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What are Non-Compendial Methods?

Analytical methods that are not standardized in the US Pharmacopeia are non-compendial methods. These methods are product specific and need to be developed based on the critical quality attributes of the product. Following tests constitute non-compendial methods.

What are Non-Compendial Methods?
Non-Compendial Methods
Non-Compendial Methods
Identity Test
Identity Test

What are Identity Tests?

Identity tests are analytical methods used to determine and/or to confirm product identity. These identity tests should be product specific and are based on unique aspects of molecular structure and/or other specific properties of the product. The identity tests can be qualitative as well as quantitative in nature. The following experiments are performed as part of identity testing.


Peptide Mapping
Charge Variants Profile
Biological Potency

What is Peptide Mapping?

Peptide mapping is a widely used analytical technique to identify and/or to verify a primary structure of biologics. It is one of the most widely used identity tests for testing in-process samples, drug substances, and drug product release.

How is Peptide Mapping performed?

The peptide mapping workflow involves endo-peptidase digestion of the protein followed by separation of peptides by reversed phase liquid chromatography. The procedure generates a 'fingerprint' or set of unique peptides of the protein that will be compared to the reference standard.

What we provide?

Using our expertise in various analytical technologies and knowledge in cGLP, cGMP, and other regulatory guidelines, we provide a wide range of analytical services to support biopharmaceutical product development, including identity testing.

What is Charge Variants Profile?

Molecular variants of biologics, with differences in their charge, are known as charge variants. These charge variants are generally acidic or basic in nature and are compared with the main species. In the identity test, the charge variants profile of the test sample is compared to the reference standard profile.

How are Charge Variants measured?

Charge variants can be separated and measured by orthogonal techniques such as ion exchange chromatography (IEX) and imaged capillary Iso-Electrophoretic focusing (icIEF). In IEX chromatography, molecules are separated based on their surface charge, while in icIEF, molecules are separated based on total charge i.e., Isoelectric point (pI) of the molecule.

What we provide?

We provide charge distribution data by both IEX and icIEF methods as per customer requests. The data obtained from these analyses demonstrate that the main peak is consistent with the expected product and hence, can be used as an identity test. Our laboratories are well equipped with broad analytical technologies such as HPLC, UPLC, icIEF analysis, and LC-MS etc. We are also capable of isolating separated peaks i.e. (single charge variant) using semi-preparative IEX columns. Isolated peaks can be further characterized by LC-MS as per regulatory requirements.

What is Potency?

Potency is a quantitative measure of biological activity of a biomolecule. Potency measures the ability of biologics to trigger specific responses in a disease-relevant system. Potency is a critical quality attribute in manufacturing process of biologics and is often determined by mechanism of action based biologically relevant assay.

How is Potency measured?

Biological activity is the specific ability of a biologic to achieve a desired biological effect. The results of biological assays should be expressed in units of activity calibrated against a reference standard, when applicable, for the assay utilised. Relative potency is measured by comparing the concentration-response curves of a test batch with that of a reference standard.

What we provide?

We provide the services to measure the biological potency of biologics including plate based binding assays, cell based binding assays and cell-based bioassays and/or as per customer request. Since binding is very specific to the molecule, this method can be used as an identity test method.


Purity Test
Purity Test

What are Purity Tests?

Purity tests are analytical methods used to measure the purity of Biologics. Purity measurement should focus on separation of the desired product from the impurities.

How to measure Purity of Biologics?

The absolute purity of biologics is difficult to measure, and results are method dependent. Consequently, the purity of drug substance and/or drug product is usually measured by a combination of analytical methods. Purity can be determined using the following analytical methods shown in the info-graphic below:



Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS)

What is Capillary Electrophoresis (CE)?

Capillary electrophoresis is a type of electrokinetic separation method in which the separation of charged biomolecules is carried out in a capillary tube that is immersed in an electrophoresis running solution.

How does CE-SDS work?

CE-SDS is a widely used technique for the purity determination of biologics. In this method, separation of biologics occurs based on their size. CE-SDS provides a general picture of both the size of target proteins and the presence of other protein-based impurities. CE-SDS analysis of biologics can be performed under reducing and non-reducing conditions. SDS is a critical component of the CE-SDS method. Protein molecules get denatured in the presence of SDS at high temperature, which causes specific interactions of individual SDS molecules with protein molecules. This process alters the secondary structure of proteins, thus allowing for their separation based on size.

What we provide?

We provide services for purity measurement of biologics, under both reducing and non-reducing conditions and/or as per customer request. We can also estimate fragments and covalently linked aggregates of the Bio-molecule and provide very high-quality data as per regulatory agency requirements.

What is Size Exclusion Chromatography (SEC)?

