SDS-PAGE analysis under non-reducing conditions is used to detect and estimate the amount of aggregates and fragments that are stable to the SDS treatment. It also yields the intact molecular weight for proteins containing covalently bound subunits. On the other hand, SDS-PAGE under reducing conditions detects the presence of covalently bound aggregates and proteins; and also yields the subunit molecular weights for proteins comprised of covalently bound subunits.

Samples are treated with SDS at elevated temperatures in the presence or absence of reducing agents, depending on the type of SDS PAGE analysis desired. The electrophoresis is carried out using Novex pre-cast gels and a Novex XCell II Mini-Cell system. The gels are stained either with Colloidal Coomassie Blue or Silver, destained, and scanned using a densitometer. Following scanning, the gels are photographed, dried and archived.

Isoelectric Focusing (IEF) analysis involves the separation of proteins/peptides on the basis of their charges in polyacrylamide gels using an electric field. It is used to detect micro-heterogeneity due to post-translational modifications, fragmentation, as well as modifications arising from the purification, handling and storage processes.

Samples are mixed with the IEF sample buffer and subjected to electrophoresis using Novex pre-cast pH gradient gels and a Novex XCell II MiniCell system. The gels are stained with Colloidal Coomassie Blue, destained, and scanned using a densitometer. Following scanning, the gels are photographed, dried and archived.

HPSEC is used to determine the amount of non-covalent and covalent aggregates as well as fragments present in a sample. This method separates proteins and peptides according to their relative molecular size and shape. The size exclusion column acts like a molecular sieve.

The samples are analyzed using a validated Waters 2690 Alliance system controlled by the Millennium³² software. The peak areas for the individual peaks obtained with each test article are expressed as a percentage of the total peak area obtained for the sample, and used to calculate the amount of aggregates and/or fragments present in the test article.

Reverse Phase HPLC is a technique that separates molecules according to their relative hydrophobicity. It is commonly used to assess the purity and characterize the impurities present in biotechnology-derived as well as traditional pharmaceuticals.

HPLC analysis will be carried out using a validated Waters 2690 HPLC system equipped with a Photodiode Array (PDA) detector and controlled by the Millennium³² chromatography software system. The peak areas for the individual peaks obtained with each test article are expressed as a percentage of the total peak area obtained for the sample, and used to calculate the percent purity and to quantitate the impurities present.

Western Blotting is a commonly used technique to detect the presence of impurities that react with specific antibodies. It may also be used to assess the immuno-reactivity profile of an active component, usually protein or peptide.

In this procedure, SDS PAGE (either reducing or non-reducing conditions) or IEF, using Protocol 38000 or Protocol 38005 respectively, may be used to separate proteins. The proteins are then transferred electrophoretically onto a nitrocellulose or polyvinylidine difluoride (PVDF) membrane. The membrane is probed with an antibody or a suitable mixture of antibodies directed against the substances that are to be detected. This is followed by a treatment with a secondary antibody which immunoreacts with the primary antibody and is labeled either with an enzyme or a reporter molecule. The complex formed between the two antibodies and the protein or polypeptide of interest is detected by the color change or chemiluminescence produced by suitable substrate or reporter molecules. The location of the complexes on the gel can be used to ascertain the molecular size (or approximate pI range if IEF is used for the primary separation) of the impurities that are detected.

This assay is used to detect Vancomycin concentrations in cell wash samples by Reverse Phase HPLC. The estimated Vancomycin levels will be used to assess the effectiveness of the cell wash procedure in reducing the concentration of this media component below the detection limit of the HPLC assay.

This assay is used to estimate the amount of tetracycline in samples provided by the sponsor.

This assay is used to estimate the amount of hydrocortisone in cell culture and cell wash solutions provided by the sponsor.

Hydrocortisone concentrations will be determined using Reverse Phase High Performance Liquid Chromatography (HPLC) utilizing a C18 column and isocratic elution.

This assay is used to estimate the amount of gentamicin in cell culture and cell wash solutions provided by the sponsor.

Gentamicin amino groups are derivatized using modified Waters AccQ· Tag method. Gentamicin derivatives will be separated using Reverse Phase High Performance Liquid Chromatography (RP-HPLC) utilizing a C18 column and gradient elution.

