DESC:The embodiment herein discloses a method and system for effective determination of the residual HCP 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, E.coli based Host cell protein.
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 E.coli HCP. In an embodiment, the captured antibodies are specifically developed as antibodies in a number of New Zealand rabbits when injected with the E.coli based HCPs.
Further, a primary detection antibody is introduced to this complex of anti-E.coli HCP. 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 Host cell proteins 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. The end product must be free of the residual contaminating traces of host cell proteins or within acceptable percentage as mentioned under suitable regulatory guidelines.
FIG. 1 is a process flow depicting preparation of ELISA kits to detect and quantify residual host cell proteins in a biopharmaceutical or biological sample, according to an embodiment herein. The process includes immunisation (102) of an animal, to produce host cell proteins antibodies, by injecting host cell proteins into an animal. In an embodiment, the animals are rabbits. The antibodies thus generated are purified and characterised (104) and incorporated into an enzyme-linked immunosorbent assay to detect host cell proteins from biological or biopharmaceutical samples. In an embodiment, the assay is able to detect host cell proteins in biological sample in an amount as low as 1.37ng/mL of a sample.
As indicated, the embodiments of the invention are directed towards the highly specific and sensitive detection of the host cell proteins. The following examples are illustrative of the invention:-

EXAMPLE 1:
Preparation of Reagents
Applicant’s Kit 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 1.4mg/ml and the required concentration was 2.5 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 Bovine serum albumin was by Sigma Aldrich. The assay diluent was 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-E.coli HCP polyclonal antibody (with Biotin) is prepared at the required concentration of 2.5 µg/ml with required volume being 100µl per well. This Biotin conjugated target affinity purified detection antibody is reactive to theE.coli HCP and is specific toE.coli based HCP 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 1:6000 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. A ready blocking buffer is provided.
The substrate –enzyme reaction is monitored to reach the highest OD within a range of ±15minutes from the time specified in the protocol.

Comparator Kit Assay (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 wash concentrate, tris buffered saline at 20X was diluted with 1L of distilled water (as per manufacturer’s instructions) and labelled with kit expiration date.
The capture antibody is anti-E.coli HCP antibody against HCP and the Detection antibody is a HRP(horseradish peroxidase) labelled detection antiE.coli HCP antibody. 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 HCP was prepared using range of 1000ng to 1.37ng/mL for Assay 1 using laboratory specific standard. Using the assay diluent the samples and standards were accordingly diluted. In case of Assay 2, the standardsE.coli HCPs in bovine albumin with preservative at 0,1,3,12,40 and 100ng/ml were provided.
Three products were tested with both the applicant’s invention ELISA kit and a comparator kit. The products were Insulin Glargine, Human Insulin and Aspart; each of them lyophilised. The aim is to detect the host cell proteins in the lyophilised products using a comparator’s kit and the invention’s method and compare the efficacy and sensitivity of the assay method/kits with each other.

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 HCP
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 samples Insulin glargine, Insulin Aspart and Human insulin were quantitated at the concentration of 1mg/ml. The absorbance was detected at 630nm. The readings are tabulated and shown below.

The samples detected average HCP ppm values at 2.950ppm, 28.526ppm and 27.645 ppm for Aspart,Human insulin and Glargine respectively.
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 12.346 ppm. The observed and the expected values for the spike were calculated to obtain the percentage recovery (Table 3). Multiple readings were obtained and spike recovery was calculated as known in standard of art for Insulin Glargine, Insulin Aspart and Human insulin sample of 1mg/ml.

The sum of ppm values for 1mg/ml of each product and values of spike 12.346 ppm yielded values which was approximately close to the expected. The observed values were 13.487,42.424 and 38.198 (all in ppm) whereas the expected values after spiking are 15.296,40.872 and 39.991 (all values in ppm) for Human insulin ,Insulin Aspart and Insulin Glargine respectively. The spike recovery percentage was 88.18 %, 103.80 % and 95.52 % for Human insulin, Insulin Aspart and Insulin Glargine.
As the percentage recovery was within the acceptable standard range of 80-120%, the assay performed was found to be high throughput and accurate.
Assay 2:
The HCP standard curve was performed as mentioned in the assay manufacturer’s instruction manual. The standard curve was run in the range of 1 ng/ml, 3 ng/ml, 12 ng/ml, 40 ng/ml and 100ng/ml) has provided. The standard readings were tabulated and standard curve was plotted as seen in Table 4.


The samples were quantitated at the concentration of 1mg/ml. The absorbance was detected at 450nm. The readings for each sample i.e. Insulin Glargine, Insulin aspart and Human insulin was observed and tabulated as seen in the Table 5.

The samples detected average HCP ppm values at 0.168 ppm, 0.648 ppm and 0.589 ppm for Human insulin, Aspart and Glargine respectively. The values obtained are not consistent and appears to be erratic.
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 12.346 ppm. The recovery percentage and readings were tabulated as seen in Table 6.

The spike recovery percentage was calculated in the finished product. The observed values were 2.811, 1.911 and 1.702ppm whereas the expected values are 12.514,12.994 and 12.935 ppm for Human insulin, Aspart and Insulin Glargine respectively. The Table 6 clearly exhibits the percentage recovery was 22.463%, 14.707% and 13.158% for Human insulin, Insulin Aspart and Insulin Glargine respectively. The sum of ppm values for 1mg/ml of each product and values of 12.346 ppm yielded values which was erratic and significantly different than the expected. 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.
The applicant’s kit thereby proves to be more efficient and accurate in terms of sensitivity and specificity of detection of residual Host cell proteins in the final product.

,CLAIMS:We claim:
1. A method or assay to detect E.coli host cell proteins (HCP) in biopharmaceutical cultures, samples or solutions, said method or assay comprising:
adding an antibody specific to E.coli host cell protein to said biopharmaceutical cultures such that antibody-E.coli HCP conjugate is formed when host cell proteins are present in said biopharmaceutical culture or product;
adding a labelled primary detection antibody such that anti HCP antibody- HCP–primary detection antibody conjugate is formed when said antibody-HCP is formed;
reacting said anti HCP-antibody- HCP–primary detection antibody conjugate, when formed, with an enzyme linked secondary antibody that forms an capture antibody-HCP-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-E.coli HCP polyclonal 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 Host cell proteins (HCPs)comprising of –
a capture antibody to bind to the target E.coli HCP and form HCP-anti HCP antibody complex;
a labelled- primary detection antibody directed to bind to this complex of HCP -anti HCP polyclonal 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-HCP antibody specifically directed to detect E.coli host cell proteins 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 4, the kit which has a limit of detection in the range of 1.37ng/ml to 1000ng/mL.