prepared or reconstituted and stored at appropriate temperature specified as per manufacturer’s instructions. The buffer solutions were prepared accordingly using Ultra Pure water or Phosphate Buffered Saline(PBS) by MP Biomedicals, as per the method requirements. The Ultra Pure water is preferred which is free of undissolved and colloidal ions and organic molecules(free of particles > 0.2µm). The water used for the assays are MilliQ Water from Merck Millipore.
The method of the invention provides capture antibody which is at a concentration of 96µg/ml and the required concentration was 2.0 µg/ml in 1X PBS with 100µl per well. The washing buffer is prepared by the addition of 0.1% of Tween 20(Sigma Aldrich) provided to 1X PBS and labelled as 1X PBST (Phosphate Buffer Saline Tween). The blocking buffer comprises of 3%BSA in 1X PBST. The Bovine serum albumin was by Sigma Aldrich. The assay diluent is prepared by addition of 1% BSA in 1X PBST. The 1X PBST is a reagent required for majority steps of washing in the assay and should be prepared accordingly.
The Detection 1 comprising of the anti-trypsin polyclonal antibody (with Biotin) is prepared at the required concentration of 1.5 µg/ml with required volume being 100µl per well. This Biotin conjugated target affinity purified detection antibody is reactive to the porcine trypsin and is and is protein specific .The Detection 2, which is a secondary antibody attached to the enzyme Streptavidin-Alkaline Phosphatase (SA-ALP) was prepared as per required concentration of 1:7000 in assay diluent. The chromogenic substrate was prepared by the combining the equal volumes of Substrate A and Substrate B reagents. A buffer blank is also prepared using the antigen diluent.
The substrate –enzyme reaction is monitored to reach the highest OD within a range of ±15minutes from the time specified in the protocol.
Assay 2 Method Reagents:
The assay reagents are prepared or reconstituted and stored at appropriate temperature specified as per manufacturer’s instructions. The buffer solutions were prepared accordingly using Ultra Pure water or Phosphate Buffered Saline(PBS), as per the method requirements. The Ultra Pure water is preferred which is free of undissolved and colloidal ions and organic molecules(free of particles > 0.2µm).
The assay included Wash Buffer preparation at 10X concentration, which was diluted to 1X using ultra pure water. The Coating buffer of 10X concentration was prepared to required volume and concentration of 1X. Similarly the Assay diluent was prepared to concentration of 1X using the provided Diluent of stock 10X.
The capture antibody was a regular antibody against porcine trypsin provided at concentration of 0.15mg/mL and the Detection antibody is a Biotin labelled detection antibody (reactive to the porcine trypsin) provided at concentration of 0.102mg/ml. The storage buffer for each of these antibodies is Phosphate buffer saline with pH7.4± 0.1 with cryoprotectant. The capture antibody and the detection antibody are prepared to the concentration of 1X using 1X coating buffer and assay diluent respectively. The secondary detection agent with Streptavidin alkaline phosphatase (SA-ALP) is not provided with this assay method. The secondary detection reagent is prepared using 1X assay diluent.
The recommended concentration for the capture antibody is 0.5-1.5 µg/ml and for the detection antibody is 0.25-1.0 µg/ml. The Substrate, blocking buffer and stop solution were provided and to be used without any dilutions or additions.
Preparation of standard curve
The standard curve of trypsin was prepared using range of 1000ng to 1.37ng/mL using laboratory specific standard. Using the assay diluent the samples and standards were accordingly diluted. The sample being tested is a batch of the final end-product carboxypeptidase which has undergone trypsinization process. The aim is to detect the residual trypsin in the lyophilised carboxypeptidase using an assay method which contains capture antibody and other using the invention’s method comprising of specific anti-trypsin antibody.
The standard curve is being set up as per the manufacturer’s instructions.

EXAMPLE 2:
Method of the assay
Both the assays were performed as per the protocol mentioned in Fig.2 and Fig.3 for the applicant’s invention and other assay respectively.
EXAMPLE 3:
Quantitation and determination of Residual trypsin
Assay 1:
The ELISA plate was read at optical density of 630nm. The standard curve had yielded reading as seen in Table 1. The standard curve was plotted accordingly.