Immobilized Protein A is frequently used to purify antibodies on the large and small scale. This method is an ideal purification technique because Protein A binds antibodies at the Fc or non-specific region. Although significant advances have been made to minimize leaching, small amounts of Protein A may co-elute with the antibody and contaminate purified antibody samples. This assay is used determine the amounts of contaminating Protein A antibody present after antibody preparations.

The assay is an ELISA employing a polyclonal anti-Protein A capture antibody and a biotinylated monoclonal anti-Protein A antibody. Color is developed using a streptavidin labeled alkaline phasphatase conjugate and substrate.

The amount of residual DNase will be determined by using UV spectrophotometry.

Samples and standards are equilibrated to 25° C, in a reaction cocktail containing sodium acetate, magnesium sulfate, sodium chloride, and deoxyribonucleic acid. The increase in A_260nm is monitored for approximately 10 minutes. The maximum linear rate will be used to measure the ∆A_260nm/min. for the samples, standards and controls. A 2000 Kunitz DNase standardization vial will be used as a reference standard to correct for the actual units/ml of DNase in the standards and test articles. The sponsor provided test sample concentration or mass will be used to calculate the total Units of DNase per unit mass, concentration, or volume of sample.

The amount of residual RNase will be determined by using UV spectrophotometry.

This assay is based upon the release of acid soluble oligonucleotides following digestion of yeast RNA. One unit releases acid soluble oligonulcleotides equivalent to one A₂₆₀ unit in the reaction mixture at 37° C and pH 7.5 under the specified conditions of this assay. Samples and standards are incubated at 37° C for an exact time interval with RNA. The reaction is stopped by the addition of an uranyl acetate-perchloric acid solution. The solutions are incubated on ice, then clarified by centrifugation. The supernatants are diluted in water, and the A₂₆₀ in read. The sponsor-provided protein/solid concentration will be used to calculate the total units of RNase per mg of solid or protein.

Protein production is frequently performed through recombinant expression in Chinese Hamster Ovary (CHO) cells. This assay provides a method to quantitate the levels of CHO host cell proteins in protein products.

Samples are added to microtiter strips pre-coated with anti-CHO affinity purified capture antibody. A second antibody, alkaline phosphatase enzyme labeled anti-CHO, is added to each well. This results in the formation of the following sandwich complex: solid phase antibody-CHO protein-alkaline phosphatase labeled anti-CHO. The microtiter strips are then aspirated and washed to remove any unbound antibody. Following the washing of wells, p-nitrophenyl phosphate (PNPP) substrate is added to each well. The resulting color intensity, which is proportional to the concentration of CHO host cell proteins in the sample, is determined by reading the wells using a validated Spectra MAX 340 plate reader.

NOTE:
If client’s sample buffer is below pH 6.0 or above pH 8.0, it should be provided with the sample as a control for the assay.

This assay uses purified polyclonal antibodies specific to BenzonaseÒ endonuclease.

The assay is initiated by adding the standards, samples, and controls to microtiter strips, which are pre-coated with affinity purified polyclonal, capture antibody. The wells are incubated for a two-hour period, then washed. A secondary horseradish peroxidase conjugated antibody, directed against BenzonaseÒ endonuclease, is added to each well, and the plate is incubated for 1 hour. This results in the formation of the following sandwich complex: solid phase antibody-BenzonaseÒ , endonuclease-HRP, conjugated antibody. The wells are washed and aspirated to remove any unbound reactants. Residual BenzonaseÒ is detected by the addition of TMB peroxidase, a HRP substrate, to each well. The wells are developed for a fifteen-minute incubation period. The resulting color intensity, which correlates to the amount of analyte, is quantitated using a calibrated Spectra MAX 340-plate reader. Accurate measurement is achieved by comparing the signal of the sample to the BenzonaseÒ endonuclease standards assayed at the same time.

Recombinant expression by the HEK 293 cell line has been used for the production of a number of proteins and a variety of gene therapy agents.