Size Exclusion Chromatography (SEC) is a method in which biomolecules in solution are separated based on their size (molecular weight) as they pass through a resin packed in a column.

How does SEC work?

SEC columns are packed with porous resin, that consists of a porous matrix of spherical particles (beads) that lack reactivity and adsorptive properties.

After sample is introduced in to the column, biomolecules larger than the resin pores are unable to diffuse into the beads, so they elute first. Molecules smaller than the beads can penetrate the pores to varying degrees based on their size. Molecules that enter the total pore volume are eluted last. The elution of samples occurs under isocratic conditions, so there is no need to use different buffers during the separation.

What we provide?

We offer services for separation and quantitation of high molecular weight species (total aggregates), monomer (main peak) and low molecular weight species (degradants) under native conditions and/or as per customer request. We provide high quality data as per regulatory requirements.

What is Hydrophobic Interaction Chromatography (HIC)?

Molecular variants of biologics, with differences in their hydrophobicity, are know as hydrophobic variants. These hydrophobic variants, generally referred to as pre peak (oxidized species), main peak and post main peak, are separated by HIC. HIC is one of the most widely used technique for separating and purifying proteins in their native state.

How does HIC work?

HIC separates biomolecules based on their surface hydrophobicity. Separation is achieved by utilizing reversible interactions between biomolecules and hydrophobic surface of the chromatographic resin. In HIC, biomolecules bind to the chromatographic resin at high salt concentration which strengthens their biomolecular interactions, while dropping the salt concentration causes separation and elution based on hydrophobicity. HIC is a useful separation technique to purify biomolecules while maintaining native state to preserve their biological activity because it uses milder and non-denaturing conditions. HIC method is very useful to determine the biological impact of protein oxidation.

What we provide?

We offer services to measure oxidation levels and identify the oxidized amino acids by collecting the fractions from each hydrophobic variant peak using our HIC and LC-MS capability. We can also determine the impact of oxidation level on the potency of the biomolecule by using our bioassay capability and provide high quality data as per customer requests and as per regulatory guidelines.

What is Reversed Phase Chromatography (RPC)?

Any liquid chromatographic method that uses a hydrophobic or non polar stationary phase in conjunction with a polar mobile phase is known as reversed-phase chromatography.

How does it work?

RP chromatography separates biomolecules based on their hydrophobicity. Protein molecules bind to the RP column resin in aqueous mobile phase which strengthen their interactions, while increasing the concentration of organic mobile phase causes separation and elution of the peaks based on hydrophobicity of protein molecules. RP chromatography is used for the separation of oxidized (pre-peaks), non-oxidized (main peak) and post main peaks.

What we provide?

We offer services to measure the oxidation levels and other post translational modifications by isolating individual variant peak using RPC and identify the oxidized amino acids using our LC-MS capability. We provide high-quality data to customers as per their request and as per regulatory guidelines

Quantity of Biologics
Quantity of Biologics

What is Quantity Measurement and Why is it required?

Quantity of biologics is the total amount of biological drug present in each sample. Measurement of accurate quantity of biologics (drug) plays an important role for the dose determination (amount of drug to be delivered to the patient). Exact drug quantity measurement is also very critical for analytical methods to generate high quality data. Careful selection of quantitative methods is essential to generate accurate quantity data.

Methods for quantity measurement of biologics are shown in the infograph below:


Extinction Coefficient Determination
Absorbance Measurement at 280 nm
Bradford Assay
BCA Assay
Quantitative Amino Acid Analysis by HPLC

What is Extinction Coefficient?

The extinction coefficient is an unique property of biologics, that measures how strongly a species reflects or absorbs UV light at a fixed wavelength. It is a natural property of biomolecules that depends on atomic, chemical and protein structural composition.

How can it be measured?

Extinction coefficient can be determined using the following two methods:

  1. Molar absorptivity or molar extinction coefficient as described by Beer's law according to the equation shown below:

    A = εbc

    where A is the absorbance

    ε is the molar extinction coefficient

    b is the path length of the cuvette

    where "A" and "C" are measured using the spectrophotometer. Molar extinction coefficient can be obtained by calculating the slope of the absorbance vs. concentration plot.

  2. Extinction coefficient as measured by Amino Acid Analysis (AAA) by HPLC.

What we provide?

We offer services for the accurate measurement of molar extinction coefficient using our analytical capabilities. We provide very precise protein concentration (quantity) of a given biological sample as per customer and regulatory agencies requirements.

What is Absorbance Measurement at 280 nm?

Measuring the UV absorbance at 280 nm is a commonly used method for quantity determination of biologics. Biomolecules show strong absorbance at 280 nm due to the presence of intrinsic chromophoric amino acids such as tryptophan, tyrosine, etc. residues.

How can it be measured?

