How and Why to calculate Percent Carbon for TOC

Total organic carbon (TOC) is a non-specific method often used in cleaning to determine if product residues have been removed to acceptable levels. Due to TOC's non-specific nature, the level of recovered product residues in TOC samples should consider the total theoretical percent carbon from the material being analyzed. This memo will provide guidance on how to correctly determine the theoretical percent carbon for a given product as well as how to apply it.

Background:

Total organic carbon (TOC) is a non-specific method that can determine if a cleaning process has removed product residues to acceptable levels. TOC measures the total amount of carbon within a sample, including product residues, cleaning agents, microbes, and any other water-soluble impurities containing carbon. When TOC is used to determine if product residues have been removed to acceptable levels, it should be assumed that all organic carbon detected is directly from the product [1].

TOC recovery studies quantitate the amount of recoverable organic residue against the amount of spiked organic residue from a coupon surface. Recovery of organic residue can be influenced by multiple factors that may contribute additional organic carbon, such as the sample container, the solvent used for analysis, the atmosphere, the surface of the coupon, and the coupon cleaning method. Therefore, to examine the accurate recovery of only process materials in the TOC sample, recovery is assessed as the theoretically calculated carbon content of the API.  

Similarly, the acceptance limits must also be corrected due to the potential for outside factors to add organic carbon to TOC samples. Acceptance limits for TOC swab and rinse samples should be corrected by multiplying the limits by the theoretical carbon content of the API so they are representative of only the process materials that may be present on equipment surface post-cleaning.   

For small molecules, the theoretical carbon content can be derived at each product stage (e.g., intermediate, API, drug product) because the molecular structure can be characterized at each stage of production.  

For an intermediate biologic, where there is limited characterization, the theoretical carbon content is typically derived using the API in its formulated state as this represents a worst-case for the maximum carbon content potentially present. If there is adequate characterization data of the intermediate biologic, the theoretical carbon content may be calculated at this product stage. 

The process for determining the theoretical percent carbon :

Consider a hypothetical API, MinQ, at its nominal concentration, where the formulation consists of 10 mg/mL of MinQ (C10H17N3O6S) in 10.0 mM of Sorbitol (C6H14O6) and 5.0 mM Sodium Acetate (CH3COONa). The theoretical carbon content for MinQ can be determined as follows: 

1. First determine the total molecular weight of each molecule by adding the molecular mass of each individual element. The percent carbon of each molecule should be individually calculated according to the equation below, where the total mass of carbon in the molecule is divided by the total molecular weight of the molecule. 

% Carbon in MinQ:

Where,

o    Total mass of carbon in MinQ: 120.11 g/mol

o    Total molecular weight of MinQ: 307.326 g/mol

o    Percent carbon in MinQ: 39.08 %

% Carbon in Sorbitol:

Where,

o    Total mass of carbon in Sorbitol: 72.066 g/mol

o    Total molecular weight: 182.174 g/mol

o    Percent carbon in Sorbitol: 39.56 %

% Carbon in Sodium Acetate:

Where,

o    Total mass of carbon in Sodium Acetate: 24.022 g/mol

o    Total molecular weight of Sodium Acetate: 82.034 g/mol

o    Percent carbon in Sodium Acetate: 29.28 %

2. For the non-API molecules (i.e., Sorbitol and Sodium Acetate) derive the concentration of these molecules in the formulation according to the following equation. Note: This step is not applicable for the API molecule, MinQ, as the target concentration (10 mg/mL ) is given.

Concentration of Sorbitol in the formulation:

Where,

o    Concentration given: 10.0 mM

o    Molecular weight: 182.172 g/mol

o    Concentration in formulation: 1.8217 mg/mL

Concentration of Sodium Acetate in the formulation:

Where,

o    Concentration given: 5.0 mM

o    Molecular weight: 82.034 g/mol

o    Concentration in formulation: 0.4102 mg/mL

3. Next, determine the carbon content for each molecule (i.e., MinQ, Sorbitol, and Sodium Acetate) in the formulation according to the following calculation:

Carbon content of MinQ:

Where,

o    Percent carbon in MinQ: 39.08 %

o    Concentration of MinQ in the formulation: 10 mg/mL

o    Carbon content of MinQ: 3.908 mg/mL

Carbon content of Sorbitol:

Where,

o    Percent carbon in Sorbitol: 39.56 %

o    Concentration of Sorbitol in the formulation is 1.8217 mg/mL

o    Carbon content of Sorbitol: 0.72 mg/mL

Carbon content of Sodium Acetate:

Where,

o    Percent carbon in Sodium Acetate: 29.28 %

o    Concentration of Sodium Acetate in the formulation: 0.4102 mg/mL

o    Carbon content of Sodium Acetate: 0.12 mg/mL

4. Sum the derived carbon content of each molecule (represented as E) to determine the total carbon content (represented as ETotal) in the product formulation according to the following calculation.