The carboxypeptidase samples were quantitated at the concentration of 1mg/ml and 0.5mg/ml. They yielded readings which were below quantitation limit (BQL). This is indicative of the negligible or not present residual trypsin in the finished product.
Spike recovery was performed to assess the reproducibility of any assay. Spiking is a term which indicates addition of a known amount of protein and its “recovery” is the concentration obtained when measured. If the recovery value is significantly different than the expected value, it is indicative of an interfering matrix element.
The spike recovery which was performed to assess and confirm the reproducibility of the data, was performed at concentration of 100ppm and 10ppm. The observed and the expected values for the spike were calculated to obtain the percentage recovery (Table 2). Performed in duplicates for a) CPB 1 mg/ml and 100ppm (parts per million) and b) CPB 0.5 mg/ml and 10 ppm. The duplicate readings obtained was 105.867As the percentage recovery was within the acceptable standard of 80-120%, the assay performed was found to be high throughput and accurate.

Assay 2:
The trypsin standard curve was performed as mentioned in the assay manufacturer’s instruction manual. The standard curve was run in the range 1000 ng,333.33 ng, 111.11ng, 37.04 ng, 12.35 ng, 4.12 ng,1.37ng , 0.46 ng( all standard concentrations are in ng/ml).

The carboxypeptidase samples were quantitated at the concentration of 1mg/ml and 0.5mg/ml. They yielded readings exhibited the values to be 3.235 ng/ml, 1.708 ng/ml. This is indicative of the present residual trypsin in the finished product.
Spike recovery was performed even for this Assay method to determine its reproducibility. The spike recovery which was performed to assess and confirm the reproducibility of the data, was performed at concentration of 12.5 ng and 6.25 ng. The residual trypsin was calculated to be 3.24 ppm and 0.85 ppm in the finished product. The Table 4 clearly exhibits the percentage recovery was 24.84 %and 26.91 % respectively for 12.5ng and 6.25ng spiking. As the expected spike recovery percentage is 80-120%, the spike recovery from the Assay Method 2 is indicative about poor specificity and sensitivity due to probable matrix interference and substandard detection efficiency since this method did not yield the expected spike recovery values.

,CLAIMS:We claim:
1. A method or assay to detect porcine trypsin in biopharmaceutical cultures, samples or solutions, said method or assay comprising:
adding an antibody specific to porcine trypsin to said biopharmaceutical cultures such that antibody-trypsin conjugate is formed when porcine trypsin is present in said biopharmaceutical culture;
adding a labelled primary detection antibody such that antitrypsin-antibody-porcine trypsin–primary detection antibody conjugate is formed when said antibody-trypsin is formed;
reacting said antitrypsin-antibody-porcine trypsin–primary detection antibody conjugate, when formed, with an enzyme linked secondary antibody that forms an capture antibody-trypsin-primary detection antibody – enzyme linked secondary antibody conjugate or complex; and
reacting said antibody – enzyme linked secondary antibody conjugate or complex with a chromogenic
substrate to bind and leading to a fluorescent reaction.

2. The method as claimed in claim 1, wherein the primary detection antibody is an anti-trypsin antibody labelled with Biotin.

3. The method as claimed in Claim 1, wherein the enzyme linked to the secondary detection antibody is Streptavidin Alkaline phosphatase.

4. A kit for Enzyme linked immunosorbent assay (ELISA) for the detection of residual trypsin comprising of –
a capture antibody to bind to the target porcine trypsin and form trypsin- anti-trypsin antibody complex;
a labelled- primary detection antibody directed to bind to this complex of trypsin- anti-trypsin antibody;
an enzyme linked secondary detection antibody to bind to the above conjugate;
a chromogenic substrate which reacts with conjugated secondary detection antibody and yields color for colorimetric detection; and
Blocking agent to prevent non-specific binding by undesired entities during the assay performed.

5. The kit as claimed in claim 4, further comprising anti-trypsin antibody specifically directed to detect porcine Trypsin in biological samples.

6. The kit as claimed in claim 4, the kit wherein the primary detection antibody is labelled with Biotin for increased affinity and detection.

7. The kit as claimed in claim 4, the kit containing secondary detection antibody linked to enzyme Streptavidin alkaline phosphatase for specific binding to substrate and detection.

8. The kit as claimed in claim 4, the kit containing chromogenic substrates such as TMB (tetramethylbenzidine),HRP(horseradish peroxidase) substrate, etc.

9. The kit as claimed in claim 4, the kit containing blocking agents such as Tween 20 and Bovine serum albumin (BSA) to prevent binding of non-specific and undesired entities.

10. The kit as claimed in claim 4s, the kit which has a limit of detection in the range of 0.415ng/ml to 1000ng/mL.