The wavelength at which a biological molecule shows maximum absorbance is used for quantity or concentration determination. Absorbance at 280 nm is measured using a spectrophotometer or microplate reader. There are two main factors that affect the absorbance: concentration of the substance and path length  of the cuvettes. The molar absorptivity (extinction coefficient) at 280 nm can also be predicted from the protein sequence. However, measurement of UV absorbance at 280 nm is necessary to obtain the accurate extinction coefficient. But, this method is only applicable to proteins that contain tryptophan or tyrosine residues.

What we provide?

We offer services for the accurate protein concentration determination of biological samples.

What is Bradford Assay?

Bradford assay is a Coomassie Brilliant blue G-250 dye-binding assay. Upon protein binding, absorbance of Coomassie Brilliant blue G-250 dye changes from 465 nm to 595 nm due to a color shift. Consequently this color shift enables the accurate determination of protein concentration.

How does it work?

Bradford assay is a quick and sensitive method for protein concentration determination at room temperature under acidic conditions. Both reference standard and samples are mixed with the dye, followed by incubation at room temperature for approximately 10 to 15 min. The resultant blue color is measured at 595 nm. This method measures the presence of the basic amino acid residues including arginine, lysine and histidine, which contribute to the formation of protein-dye complex.

What we provide?

We offer services to measure the total protein concentration in a given biological samples using Bradford Assay dye. We provide high-quality protein concentration data as per customer and regulatory agencies requirements.

What is BCA assay?

Bicinchoninic acid (BCA) assay is a copper based colorimetric assay for the quantitation of total protein. In this assay, proteins cause reduction of Cu2 + to Cu+ under alkaline condition. Reduced Cu+ forms a complex with two molecules of BCA, which causes change in color of reagent solution from green to purple. This color shift shows a strong absorbance at 562nm. The color shift from green to pueple is directly proportional to the amount of protein present in the sample.

How do we measure the Protein Concentration by BCA Assay?

In BCA assay, two molecules of BCA chelate with one Cu+ ion which causes a change in color of the reagent solution from green to purple. The BCA-Cu+  complex is influenced by, the number of peptide bonds and presence of amino acids cysteine, cystine, tyrosine, and tryptophan side chains. To increase the sensitivity, the assay is performed at elevated temperatures (e.g. 60°C) so that the protein molecules get denatured, thereby increasing exposure of the reactive amino acids.

What we provide?

We offer services to measure the total protein quantification in a given biological samples using BCA Assay. We provide highly quality protein quantity data as per customer and regulatory agencies requirements.

What is Quantitative Amino Acid Analysis (AAA) by HPLC?

Amino Acid analysis is a gold standard method for quantification of proteins in each biological sample. 

This method includes two steps:

  1. Hydrolysis of an accurate amount of biological sample. For example, incubation of samples in 6N HCl at 110°C for 24 hr.

  2. Separation and gradient elution of the released amino followed by reaction with ninhydrin to yield highly colored (purple) products. The derivatized individual amino acids are detected and quantitated by an ultraviolet or fluorescent detector. This method is highly accurate because it measures individual amino acids, thereby avoids the potential for overestimation of protein, reported with other protein quantitation methods. AAA also provides information on the amino acid content of products and samples containing other non-protein components.

What we provide?

We offer golden standard services for an accurate quantification of protein in each biological samples using quantitative amino acid analysis by HPLC. We provide high-quality protein concentration data as per customer and regulatory agencies requirements.


What are Impurities?

Impurities are undesired components present in the drug substance or drug product. These Impurities may derive from the product or from the process. The impurities derived from product are called product related impurities and those derived from the process are known as process related impurities. These impurities often present at very low or trace levels in highly complex sample matrices. Consequently, to measure these impurities, highly sensitive and specific analytical methods are required. 

What are process related Impurities?
What are product related Impurities?

Impurities derived from the manufacturing processes are known as process-related impurities.

Product related impurities are molecular variants derived from the product during translation, post-translation, processing, storage etc. These impurities may affect the safety and efficacy of the product.

Potency of Biologics
Potency of Biologics
What is potency of Biologics?

Potency assays are used to measure the specific ability or capacity of a drug to elicit a particular response at a certain dose in a relevant biological system. Potency assays of biologics are a regulatory agencies requirement for release of drug substance and/or product under cGMP conditions.

Potency testing involves comparison of a product's biological activity to that of a reference standard. 

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bd_global@crystalpharmatech.com +1-609-604-8303
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Suite B4-101, Biobay, 218 Xinghu Street, Suzhou Industrial Park, Suzhou, China, 215123
Suite 500-B, 3000 Eastpark Blvd, Cranbury, New Jersey 08512, USA
bd_global@crystalpharmatech.com +1-609-604-8303