Total carbon content of MinQ in the final formulation buffer:

Where,

o    Carbon content of MinQ: 3.908 mg/mL

o    Carbon content of Sorbitol: 0.72 mg/mL

o    Carbon content of Sodium Acetate: 0.12 mg/mL

o    Total carbon content: 4.748 mg/mL

5. Sum the concentration of each molecule (represented as D) to determine the total molecular content (represented as DTotal) of the product formulation according to the following calculation.

Total molecular content of MinQ in the final formulation buffer:

Where,

o    Concentration of MinQ: 10 mg/mL

o    Concentration of Sorbitol: 1.8217 mg/mL

o    Concentration of Sodium Acetate: 0.4102 mg/mL

o    Total molecular content: 12.232 mg/mL

6. Lastly, to determine the total percent carbon of the product formulation, divide the total carbon content (step 4), by the total molecular content (step 5), and convert it to a percent according to the following calculation.

Total percent carbon of MinQ in the final formulation buffer:

The theoretical percent carbon for MinQ at its nominal concentration of 10 mg/mL in the final formulation buffer is 38.82 %. As previously discussed, the theoretical percent carbon should be accounted for in TOC recovery studies and in the establishment of swab and rinse limits. Continuing with our example:

1. During a recovery study, if 10 mg of MinQ is spiked onto the coupon, and the theoretical percent carbon is 38.82 %, then the amount of organic carbon deposited from MinQ is 3.882 mg.

2. When calculating cleaning limits, if the health-based swab MACO limit is 15 ppm, the swab recovery factor has been established as 0.75 and the theoretical percent carbon is 38.82 %, then the corrected TOC swab limit will be 4.37 ppm[A]. The same correction factors should be used when calculating TOC rinse limits.

[A] For more information on limits, refer to How to Establish Swab and Rinse Limits for Product Residues

Typically, documentation for product-specific carbon content calculations is summarized such as in the tables shown below, using the same example.

MinQ Molecular Formula	C10H17N3O6S
	Element	C	H	N	O	S
	Mass	12.011	1.008	14.007	15.999	32.065
	Content	10	17	3	6	1
	Mass Totals (A)	120.11	17.136	42.021	95.994	32.065
Molecular Weight   (Sum of A Values)	g/mol (B)	307.326
Carbon Content  (Carbon A value / B) x 100	% C (E)	39.08
Target Concentration	mg/mL (D)	10.0
Carbon in Formulation   (C / 100) x D	mg/mL (E)	3.908

Sorbitol 10 mM Molecular Formula	C6H14O6
	Element	C	H	O
	Mass	12.011	1.008	15.999
	Content	6	14	6
	Mass Totals (A)	72.066	14.112	95.994
Molecular Weight   (Sum of A Values)	g/mol (B)	182.172
Carbon Content  (Carbon A value / B) x 100	% C (E)	39.56
Concentration in Formulation (10mM) x B)	mg/mL (D)	1.8217
Carbon in Formulation   (C / 100) x D	mg/mL (E)	0.72

Sodium Acetate 5.0 mM Molecular Formula	CH3COONa

	Element	C	H	O	Na
	Mass	12.011	1.008	15.999	22.989
	Content	2	3	2	1
	Mass Totals (A)	24.022	3.024	31.998	22.989
Molecular Weight   (Sum of A Values)	g/mol (B)	82.033
Carbon Content  (Carbon A value / B) x 100	% C (E)	29.28
Concentration in Formulation(5mM) x B)	mg/mL (D)	0.4102
Carbon in Formulation ((C / 100) x D)		0.12

Carbon Content of Product MinQ
Total carbon content in final formulation (sum of E values)	4.748
Total molecular content in final formulation (sum of D values)	12.232
% carbon content in final formulation (sum of E values / sum of D values) x 100	38.82 %

Utilizing the methods shown in this memo will ensure the theoretical carbon content is correctly calculated further supporting TOC swab and rinse recovery values and establishment of acceptance limits. By accounting for theoretical carbon content, our TOC samples will accurately reflect the level of recovered product residue on equipment surfaces.

References -

1. Parenteral Drug Association Technical Report 29, Points to Consider for Cleaning Validation

2.  European Medicines Agency, Guideline on setting health based exposure limits for use in risk identification in the manufacture of different medicinal products in shared facilities

3. Parenteral Drug Association Technical Report 49, Points to Consider for Biotechnology Cleaning Validation

Contributors: Harrison Sweeney, Joanna Joseph, and Jenna Carlson