The HEK 293 HCP assay is a two-site immunoenzymetric assay. Samples, which may contain HEK 293 HCPs, are reacted in microtiter strips coated with an affinity purified capture antibody. A secondary horseradish peroxidase (HRP) conjugated enzyme is reacted simultaneously, resulting in the formation of a sandwich complex of solid phase antibody-HCP-enzyme labeled antibody. The microtiter strips are washed to remove any unbound reactants. The quantity of HEK 293 HCPs is detected by the addition of TMB peroxidase, a HRP substrate, to each well. The amount of hydrolyzed substrate is read on a validated Spectra MAX 340-plate reader and is directly proportional to the concentration of HEK 293 HCPs present.

This assay is used to determine the compatibility of insulin by determining the pH, potency and desamido content, and to estimate the amount of aggregates and/or fragments present in insulin samples.

The insulin sample is tested according to the United States Pharmacopeia procedures for related compounds and high molecular weight proteins. HPLC analysis is carried out using a validated Waters 2690 HPLC system controlled by Millennium³² chromatography software system. The methods use USP system suitability controls and/or client controls if requested.

This method provides a direct procedure for determination of protein and peptide concentration, which is useful in evaluating potency and strength. It is also useful in establishing the identity of peptide products. In addition, it can be utilized to validate colorimetric methods for determining protein concentration and in determining or confirming extinction coefficients of proteins and peptides.

Proteins and peptides are hydrolyzed to their constituent amino acids at elevated temperatures using 6N HCl in the vapor phase under vacuum using a validated Waters Pico-Tag system. All the amino acids present in the hydrolysate, including trytophan, cystine and cysteine, are quantitated using the Waters AccQ-Tag system. In this method, the mixture of amino acids is reacted with AccQ-Fluor reagent, which forms stable derivatives. These derivatives are separated by reverse phase HPLC using a validated Waters 2690 HPLC equipped with an UV and fluorescence detector and controlled by the Millennium³² software. The fluorescence detector is used to detect and quantitate all the amino acids, except tryptophan, which is monitored and quantitated using the UV detector. Tryptophan is stabilized during the HCI hydrolysis using dodecanethiol. Cysteine and cystine are converted to their mercaptopropionic acid derivatives during the hydrolysis for more accurate quantitation. The concentra-tion of each amino acid in the hydrolysate is determined by comparing the response obtained for that amino acid with standards containing known amounts of amino acids. The standards are subjected to the same hydrolysis conditions as the samples to correct for loss of labile amino acids like Threonine and Serine. This data is used to obtain the amino acid composition and concentration of proteins and peptides.

The theoretical extinction coefficient of a protein or peptide can be calculated from the relative amounts of the aromatic amino acids tryptophan, tyrosine and phenylalanine, using the literature extinction coefficient values for these amino acids. However, the value thus obtained is sometimes different from the actual value due to the effect of the secondary and tertiary structure of the molecule, as well as solvent and pH effects. Hence, it is crucial to confirm the theoretical extinction coefficient of the molecule under the conditions to be used for concentration determination.

A set of samples with UV absorbance of about 1 at 280 nm is prepared and their absorbances are determined using a validated Perkin Elmer Lambda 40 Spectrometer. The protein/peptide concentrations of these set of samples is determined by amino acid analysis. (See Protocol 38007.) The results obtained are used to obtain average values for the concentration (in mg/ml) as well as the absorbance at 280 nm which in turn is used to calculate the extinction coefficient for the molecule, i.e., the absorbance at 280 nm of a 1mg/ml solution.

This is a custom assay that can be developed to quantitate the immunoreactivity of
a variety of active components within a sample. The assay can be developed as a GLP or GMP test.

A variety of ELISA systems can be used for the detection of antigen and/or antibodies, including indirect ELISAs; direct competitive ELISAs; antibody-sandwich ELISAs; double antibody-sandwich ELISAs; direct and indirect cellular ELISAs to detect cell surface antigens; and ELISAs for measuring serum antibody titer. In each test system, the solid phase reagents are incubated with secondary or tertiary reactants covalently coupled to an enzyme. Unbound conjugates are removed by washing and the appropriate substrate is added. The amount of hydrolyzed substrate is read on a validated microtiter plate reader and is directly proportional to the amount of analate present in the sample.